WO2025006460A1 - Systems and methods of sequencing polynucleotides with modified bases - Google Patents

Abstract

Described are DNA sequencing systems and methods. Systems and methods for determining the nucleotide sequence of a polynucleotide may include determining the sequence of a polynucleotide with more than four types of bases by increasing the number of encoding states in sequencing by synthesis (SBS) systems. The systems and methods may expand the encoding space of current systems by discretizing the amplitude space in a channel into more than two states, and/or by adding additional channels.

Description

ILLINC.763WO / IP-2525-PCT PATENT SYSTEMS AND METHODS OF SEQUENCING POLYNUCLEOTIDES WITH MODIFIED BASES INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No.63/511,366 filed June 30, 2023, the content of which is incorporated by reference in its entirety. BACKGROUND Field [0002] The present disclosure relates to DNA sequencing systems and methods. In particular, this disclosure relates to improved detection methods for detecting more than four nucleotides by increasing the types of nucleotides detectable in an image, and by increasing the number of images detecting nucleotides. Background [0003] Current sequencing technologies involve determining DNA or RNA sequences by deciphering four natural bases in the genome: A, T (U), G, and C. However, many of these DNA or RNA sequences have modified nucleotide bases. These modified bases play essential roles in biological processes such as epigenetic studies, epi-transcriptomics, human diseases, and cancer. A common form of DNA modification is a methylated C (5-methylcytosine or 5-MeC) base found in CpG dinucleotides. RNA modifications can also arise from noncoding RNA, such as ribosomal RNA and transfer RNA. The current standard for DNA methylation analysis typically uses genome sequencing of bisulfite-converted DNA. Since uracil, read as thymine, will bind to complementary adenosine, 5-MeC can be partially inferred with existing four base detection systems. However, as the number and complexity of chemical base modifications continue to grow, it may be advantageous to be able to detect more than the four unmodified bases during a DNA sequencing process. [0004] Current base calling schemes for four bases generally include either two or four- channel base calling. Some sequencing systems, such as those from Illumina, Inc. (San Diego, CA) use onboard real-time analysis (RTA) to turn raw image data into base calls. This process can be massively parallelized in order to occur in real-time on the instrument. The number of images fed into the RTA base calling software could be either four images (referred to as four-channel base calling) or two images (referred to as two-channel base calling). [0005] For two-channel base calling systems, the four DNA bases are encoded using two bits of information from the two channels (“on and “off” states). After extracting the signal from the images, the intensities are first normalized and scaled through various correction algorithms. A probabilistic model that uses an assumption of equal base diversity is typically used during base-calling to map a probability density distribution of detecting each base based on the observed fluorescence emissions. Once this probabilistic model is determined, the base calls can then be assigned based on the inference outcome of the probabilistic model (i.e., the most likely base). In contrast, a typical four channel base calling system might simply associate each color channel with a specific fluorescently labeled nucleotide base. Using a corrected intensity, the base- calling step assigns a base call label based on the channel with the highest amplitude. Both processes may be repeated for every well on the flow cell surface and every chemistry cycle in the sequencing instrument. [0006] Typical two channel and four channel systems are designed to detect only four distinct fluorescent signals corresponding to the four natural bases, and as configured, cannot determine more than four types of nucleotides. Existing RTA models assume equal base diversity and are trained on the four natural bases and are not currently suitable for detecting more than four bases. The dyes and ratios of dyes used for typical two channel and four channel systems are optimized to resolve four distinct fluorescent signals. Accordingly, typical systems and methods currently do not distinguish between any additional bases and the four natural bases, leading to errors in base identification. SUMMARY [0007] An aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting the absence of fluorescent emissions from a fourth labeled nucleotide; detecting fluorescent emissions from a fifth labeled nucleotide at a third wavelength; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions wherein at least one of the labeled nucleotides is a modified nucleotide. [0008] In some embodiments, the one or more modified nucleotides can include a 5- methylcytosine, a N6-methyladenine, or an inosine. In some embodiments, a method may include the step of detecting the fluorescent emissions at the third wavelength includes detecting the fluorescent emissions at a first intensity and at a second intensity, wherein the first intensity corresponds to a first modified nucleotide and wherein the second intensity corresponds to a second modified nucleotide. [0009] In some embodiments, the flow cell includes wells configured to bind polynucleotides; further wherein the incorporation of one of the at least four labelled nucleotide conjugates into a well is detected from at least one signal state. In some embodiments, the presence of an empty well is determined from a dark state. In some embodiments, base calling may be performed by a clustering model where each of the at least four labelled nucleotides are distributed as a cloud in an X-Y scatterplot with an intensity level of at least one signal state such that each labelled nucleotide is more similar to nucleotides of the same label than to those from different labelled nucleotides. In some embodiments, at least four labelled nucleotides are distributed as a cloud with an intensity level in at least two signal states. [0010] In some embodiments, empty wells are distributed as a cluster with an intensity of at least one signal state such that each empty well is more similar to empty wells than to those from the different detectable nucleotide conjugate types, and there are at least five clusters. In some embodiments, assigning empty wells is performed by analyzing if a dark state exists for the first ten cycles of a sequencing run. [0011] In some embodiments, a method may include the step of labelling of empty wells by analyzing if a series of a single nucleotide exists in the first ten cycles of a sequencing reaction, and relabeling subsequent nucleotides as an empty well. In some embodiments, signals from all nucleotides and empty wells are segmented into separate populations. In some embodiments, a method may include the step of segmenting the population of a cloud in an X-Y scatterplot using Otsu's method. [0012] In some embodiments, a method may include the step of segmenting the population of a cloud in an X-Y scatterplot using a k-means algorithm. In some embodiments, a method may include the step of segmenting the population of a cloud in an X-Y scatterplot using an expectation maximization algorithm. In some embodiments, a method may include a determining step that is performed for at least one of a single cycle, a few early cycles, or every cycle in a sequencing run. In some embodiments, a method may include a determining step that is present for every cycle in a sequencing run, and the determining step is further used to detect that an insert was completely sequenced. [0013] In some aspects, the techniques described herein relate to a non-transitory computer-readable medium storing a polynucleotide sequencing program including instructions that, when executed by a processor, causes a polynucleotide sequencing apparatus to: detect fluorescent emissions from a first labeled nucleotide at a first wavelength; detect fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detect fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detect the absence of fluorescent emissions from the fourth labeled nucleotide; detect fluorescent emissions from one or more modified nucleotides at a third wavelength; and determine the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions. [0014] In some aspects, the techniques described herein relate to a system for sequencing polynucleotides bound to a flow cell, including: a machine-readable memory; and a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting the absence of fluorescent emissions from the fourth labeled nucleotide; detecting fluorescent emissions from one or more modified nucleotides at a third wavelength; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions. [0015] An aspect of the disclosure is directed to amplitude multiplexing. For example, an aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flow cell, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting fluorescent emissions from a fourth labeled nucleotide at the first wavelength and a third intensity that is less than the first intensity; detecting fluorescent emissions from a fifth labeled nucleotide at the second wavelength and a fourth intensity that is less than the second intensity; and determining the sequence of the polynucleotides based on the detected fluorescent emission and intensity. [0016] In some embodiments, a method may include the step of detecting fluorescent emissions from a sixth labeled nucleotide at the first wavelength and a fifth intensity that is less than the third intensity, and at the second wavelength and a sixth intensity that is less than the fourth intensity. [0017] In some embodiments, a method may include the step of detecting fluorescent emissions from a seventh labeled nucleotide at the first wavelength and a seventh intensity that is greater than the third intensity, and at the second wavelength and an eighth intensity that is less than the second intensity; wherein detecting fluorescent emissions from a third labeled nucleotide at the first wavelength is greater than the third intensity and at the second wavelength is greater than the fourth intensity. [0018] In some embodiments, a method may include the step of detecting fluorescent emissions from an eighth labeled nucleotide at the first wavelength and a ninth intensity that is less than the first intensity, and at the second wavelength and tenth intensity that is greater than the fourth intensity. In some embodiments, a method may include the step of detecting the absence of fluorescent emissions at the first wavelength and at the second wavelength and determining the absence of a polynucleotide. In some embodiments, a method may include the step of detecting the absence of fluorescent emissions at the first wavelength and at the second wavelength and determining the absence of a ninth labeled polynucleotide. [0019] In some embodiments, the intensity of fluorescent emissions at a wavelength for a polynucleotide is reduced, relative to other fluorescent emissions at the wavelength for a different polynucleotide, by using smaller stoichiometries of a fluorophore dye for the nucleotide detected at a lower intensity. In some embodiments, the intensity of fluorescent emissions at a wavelength for a polynucleotide is reduced, relative to other fluorescent emissions at the wavelength for a different polynucleotide, by using a different fluorophore dye with lower emission at the wavelength for the nucleotide detected at a lower intensity. [0020] An aspect of the disclosure is directed to base calling of five types of nucleotides. For example, an aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting the absence of fluorescent emissions from the fourth labeled nucleotide; detecting fluorescent emissions from a fifth nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions. [0021] An aspect of the disclosure is directed to four channel sequencing systems and methods that employ four channels and a dark state to identify five nucleotides. For example, an aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths; detecting fluorescent emissions from a fourth labeled nucleotide at a fourth wavelength, wherein the fourth wavelength is different from the first, second, and third wavelengths; detecting the absence of fluorescent emissions from a fifth labeled nucleotide; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions. [0022] In some embodiments, a method may include the step of detecting fluorescent emissions from a fifth nucleotide at a third wavelength occurs after a chemical processing step. [0023] In some aspects, the techniques described herein relate to a method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths; detecting fluorescent emissions from a fourth labeled nucleotide at a fourth wavelength, wherein the fourth wavelength is different from the first, second, and third wavelengths; detecting the absence of fluorescent emissions from a fifth labeled nucleotide; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions. [0024] An aspect of the disclosure relates to mitigating potential issues with assigning bases (or empty wells) that have a low probability of occurring, due to, for example, low base diversity. By labeling more than five nucleotides, more than five clouds may be present in a two- dimensional plot of intensities in 2-channel SBS. An aspect of the disclosure is directed to addressing the more than five clouds in RTA methods. In some embodiments, two channel and four channel systems may be adapted to determine more than four types of nucleotides. RTA models according to the disclosure may address equal base diversity assumptions and may train on more than the four natural bases. The disclosure provides for dyes and ratios of dyes resulting in distinct fluorescent signals and intensities to resolve more than four bases. BRIEF DESCRIPTION OF THE DRAWINGS [0025] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear. While the disclosure has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The disclosure is not limited to the disclosed embodiments. Variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed disclosure, from a study of the drawings, the disclosure and the appended claims. [0026] FIG. 1A schematically illustrates an example sequencing system which can perform embodiments of the disclosed sequencing technology. [0027] FIG. 1B schematically illustrates an example imaging system to be used in embodiments of the disclosed sequencing technology. [0028] FIG.2 is a flowchart illustrating a process of sequencing polynucleotides bound to a flow cell with two or more detection channels. [0029] FIG.3 is a flowchart illustrating a process of sequencing polynucleotides bound to a flow cell with two or more encoding states in per channel, according to some embodiments of the present disclosure. [0030] FIG. 4 shows X-Y scatterplots in three panels of the fluorescent emissions for a two channel system with two or more encoding states in per channel, according to some embodiments of the present disclosure. [0031] FIG.5 is a flowchart illustrating a process of sequencing polynucleotides bound to a flow cell, according to some embodiments of the present disclosure. [0032] FIG. 6 shows two panels illustrating examples of base calling for five bases, with the left panel showing an example X-Y axis scatterplot of the fluorescence emissions in two channels, and the right panel showing an example X-Y axis graph of the probability density distribution for detecting fluorescent emissions over channel three for the five nucleotides in the left panel. [0033] FIG. 7 shows a three-dimensional X-Y-Z example scatterplot of the detected fluorescence emissions in three channels, according to some embodiments of the present disclosure. [0034] FIG.8 is a flowchart illustrating a process of sequencing polynucleotides bound to a flow cell, according to some embodiments of the present disclosure. [0035] FIG. 9 illustrates in three bar charts, which provide three examples of base calling in a system configured to detect five bases with four channels, where each panel of summarizes a different base calling scenario through a X-Y histogram of emissions intensities in four channels. The left panel shows a T basecall, the middle panel shows a C basecall and the right panel shows a G basecall. [0036] FIG. 10 shows two X-Y axis scatterplot panels of the fluorescence emissions of eight nucleotides in a total of four channels, according to some embodiments of the present disclosure. The left panel shows a first two color channel, bi-level encoding scheme. The right panel shows a second two color channel, bi-level encoding scheme. [0037] FIG. 11 schematically illustrates a system including a memory comprising a sequencing module. DETAILED DESCRIPTION [0038] All patents, applications, published applications and other publications referred to herein are incorporated herein by reference to the referenced material and in their entireties. If a term or phrase is used herein in a way that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the use herein prevails over the definition that is incorporated herein by reference. [0039] One aspect of the disclosure provides for systems and methods for determining the sequence of a polynucleotide with more than four types of bases by increasing the number of encoding states in a sequencing by synthesis (SBS) system. In order to differentiate between bases, each base produces a unique encoding state that allows the system to distinguish the fluorescent signals coming from clusters of nucleic acids so that they can be detected and used to accurately identify the base. Current systems, including two and four channel systems, are typically designed to detect only four distinct fluorescent signals, where each signal is assigned to one of the four natural bases. The systems and methods of this disclosure expand upon the encoding space of current systems by discretizing the amplitude space in a channel into more than two states, and/or by adding additional channels. [0040] Expanding the encoding space of current systems can be done by assigning unique labels to each sample so they can be multiplexed. This allows for greater than four bases to be identified in a single sequencing experiment. One such example is “amplitude multiplexing”, where one channel may include an “off” state, an “on” state, and in addition, an intermediate state that is “half on, half off” state where the fluorescent label provides approximately 50% intensity of the full “on” state. One example of channel multiplexing is where a three channel system (two plus one channels) may identify the four natural bases with two of the three channels, leaving the third channel to identify a fifth modified base. In some embodiments, amplitude multiplexing and channel multiplexing may be combined, as will be disclosed more fully with reference to Fig.7 and Table 1 below. [0041] The disclosure provides for systems and methods for determining the sequence of a polynucleotide by labeling at least four nucleotides in a sequencing by synthesis (SBS) system. In some embodiments, the polynucleotides have four natural bases, and a modified base. In this embodiment, a two-dimensional scatterplot showing the measured wavelengths and intensities from each fluorescent label will have at least five distinct cloud formations, one for each fluorescent label. For example, a first base A may be labeled with a first fluorophore within a cluster bound to all of the A bases which are to be incorporated into an insert during SBS sequencing reactions. A second base may be labeled with a second fluorophore bound to all of the C bases which are to be incorporated into the insert during the SBS sequencing reactions. A third base T may be labeled with both the first and the second fluorophore on each of the T bases to be incorporated into the insert during SBS sequencing reactions. The fourth base G may be a dark base. And finally, the modified base “X” may be labeled with the first fluorophore at a reduced intensity. Thus, only a percentage of the labeled X bases incorporated into the insert during SBS sequencing reactions would be labelled bases, so that the intensity of the fluorescence from this modified base would be detectibly lower than the intensity of a base that was fully labeled. For example, only 50% of the X bases may be labeled with the first fluorophore. In this way, the intensity of the X nucleotides would be half of the intensity of any base that was 100% labeled with the first fluorophore even though they are both labeled with the same fluorophore. [0042] It should be realized that the intensity of any of the fluorophores used in embodiments of the invention can be any detectable percentage of a full-intensity fluorophore by mixing labeled and unlabeled nucleotides together prior to contact with the flow cell. For example, a mixture of 50% labeled C nucleotides and 50% unlabeled C nucleotides will result in the sequencing system detecting an intensity of C nucleotides that is 50% of the expected full fluorescence of a cluster of sequences as compared to a cluster of all labeled C nucleotides. In this example, the nucleotide “C” is encoded in an intermediate state that is “half on, half off” state with approximately 50% intensity of the full “on” state. Of course, any percentage mixture of labeled and unlabeled nucleotides may be used so that a desired percentage intensity may come from a cluster during sequencing runs. For example, 30, 40, 50, 60, 70, or 80 percent of labeled nucleotides may be used, along with unlabeled nucleotides, to result in an intensity that is 30, 40, 50, 60, 70, or 80 percent, respectively, of a full intensity signal for a cluster having that particular fluorophore. [0043] One other aspect of the disclosure is a method of sequencing polynucleotides bound in clusters to a flow cell by using increasingly bright dyes, or dyes with high quantum yields for some of the nucleotides. Accordingly, an increased intensity dye may be used to increase the intensity of a nucleotide that may be encoded within a channel. in some embodiments, the method may produce nucleotide clouds in a two dimensional scatterplot which form an L-shaped configuration, where one channel includes bi-level encoding and the other channel includes three states. For example, the method may include labeling a first labeled nucleotide resulting in emissions at a first wavelength and a first intensity. In this example, a first reversibly terminated nucleotide T is labeled with a green dye which emits light at a first wavelength. The method then includes detecting fluorescent emissions from a second reversibly terminated nucleotide A which is labeled with a green dye which emits light at the first wavelength, but at a second intensity that is less than the first intensity. The method then detects fluorescent emissions from a third reversibly terminated nucleotide C which is labeled with a blue dye that emits light at a second wavelength that is different from the first wavelength. The method then includes detecting the absence of fluorescent emissions from an unlabeled fourth reversibly terminated nucleotide G. Moreover, during the library preparation process, particularly modified nucleotides, such as met-C, may have a specific percentage of the modified nucleotides labeled such that a detectable reduction in the light being emitted at the first wavelength can be detected. [0044] Moreover, in some aspects, the disclosure herein relates to a system for performing the above methods of sequencing polynucleotides bound to a flow cell. The system would include a memory linked to one or more processors which are configured to execute machine-readable instructions, which, when executed by the one or more processors, cause the system to perform the above method steps. [0045] Another embodiment is related to the above method, but instead of using nucleotides which have predetermined ratios of fluorophores for some channels, to instead use additional fluorescent channels. For example, four channel base calling may be extended to determine five bases by using the “dark” base encoding, where one of the color channels is used to detect a modified nucleotide, and a natural base is assigned to a dark state. Accurate base calls for four of the five bases may be performed by a “maximum amplitude” approach. To distinguish the fifth base, here one of the natural bases, truly dark wells are distinguished from wells that are just temporarily dark using an empty detection algorithm. In this embodiment, some wells will be empty and not contain any clusters, as cluster formation does not always occur in every well. In these empty wells, a dark state cloud may be formed in a four channel histogram, as will be disclosed more fully with reference to Fig.10 below. [0046] Base calling using these multiplexing encoding schemes may operate in a manner that is similar to two-channel, binary level encoding schemes for the four natural bases. However, one aspect of the disclosure is directed to addressing poor performance associated with identifying bases that are present less often than the four natural bases. Some base calling models operate with an underlying assumption of “base call diversity” baked into the base calling model, where the model assumes that at any given cycle for any given population of clusters, the base representation amongst the four or more bases is roughly equal in ratio. While this assumption holds for naturally occurring DNA bases, introducing additional bases to denote specific features in the genome, this equal diversity assumption might not be realistic for other features in the genome (such as methylated bases). This base diversity challenge may mitigated by, for example, introducing known sequences, including an excess of the less abundant 5th or 6th base, to train RTA and achieve higher accuracy of calling a fifth base. Definitions [0047] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [0048] It is noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless expressly and unequivocally limited to one referent. It will be apparent to those skilled in the art that various modifications and variations can be made to various embodiments described herein without departing from the spirit or scope of the present teachings. Thus, it is intended that the various embodiments described herein cover other modifications and variations within the scope of the appended claims and their equivalents. [0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have”, “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. [0050] All references cited herein are incorporated herein by reference in their entirety. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. [0051] As used herein, common organic abbreviations are defined as follows: °C Temperature in degrees Centigrade dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate ddNTP Dideoxynucleotide triphosphate ffA Fully functionalized A nucleotide ffC Fully functionalized C nucleotide ffG Fully functionalized G nucleotide ffN Fully functionalized nucleotide ffT Fully functionalized T nucleotide h Hour(s) RT Room temperature SBS Sequencing by Synthesis A Adenosine (may refer to nucleotide base calls) C Cytosine G Guanine T Thymine [0052] As used herein, a “peptide” refers to two or more amino acids joined together by an amide bond (that is, a “peptide bond”). Peptides comprise up to or include 50 amino acids. Peptides may be linear or cyclic. Peptides may be Į, ȕ, Ȗ, į, or higher, or mixed. Peptides may comprise any mixture of amino acids as defined herein, such as comprising any combination of D, L, Į, ȕ, Ȗ, į, or higher amino acids. [0053] As used herein, a “protein” refers to an amino acid sequence having 51 or more amino acids. [0054] As used herein, “nucleobase” is a heterocyclic base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof. A nucleobase can be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted at the 8 position with methyl or bromine, 9-oxo-N6-methyladenine, 2- aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine, 2,6- diaminopurine, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)- alkynylcytosine, 5- fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4- triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6- dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturally occurring nucleobases described in U.S. Pat. Nos. 5,432,272 and 6,150,510 and PCT applications WO 92/002258, WO 93/10820, WO 94/22892, and WO 94/24144, and Fasman ("Practical Handbook of Biochemistry and Molecular Biology", pp.385-394, 1989, CRC Press, Boca Raton, LO), all herein incorporated by reference in their entireties. [0055] As used herein, the term “nucleotide” is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some examples also includes a nucleobase. A nucleotide that lacks a nucleobase may be referred to as “abasic.” In some embodiments, a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA), the sugar is a ribose, and in deoxyribonucleotides (DNA), the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present at the 2' position in ribose. The nitrogen containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. The phosphate groups may be in the mono-, di-, or tri-phosphate form. These nucleotides are natural nucleotides, but it is to be further understood that non-natural nucleotides, modified nucleotides or analogs of the aforementioned nucleotides can also be used. [0056] Examples of nucleotides may include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP). [0057] Examples of nucleotides may also be intended to encompass any nucleotide analogue which is a type of nucleotide that includes a modified nucleobase, sugar, backbone, and/or phosphate moiety compared to naturally occurring nucleotides. Nucleotide analogues also may be referred to as “modified nucleic acids.” Example modified nucleobases include inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5- hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5- propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4- thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8- thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3-deazaguanine, 3-deazaadenine or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5'-phosphosulfate. Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates. Nucleotide analogues also include locked nucleic acids (LNA), peptide nucleic acids (PNA), and 5-hydroxylbutynl-2'- deoxyuridine (“super T”). [0058] In some embodiments, the term “modification” as used herein is intended to refer not only to a chemical modification of a nucleic acids, but also to a variation in nucleic acid conformation or composition, interaction of an agent with a nucleic acid (e.g., bound to the nucleic acid), and other perturbations associated with the nucleic acid. As such, a location or position of a modification is a locus (e.g., a single nucleotide or multiple contiguous or noncontiguous nucleotides) at which such modification occurs within the nucleic acid. For a double-stranded template, such a modification may occur in the strand complementary to a nascent strand synthesized by a polymerase processing the template or may occur in the displaced strand. For example, modified nucleotides may include 5-methylcytosine, N6-methyladenosine, N3- methyladenosine, N7-methylguanosine, 5-hydroxymethylcytosine, pseudouridine, thiouridine, isoguanosine, isocytosine, dihydrouridine, queuosine, wyosine, inosine, triazole, diaminopurine, ȕ-D-glucopyranosyloxymethyluracil (a.k.a., ȕ-D-glucosyl-HOMedU, ȕ-glucosyl- hydroxymethyluracil, “dJ,” or “base J”), 8-oxoguanosine, and 2ƍ-O-methyl derivatives of adenosine, cytidine, guanosine, and uridine. Modified DNA and RNA bases are further described, for example, in Narayan P, et al. (1987) Mol Cell Biol 7(4):1572-5; Horowitz S, et al. (1984) Proc Natl Acad Sci U.S.A. 81(18):5667-71; “RNA's Outfits: The nucleic acid has dozens of chemical costumes,” (2009) C&EN; 87(36):65-68; Kriaucionis, et al. (2009) Science 324 (5929): 929-30; and Tahiliani, et al. (2009) Science 324 (5929): 930-35; Matray, et al. (1999) Nature 399(6737):704-8; Ooi, et al. (2008) Cell 133: 1145-8; Petersson, et al. (2005) J Am Chem Soc. 127(5):1424-30; Johnson, et al. (2004) 32(6):1937-41; Kimoto, et al. (2007) Nucleic Acids Res. 35(16):5360-9; Ahle, et al. (2005) Nucleic Acids Res 33(10):3176; Krueger, et al., Curr Opinions in Chem Biology 2007, 11(6):588); Krueger, et al. (2009) Chemistry & Biology 16(3):242; McCullough, et al. (1999) Annual Rev of Biochem 68:255; Liu, et al. (2003) Science 302(5646):868-71; Limbach, et al. (1994) Nucl. Acids Res.22(12):2183-2196; Wyatt, et al. (1953) Biochem. J.55:774-782; Josse, et al. (1962) J. Biol. Chem.237:1968-1976; Lariviere, et al. (2004) J. Biol. Chem. 279:34715-34720; and in International Application Publication No. WO/2009/037473, the disclosures of which are incorporated herein by reference in their entireties. [0059] Modifications may further include the presence of non-natural base pairs in the nucleic acid, including but not limited to hydroxypyridone and pyridopurine homo- and hetero- base pairs, pyridine-2,6-dicarboxylate and pyridine metallo-base pairs, pyridine-2,6- dicarboxamide and a pyridine metallo-base pairs, metal-mediated pyrimidine base pairs T-Hg(II)- T and C-Ag(I)-C, and metallo-homo-basepairs of 2,6-bis(ethylthiomethyl)pyridine nucleobases Spy, and alkyne-, enamine-, alcohol-, imidazole-, guanidine-, and pyridyl-substitutions to the purine or pyridimine base (Wettig, et al. (2003) J Inorg Biochem 94:94-99; Clever, et al. (2005) Angew Chem Int Ed 117:7370-7374; Schlegel, et al. (2009) Org Biomol Chem 7(3):476-82; Zimmerman, et al. (2004) Bioorg Chem 32(1):13-25; Yanagida, et al. (2007) Nucleic Acids Symp Ser (Oxf) 51:179-80; Zimmerman (2002) J Am Chem Soc 124(46):13684-5; Buncel, et al. (1985) Inorg Biochem 25:61-73; Ono, et al. (2004) Angew Chem 43:4300-4302; Lee, et al. (1993) Biochem Cell Biol 71:162-168; Loakes, et al. (2009), Chem Commun 4619-4631; and Seo, et al. (2009) J Am Chem Soc 131:3246-3252, the disclosures of which are incorporated herein by reference in their entireties). Other types of modifications include, e.g, a nick, a missing base (e.g., apurinic or apyridinic sites), a ribonucleoside (or modified ribonucleoside) within a deoxyribonucleoside-based nucleic acid, a deoxyribonucleoside (or modified deoxyribonucleoside) within a ribonucleoside-based nucleic acid, a pyrimidine dimer (e.g., thymine dimer or cyclobutane pyrimidine dimer), a cis-platin crosslinking, oxidation damage, hydrolysis damage, other methylated bases, bulky DNA or RNA base adducts, photochemistry reaction products, interstrand crosslinking products, mismatched bases, and other types of “damage” to the nucleic acid. Modified nucleotides can be caused by exposure of the DNA to radiation (e.g., UV), carcinogenic chemicals, crosslinking agents (e.g., formaldehyde), certain enzymes (e.g., nickases, glycosylases, exonucleases, methylases, other nucleases, glucosyltransferases, etc.), viruses, toxins and other chemicals, thermal disruptions, and the like. [0060] As used herein, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are bonded to one another. A polynucleotide is one nonlimiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues thereof such as locked nucleic acids (LNA) and peptide nucleic acids (PNA). A polynucleotide may be a single stranded sequence of nucleotides, such as RNA or single stranded DNA, a double stranded sequence of nucleotides, such as double stranded DNA, or may include a mixture of a single stranded and double stranded sequences of nucleotides. Double stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice-versa. Polynucleotides may include non-naturally occurring DNA, such as enantiomeric DNA, LNA, or PNA. The precise sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the foregoing. [0061] The terms “oligonucleotide” and “polynucleotide” may be used interchangeably herein. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description, the terms may be used to distinguish one species of polynucleotide from another when describing a particular method or composition that includes several polynucleotide species. [0062] The term “nucleic acid” and “polynucleotide” may be used interchangeably to refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and phosphorothioate DNA. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof. Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2- amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate, pyrrolo- pyrimidine triphosphate, and 2-thiocytidine, as well as the alphathiotriphosphates for all of the above, and 2ƍ-O-methyl-ribonucleotide triphosphates for all the above bases. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP. [0063] As used herein, a “nucleoside” is structurally similar to a nucleotide, but is missing the phosphate moieties. An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule. The term “nucleoside” is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and/or a carbon has been replaced with a sulfur or an oxygen atom. A “nucleoside” is a monomer that can have a substituted base and/or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and/or RNA polymers and oligomers. [0064] The term “purine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term “pyrimidine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g. 7-methylguanine), theobromine, caffeine, uric acid and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (e.g., 5-methylcytosine). [0065] The term “nucleobase” as used herein, is a purine base or a pyrimidine base. Non-limiting examples of purine nucleobases include adenine (A), guanine (G), and derivatives or analogs thereof. Non-limiting examples of pyrimidine nucleobases include cytosine (C), thymine (T), uracil (U), and derivatives or analogs thereof. [0066] As used herein, when an oligonucleotide or polynucleotide is described as “comprising” a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. Similarly, when a nucleoside or nucleotide is described as part of an oligonucleotide or polynucleotide, such as “incorporated into” an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is formed between a 3^ hydroxy group of the oligonucleotide or polynucleotide with the 5^ phosphate group of a nucleotide described herein as a phosphodiester bond between the 3^ carbon atom of the oligonucleotide or polynucleotide and the 5^ carbon atom of the nucleotide. [0067] As used herein, the term “array” refers to a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be differentiated from each other according to relative location. An array can include different probe molecules that are each located at a different addressable location on a substrate. Alternatively, or additionally, an array can include separate substrates each bearing a different probe molecule, wherein the different probe molecules can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in U.S. Patent No. 6,1055,331 B1, US 2002/0102578 and PCT Publication No. WO 00/63437. Exemplary formats that can be used in the invention to distinguish beads in a liquid array, for example, using a microfluidic device, such as a fluorescent activated cell sorter (FACS), are described, for example, in US Pat. No. 6,524,793. Further examples of arrays that can be used in the invention include, without limitation, those described in U.S. Pat Nos. 5,329,807; 5,336,1027; 5,561,071; 5,583,911; 5,658,734; 5,837,858; 5,874,919; 5,919,523; 6,836,969; 6,987,768; 6,987,776; 6,988,920; 6,997,006; 6,991,893; 6,1046,313; 6,316,949; 6,382,591; 6,514,751 and 6,610,382; and WO 93/17126; WO 95/11995; WO 95/35505; EP 742987; and EP 799897. [0068] A nucleotide analog may be attached to or associated with one or more photo- detectable labels to provide a detectable signal. In some embodiments, a photo-detectable label may be a fluorescent compound, such as a small molecule fluorescent label. Fluorescent molecules (fluorophores) suitable as a fluorescent label include, but are not limited to: 1,5 IAEDANS; 1,8- ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); fluorescein amidite (FAM); 5-carboxynapthofluorescein; tetrachloro-6-carboxyfluorescein (TET); hexachloro-6-carboxyfluorescein (HEX); 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE); VIC®; NED™; tetramethylrhodamine (TMR); 5-carboxytetramethylrhodamine (5- TAMRA); 5-HAT (Hydroxy Tryptamine); 5-hydroxy tryptamine (HAT); 5-ROX (carboxy-X- rhodamine); 6-carboxyrhodamine 6G; 6-JOE; Light Cycler® red 610; Light Cycler® red 640; Light Cycler® red 670; Light Cycler® red 705; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine; 6-methoxy-N- (4-aminoalkyl)quinolinium bromide hydrochloride (ABQ); Acid Fuchsin; ACMA (9-amino-6- chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; AFPs-AutoFluorescent Protein-(Quantum Biotechnologies); Texas Red; Texas Red-X conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (TetramethylRodamine-lsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; X-Rhodamine-5-(and-6)-Isothiocyanate (5(6)-XRITC); Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; interchelating dyes such as YOYO-3, Sybr Green, Thiazole orange; members of the Alexa Fluor® dye series (from Molecular Probes/Invitrogen) which cover a broad spectrum and match the principal output wavelengths of common excitation sources such as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750; members of the Cy Dye fluorophore series (GE Healthcare), also covering a wide spectrum such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7; members of the Oyster® dye fluorophores (Denovo Biolabels) such as Oyster- 500, -550, -556, 645, 650, 656; members of the DY-Labels series (Dyomics), for example, with maxima of absorption that range from 418 nm (DY-415) to 844 nm (DY-831) such as DY-415, - 495, -505, -547, -548, -549, -550, -554, -555, -556, -560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700, -701, -730, -731, -732, -734, -750, -751, -752, -776, -780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL; members of the ATTO series of fluorescent labels (ATTO-TEC GmbH) such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740; members of the CAL Fluor® series or Quasar® series of dyes (Biosearch Technologies) such as CAL Fluor® Gold 540, CAL Fluor® Orange 560, Quasar® 570, CAL Fluor® Red 590, CAL Fluor® Red 610, CAL Fluor® Red 635, Quasar® 570, and Quasar® 670. In some embodiments, a first photo-detectable label interacts with a second photo-detectable moiety to modify the detectable signal, e.g., via fluorescence resonance energy transfer (“FRET”; also known as Förster resonance energy transfer). [0069] The fluorescent labels utilized by the systems and methods disclosed herein can have different peak absorption wavelengths, for example, ranging from 400 nm to 800 nm. In some embodiments, the peak absorption wavelengths of the fluorescent labels can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments the peak absorption wavelengths of the fluorescent labels can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. [0070] The fluorescent labels can have different peak emission wavelength, for example, ranging from 400 nm to 800 nm. In some embodiments, the peak emission wavelengths of the fluorescent labels can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments the peak emission wavelengths of the fluorescent labels can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. [0071] The fluorescent labels can have different Stokes shift, for example, ranging from 10 nm to 200 nm. In some embodiments, the stoke shift can be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a number or a range between any two of these values. In some embodiments, the stoke shift can be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. [0072] In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can vary, for example, ranging from 10 nm to 200 nm. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a number or a range between any two of these values. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. [0073] A “light source” may be any device capable of emitting energy along the electromagnetic spectrum. A light source may be a source of visible light (VIS), ultraviolet light (UV) and/or infrared light (IR). “Visible light” (VIS) generally refers to the band of electro- magnetic radiation with a wavelength from about 400 nm to about 750 nm. “Ultraviolet (UV) light” generally refers to electromagnetic radiation with a wavelength shorter than that of visible light, or from about 10 nm to about 400 nm range. “Infrared light” or infrared radiation (IR) generally refers to electromagnetic radiation with a wavelength greater than the VIS range, or from about 750 nm to about 50,000 nm. A light source may also provide full spectrum light. Light sources may output light from a selected wavelength or a range of wavelengths. In some embodiments of the invention, the light source may be configured to provide light above or below a predetermined wavelength, or may provide light within a predetermined range. A light source may be used in combination with a filter, to selectively transmit or block light of a selected wavelength from the light source. A light source may be connected to an intensity source by one or more electrical connectors; an array of light sources may be connected to an intensity source in series or in parallel. An intensity source may be a battery, or a vehicle electrical system or a building electrical system. The light source may be connected to an intensity source via control electronics (control circuit); control electronics may comprise one or more switches. The one or more switches may be automated, or controlled by a sensor, timer or other input, or may be controlled by a user, or a combination thereof. For example, a user may operate a switch to turn on a UV light source; the light source may be applied on a constant basis until it is turned off, or it may be pulsed (repeated on/off cycles) until it is turned off. In some embodiments, the light source may be switched from a continuously-on state to a pulsed state, or vice versa. In some embodiments, the light source may be configured to be brightening or darkening over time. [0074] For operation, the light source may be connected to an intensity source capable of providing sufficient intensity to illuminate the sample. Control electronics may be used to switch the intensity on or off based on input from a user or some other input, and can also be used to modulate the intensity to a suitable level (e.g. to control brightness of the output light). Control electronics may be configured to turn the light source on and off as desired. Control electronics may include a switch for manual, automatic, or semi-automatic operation of the light sources. The one or more switches may be, for example, a transistor, a relay or an electromechanical switch. In some embodiments, the control circuit may further comprise an AC-DC and/or a DC-DC converter for converting the voltage from the voltage source to an appropriate voltage for the light source. The control circuit may comprise a DC-DC regulator for regulation of the voltage. The control circuit may further comprise a timer and/or other circuitry elements for applying electric voltage to the optical filter for a fixed period of time following the receipt of input. A switch may be activated manually or automatically in response to predetermined conditions, or with a timer. For example, control electronics may process information such as user input, stored instructions, or the like. [0075] One or more of a plurality of light sources may be provided. In some embodiments, each of the plurality of light sources may be the same. Alternatively, one or more of the light sources may vary. The light characteristics of the light emitted by the light sources may be the same or may vary. A plurality of light sources may or may not be independently controllable. One or more characteristic of the light source may or may not be controlled, including but not limited to whether the light source is on or off, brightness of light source, wavelength of light, intensity of light, angle of illumination, position of light source, or any combination thereof. [0076] In some embodiments, light output from a light source may be from about 350 to about 750 nm, or any amount or range therebetween, for example from about 350 nm to about 360, 370, 380, 390, 400, 410, 420, 430 or about 450 nm, or any amount or range therebetween. In other embodiments, light from a light source may be from about 550 to about 700 nm, or any amount or range therebetween, for example from about 550 to about 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690 or about 700 nm, or any amount or range therebetween. In some embodiments, the wavelength of the light generated by the light source can vary, for example, ranging from 400 nm to 800 nm. In some embodiments, the wavelength of the light generated by the light source can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments, the wavelength of the light generated by the light source can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. The light source may be capable of emitting electromagnetic waves in any spectrum. In some embodiments, the light source may have a wavelength falling between 10 nm and 100 ^m. In some embodiments, the wavelength of light may fall between 100 nm to 5000 nm, 300 nm to 1000 nm, or 400 nm to 800 nm. In some embodiments, the wavelength of light may be less than, and/or equal to 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1100 nm, 1300 nm, 1500 nm, 1750 nm, 2000 nm, 2500 nm, 3000 nm, 4000 nm, or 5000 nm. [0077] In one example, a light source may be a light-emitting diode (LED) (e.g., gallium arsenide (GaAs) LED, aluminum gallium arsenide (AlGaAs) LED, gallium arsenide phosphide (GaAsP) LED, aluminum gallium indium phosphide (AlGaInP) LED, gallium(III) phosphide (GaP) LED, indium gallium nitride (InGaN)/gallium(III) nitride (GaN) LED, or aluminum gallium phosphide (AlGaP) LED). In another example, a light source can be a laser, for example a vertical cavity surface emitting laser (VCSEL) or other suitable light emitter such as an Indium-Gallium-Aluminum-Phosphide (InGaAIP) laser, a Gallium-Arsenic Phosphide/Gallium Phosphide (GaAsP/GaP) laser, or a Gallium-Aluminum-Arsenide/Gallium-Aluminum-Arsenide (GaAIAs/GaAs) laser. Other examples of light sources may include but are not limited to electron stimulated light sources (e.g., Cathodoluminescence, Electron Stimulated Luminescence (ESL light bulbs), Cathode ray tube (CRT monitor), Nixie tube), incandescent light sources (e.g., Carbon button lamp, Conventional incandescent light bulbs, Halogen lamps, Globar, Nernst lamp), electroluminescent (EL) light sources (e.g., Light-emitting diodes—Organic light-emitting diodes, Polymer light-emitting diodes, Solid-state lighting, LED lamp, Electroluminescent sheets Electroluminescent wires), gas discharge light sources (e.g., Fluorescent lamps, Inductive lighting, Hollow cathode lamp, Neon and argon lamps, Plasma lamps, Xenon flash lamps), or high-intensity discharge light sources (e.g., Carbon arc lamps, Ceramic discharge metal halide lamps, Hydrargyrum medium-arc iodide lamps, Mercury-vapor lamps, Metal halide lamps, Sodium vapor lamps, Xenon arc lamps). Alternatively, a light source may be a bioluminescent, chemiluminescent, phosphorescent, or fluorescent light source. [0078] Optical filters may be tuned in terms of clarity or haze, translucency, transparency or opacity, light transmittance (LT), switching speed, durability, photostability, contrast ratio, state of light transmittance (e.g. dark state or light state). “Light transmittance” (LT) refers to the quantity of light that is transmitted or passes through an optical filter, or device or apparatus comprising same. LT may be expressed with reference to a change in light transmission and/or a particular type of light or wavelength of light (e.g. from about 10% visible light transmission (LT) to about 90% LT, or the like). LT may alternately be expressed as absorbance, and may optionally include reference to one or more wavelengths that are absorbed. According to some embodiments, an optical filter may be selected, or configured to have in one state, a LT of less than 80%, or less than 70%, or less than 60%, or less than 50%, or less than 40%, or less than 30%, or less than 20% or less than 10%, or any amount or range therebetween. According to some embodiments, an optical filter may be selected, or configured to have in another state, a LT of greater than 80%, or greater than 70%, or greater than 60%, or greater than 50%, or greater than 40%, or greater than 30%, or greater than 20% or greater than 10%, or any amount or range therebetween. [0079] A filter can be a bandpass filter and can have peak transmittance of varying wavelength, ranging from 400 nm to 800 nm. In some embodiments, the peak transmittance can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments, the peak transmittance can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. The width of the transmission window of a filter can vary, for example, ranging from 1 nm to 50 nm. In some embodiments, the width of the filter can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nm, or a number or a range between any two of these values. In some embodiments, the width of the filter can be at least, or at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nm. A shortpass filter may be considered a special bandpass filter having the lower limit of the transmission window close to 0 nm. A longpass filter may be considered a special bandpass filter having the upper limit of the transmission window close to infinity. A bandstop filter may be defined as complementary to some bandpass filter. [0080] As used herein, an “optical channel” is a predefined profile of optical frequencies (or equivalently, wavelengths). For example, a first optical channel may have wavelengths of 500 nm–600 nm. To take an image in the first optical channel, one may use a detector which is only responsive to 500 nm–600 nm light, or use a bandpass filter having a transmission window of 500 nm–600 nm to filter the incoming light onto a detector responsive to 300 nm–800 nm light. A second optical channel may have wavelengths of 300 nm–450 nm and 850 nm–900 nm. To take an image in the second optical channel, one may use a detector responsive to 300 nm–450 nm light and another detector responsive to 850 nm–900 nm light and then combine the detected signals of the two detectors. Alternatively, to take an image in the second optical channel, one may use a bandstop filter which rejects 451 nm–849 nm light in front of a detector responsive to 300 nm–900 nm light. [0081] As used herein, the term “cluster” may refer to either a cluster of polynucleotides bound to a flow cell, or to a cluster of data points in an X-Y scatterplot, which is also referred to herein as a “cloud.” The distinction between the uses of the term will be obvious from the context, and in any scenario where the use of the term cluster could equally apply to either interpretation, the description may encompass both uses of this term. Example Sequencer [0082] In FIG. 1A, an example sequencing system 100 which can perform the disclosed sequencing technology is illustrated. The sequencing system 100 can be configured to utilize disclosed sequencing methods based on a single optical excitation and a single detection channel. Non-limiting examples of the sequencing reactions utilized can include variations of sequencing-by-synthesis processes, such as those used in Illumina® dye sequencing or HeliScope® single molecule sequencing. [0083] The sequencing system 100 can include an optics system 102 configured to generate raw sequencing data using sequencing reagents supplied by a fluidics system 104 that is part of the sequencing system 100. The raw sequencing data can include fluorescent images captured by the optics system 102. The sequencing system 100 can further include a computer system 106 that can be configured to control the optics system 102 and the fluidics system 104 via communication channels 108a and 108b. For example, a computer interface 110 of the optics system 102 can be configured to communicate with the computer system 106 through the communication channel 108a. [0084] During sequencing reactions, the fluidics system 104 can direct the flow of reagents through one or more reagent tubes 112 to and from a flow cell 114 positioned on a mounting stage 116. The reagents can include, for example, fluorescently labeled nucleotides, buffers, enzymes, and cleavage reagents. The flow cell 114 can include at least one fluidic channel. The flow cell 114 can be a patterned array flow cell or a random array flow cell. The flow cell 114 can include multiple clusters of single-stranded polynucleotides to be sequenced in the at least one fluidic channel. The lengths of the polynucleotides can vary ranging, for example, from about 50 bases, 100 bases, 150 bases, 200 bases, 300 bases, 500 bases, to about 1000 bases. The polynucleotides can be attached to one or more fluidic channels of the flow cell 114, where fluidic channel is distinct from an image channel detected by detectors 126. In some embodiments, the flow cell 114 can include a plurality of wells, wherein each well can include a cluster comprising multiple identical copies of a target polynucleotide to be sequenced. The mounting stage 116 can be configured to allow proper alignment and movement of the flow cell 114 in relation to the other components of the optics system 102. In one embodiment, the mounting stage 116 can be used to align the flow cell 114 with a lens 118. [0085] The optics system 102 can include two light sources 120, such as lasers or a LEDs, with each light source configured to generate light having wavelengths distributed at around a predetermined wavelength. However, embodiments are not limited to any particular wavelength of light. The light source only needs to be configured to generate the correct wavelength of light which excites the fluorescent labels attached to the nucleotides on the flow cell. [0086] The light generated by the light source 120 can pass through fiber optic cables 122 to excite fluorescent labels in the flow cell 114. The lens 118, mounted on a focuser 124, can move along the z-axis. The focused fluorescent emissions can be detected by detectors 126, for example charge-coupled device (CCD) sensors or a complementary metal oxide semiconductor (CMOS) sensors. In some embodiments, there may by two detectors 126, but in other embodiments there may be four or more detectors 126. In some embodiments, nucleotide incorporations can be detected with zeromode waveguides as described, for example, in Levene et al. Science 299, 682- 686 (2003); Lundquist et al. Opt. Lett.33, 1026-1028 (2008); and Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties. [0087] A filter assembly 128 of the optics system 102 can be configured to filter the fluorescent emissions from the fluorescent labels in the flow cell 114. The filter assembly 128 can include a plurality of optical filters, where a correct filter can be selected depending on the particular fluorophores used in a sequencing reaction. In one alternate embodiment, the computer system 106 may automatically determine which optical filter should be used for a sequencing reaction, e.g., by scanning labels and/or barcodes attached to a sample vial and determining the particular fluorophores to be used in a sequencing reaction based on the labels and/or barcodes, or by retrieving information stored in the memory relating to previous sequencing reactions, and then control the filter assembly 128 to select and use the desired optical filter. The selected filter can be a longpass filter, a shortpass filter, a bandstop filter, or a bandpass filter, depending on the types of fluorescent molecules being used in the system. For example, the selected filter can be a bandpass filter selected to match the peak of the emission spectrum of a particular fluorescent label. [0088] In some embodiments, the detectors 126 include one or more sub-detector while the filters of the filter assembly 128 may be mechanically switched or rotated in front of the sub- detector, such that differently filtered images can be taken by the sub-detector sequentially. In some embodiments, the detectors 126 include one sub-detector and the filter assembly 128 may include at least one layer of switchable material which has a light transmittance that is variable upon application of a stimulus, where the stimulus may be light, electricity, temperature, or any combination thereof. As a result, the filter assembly 128 can provide a plurality of optical filters such that differently filtered images can be taken by the sub-detector sequentially. In some embodiments, the detectors 126 may each include one sub-detector and the filter assembly 128 may include one or more switchable filters base on the micro-electromechanical system technology, such that differently filtered images can be taken by the sub-detector sequentially. [0089] In some embodiments, the detectors 126 can include two or more sub-detectors to be selected depending on the set of fluorophores used, for example a first detector coupled with a first filter and a second detector coupled with a second filter. In some embodiments, the optics system 102 may include two or more dichroic mirrors/beamsplitters configured to split the fluorescent emissions, such that after splitting the fluorescent emissions with the dichroic mirrors, the detectors 126 can take two differently filtered images simultaneously (or close in time) using the two sub-detectors coupled with two different filters. In some embodiments, the detectors 126 can include two or more sub-detectors stacked along the incoming direction of the fluorescent emissions. Different wavelengths of the fluorescent emissions may differentially decay or be differentially absorbed along the incoming direction, such that sub-detectors at different positions along the incoming direction can be selected depending on the set of fluorophores used, or be configured to take differently filtered images simultaneously (or close in time). [0090] In use, a sample having a polynucleotide to be sequenced may be loaded into the flow cell 114 and placed in the mounting stage 116. The computer system 106 may then activate the fluidics system 104 to begin a sequencing cycle. During sequencing reactions, the computer system 106 may instruct the fluidics system 104, through the communication interface 108b, to supply reagents, for example labeled nucleotide analogs, to the flow cell 114. Through the communication interface 108a and the computer interface 110, the computer system 106 may control the light source 120 of the optics system 102 to generate light at around a predetermined wavelength and excite nucleotide analogs incorporated into growing primers hybridized to the polynucleotide being sequenced, for example. The computer system 106 may control the detector 126 of the optics system 102 to capture images of the diffraction-limited spots of DNA clusters having the fluorescently labeled nucleotide analogs. The computer system 106 can receive the fluorescent images from the detector 126 and process the fluorescent images received to determine the nucleotide sequence of the polynucleotide being sequenced. [0091] In FIG.1B, an example of an imaging system 10000 to be used in the disclosed sequencing technology is illustrated. For example, the imaging system 10000 may be used in the example sequencing system 100 illustrated in FIG. 1A. The imaging system 10000 may include a light source 11000 that can provide light at one or more wavelengths to excite fluorophores at targeted points on a sample. The light source 11000 can include one or more lasers, light-emitting diodes, or other optical sources, such that the light source 11000 can provide a variety of wavelengths of light. In some embodiments, the light source 11000 can be configured to selectively provide light with a predetermined range of wavelengths that are tuned to the set of fluorophores being used. In some embodiments, the light source 11000 can be configured to output light at an optical frequency corresponding to a wavelength in a predefined range of wavelengths of light. In some embodiments, a user of the disclosed sequencing systems may choose a specific optical frequency to be output from the light source 11000, depending on the particular fluorophores used in a sequencing reaction. In one alternate embodiment, the computer system 106 may automatically determine which optical frequency should be output from the light source 11000, e.g., by scanning labels and/or barcodes attached to a sample vial and determining the particular fluorophores to be used in a sequencing reaction based on the labels and/or barcodes, or by retrieving information stored in the memory relating to previous sequencing reactions, and then control the light source 11000 to select and output the desired optical frequency. [0092] The imaging system 10000 may include an optical path 12000 from the light source 11000 to the sample 13000, e.g., a microfluidic device including one or more flow chambers where one or more sequencing reactions occur. In some embodiments, the optical path 12000 can include a combination of one or more of mirrors, lenses, prisms, quarter wave plates, half wave plates, polarizers, filters, dichroic mirrors, beam splitters, beam combiners, objective lenses, wide field optics configured to spread light from a light source over a relatively large region of a sample, etc. The optical path 12000 can be configured to direct light from the light source 11000 to the sample 13000. In addition, the optical path 12000 may include optical components which can be configured to direct light emitted from the sample 13000 to an integration detection system 15000. In some embodiments, a portion of the optical elements that are used to direct light from the light source 11000 to the sample 13000 are also used to direct light from the sample 13000 to the integration detection system 15000. Further examples of optical paths and optical systems may be found in U.S. Pat. No. 7,589,315, U.S. Pat. No. 8,951,781, or U.S. Pat. No. 9,193,996, each of which is incorporated by reference herein in its entirety. [0093] The imaging system 10000 may include a scanning system 14000 to effectively move light relative to the sample 13000 to scan the sample to generate an image. In some embodiments, the scanning system 14000 can be implemented within the optical path 12000. For example, the scanning system 14000 can include one or more scanning mirrors that move relative to one another within the optical path 12000 to effectively move the light from the light source 11000 across the sample. In some embodiments, the scanning system 14000 can be implemented as a mechanical system that physically moves the sample 13000 so that the sample moves relative to the light from the light source 11000. In some embodiment, the scanning system 14000 can be a combination of optical components in the optical path 12000 and a mechanical system for physically moving the sample 13000 so that the light from the light source 11000 and the sample 13000 move relative to one another. [0094] The imaging system 10000 may include an integration detection system 15000 that includes one or more light detectors as well as associated electronic circuitry, processors, data storage, memory, and the like to acquire and process image data of the sample 13000. In some embodiments, the integration detection system 15000 can include photomultiplier tubes, avalanche photodiodes, image sensors (e.g., CCDs, CMOS sensors, etc.), and the like. In some embodiments, the light detectors of the integration detection system 15000 can include components to amplify light signals and may be sensitive to single photons. In some embodiments, the light detectors of the integration detection system 15000 can have a plurality of channels or pixels. The integration detection system 15000 can acquire one or more images based on the light detected from the sample 13000. [0095] In some embodiments, the optical path 12000 may include an array generator 12100 that can generate a plurality of light exposure regions on the sample 13000. In some embodiments, the array generator 12100 can generate a certain light exposure pattern on the sample 13000. These light exposure regions can be scanned over the sample 13000 using the scanning system 14000 to selectively illuminate areas of the sample 13000 for imaging. The integration detection system 15000 can integrate signals corresponding to particular points on the sample 13000 as the plurality of light exposure regions are scanned over the sample 13000. For example, for an individual point on the sample 13000, the integration detection system 1500 can selectively aggregate detected signals corresponding to the individual point where the individual point is illuminated at different times by different light exposure regions. In some embodiments, the combination of the array generator 12100 and the integration detection system 15000 can detect light simultaneously, or near-simultaneously, from a plurality of points on the sample 13000. In some embodiments, the combination of the array generator 12100 and the integration detection system 15000 can integrate the detected light from a plurality of points on the sample over time. [0096] In some embodiments, a plurality of sequencing reactions may be run parallelly in a plurality of flow chambers of the sample 13000. For example, a plurality of sequencing reactions may be performed for a plurality of biological specimen. In some embodiments, the plurality of sequencing reactions may use different sets of fluorophores. In some embodiments, the light source 11000, the array generator 12100, and the scanning system 14000 can be configured to selectively illuminate different areas of the sample 13000 with different optical frequencies of light, depending on the different sets of fluorophores used for the sequencing reactions occurring in different areas of the sample 13000. Two Or More Channel Base Calling with Modified Nucleotides [0097] As mentioned above, one aspect of the disclosure is directed to systems and methods that utilize two, three, or more fluorescent dyes to perform SBS sequencing of at least five nucleotides. In some embodiments, systems and methods may utilize two dye sequencing with four labeled nucleotides, and a dark nucleotide in a two excitation, two channel system. In some embodiments, systems and methods may utilize three dye sequencing with five labeled nucleotides in a three-channel system. In some embodiments, a three-channel system may utilize two excitation sources or three excitation sources. Using five nucleotides labeled with fluorescent dyes allows for an additional encoding space as compared to traditional two excitation, two channel systems. [0098] In some embodiments, distinguishing five different nucleotides in three channels will form at least five clouds in a three-dimensional scatter plots. In some embodiments, unlabeled wells may be identified as a cloud near the origin of a three-dimensional scatter plot. In other embodiments, a fifth base, such as a modified base, may be identified as a fifth cloud in a three-dimensional scatter plot. [0099] An aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting the absence of fluorescent emissions from a fourth labeled nucleotide; detecting fluorescent emissions from a fifth labeled nucleotide at a third wavelength; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions wherein at least one of the labeled nucleotides is a modified nucleotide.. [0100] Figure 2 shows a flowchart of a method 200 of sequencing clusters of labeled polynucleotides bound to a flow cell according to one embodiment. The method may begin at a start step 202, by, for example, gathering DNA samples, and putting the sample into a sequencing system, such as described above. Sequencing systems according to the disclosure may include flow cell sequencers such as those produced by ILLUMINA®, INC. (San Diego, CA). For example, when the method 200 begins at step 202, a flow cell may have been prepared by embedding the flow cell with fragmented polynucleotides (e.g., fragmented single- or double- stranded polynucleotide fragments). Fragmented polynucleotides may be generated from a deoxyribonucleic acid (DNA) sample. DNA samples may be from various sources, for example, a biological sample, a cell sample, an environmental sample, or any combination thereof. The lengths of fragmented polynucleotide fragments may range from, for example, 100 bases to 1000 bases, or more. [0101] Polynucleotide fragments may be bridge-amplified into clusters of polynucleotide fragments attached to the inside surface of one or more channels of a flow cell. An inside surface of the one or more flow cell channels may include two types of primers, for example a first primer type (P1) and a second primer type (P2) and the DNA fragments may be amplified by well-known methods to generate clusters. In some embodiments the flow cells are patterned with wells and each well contains a single cluster. Note that as used herein, “labeled” polynucleotides refer to polynucleotides that may be labeled directly with a flurophore, or indirectly by a fluorophore on a nucleotide that is incorporated into the complementary strand during a sequence by synthesis process. Note that when first bound to a flowcell surface, the polynucleotides are not typically fluorescently labeled. [0102] After generating clusters within the flow cell, the method 200 may begin a Sequencing by Synthesis process. During each sequencing cycle, five or more types of nucleotide analogs are added and incorporated onto the growing primer-polynucleotides. The four or more types of nucleotide analogs may have different modifications. For example, a first type of nucleotide may be an analog of deoxyguanosine triphosphate (dGTP), which is partially conjugated via a linker with a first type of fluorescent label that, after an excitation, can emit light at a first emission wavelength. In some embodiments, only a fraction of the dGTP nucleotides are labeled so that each cluster which has a dGTP as the next incorporated nucleotide will fluoresce with a reduced intensity as compared to a cluster where every incorporated nucleotide was labeled. For example, only 10, 20, 30, 40, 50, 60, or 70 percent of the nucleotides in a dGTP mixture that is used during SBS may be labeled. Thus, the intensity of the fluorescence coming from a cluster that is labeled with dGTP would be detectibly lower than clusters having every growing strand labeled with a fluorescent dye. This allows the same dye to be used on dGTP nucleotides as on another nucleotide, but still have the dGTP clusters be detectibly different from the other clusters. [0103] A second type of nucleotide may be an analog of deoxythymidine triphosphate (dTTP), which may be labeled with the same fluorescent label as dGTP, but with every nucleotide being labeled. Thus, as compared to dGTP, each cluster which is labeled with dTTP would have a higher intensity than clusters labeled with dGTP. A third type of nucleotide may be an analog of deoxycytidine triphosphate (dCTP), which is conjugated via a linker with the second type fluorescent label that, after an excitation, can emit at a second emission wavelength. A fourth type of nucleotide may be an analog of deoxyadenosine triphosphate (dATP), which is conjugated via a linker with both the first and second types of fluorescent labels, and can, after a corresponding excitation, emit at both first and second emission wavelengths. A fifth type of nucleotide may be an analog of 5-methylcytosine, which may not be conjugated with any fluorescent label, but still may be conjugated with a linker and will not, after a corresponding excitation, emit at either the first or second emission wavelengths. [0104] In a separate embodiment, the dGTP may be unlabeled and the modified base, such as 5-methylcytosine may be labeled with a third fluorescent label that can emit at a third emission wavelength that is different than either the first or second emission wavelengths. Thus, in this embodiment, detecting a well with a dark state would indicate the presence of the dGTP nucleotide and detection of emission of the third fluorescent label would indicate the presence of a modified nucleotide. [0105] The linkers may include one or more cleavage groups. Prior to the subsequent sequencing cycle, the fluorescent labels may be removed from the nucleotide analogs. For example, a linker attaching a fluorescent label to a nucleotide analog may include an azide and/or an alkoxy group, for example on the same carbon, such that the linker may be cleaved after each incorporation cycle by a phosphine reagent, thereby releasing the fluorescent label from subsequent sequencing cycles. [0106] Once the clusters are created on the flow cell, the method 200 moves to a step 210 of detecting fluorescent emissions from a first labeled nucleotide at a first wavelength. For example, step 210 may include exciting all of the clusters of labeled polynucleotides on a flow cell at a first excitation wavelength. A single light source, such as a laser or an LED source, may excite a fluorescent label at the predetermined excitation wavelength. In some embodiments, the single laser or the LED source may be non-tunable. After exciting all of the clusters, Step 210 may include detecting fluorescent emissions from the clusters at a first detection wavelength to detect the presence of a first labeled nucleotide. In general, a first detection wavelength will be red-shifted to a longer wavelength relative to a first excitation wavelength. [0107] Image processing yields base calling, described in further detail below, where the complementary nucleotides added to the molecules in a cluster during a cycle are identified. In some embodiments, the fluorescent images may be stored for later processing. In some embodiments, the fluorescent images may be processed to determine the sequence of the growing primer-polynucleotides in each cluster in real time. After detecting the fluorescence emissions from the clusters at step 210, the method 200 moves to a step 220, where the method detects fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength. [0108] When the method detects fluorescent emissions from a first wavelength in step 210 different than a second wavelength in step 220, the method may be described as employing at least two channel base calling. A “channel” may be used to describe an emission/detection process for a labeled or unlabeled base that is specific for a particular excitation wavelength and detection wavelength pair. Thus, a channel may include a particular first range of wavelengths of light used to excite a fluorescent dye and a second range of wavelengths of light used to detect the fluorescent emissions from the excited dyes. The disclosure provides for, inter alia, two, three, and/or more channel base calling for five or more bases. Some embodiments may be configured in a one excitation, two channel configuration, where a single excitation wavelength to excite a first type of fluorescent dye that emits at a first wavelength range and, at the same time, excite a second type of fluorescent dyes that can emit at a second wavelength range. For example, other embodiments may be configured in a two excitation, three channel configuration, where the first two channels are in a one excitation, two channel configuration, and the third channel corresponds to a fluorophore excited with a second excitation wavelength and a third emission wavelength. [0109] After detecting the fluorescence emissions from the clusters at step 210, the method 200 moves to a step 230, where the method detects fluorescent emissions from a third labeled nucleotide at the first and second wavelengths. For example, at step 230 fluorescent emissions from a third labeled nucleotide may be detected at the second wavelength at some intensity and at the first wavelength may be detected at the same or different intensity. In some embodiments, fluorescent emissions from a third labeled nucleotide at the first and second wavelength may be detected at some non-zero intensity. The intensity of the detected emissions at the first wavelength may be quantified as an absolute measurement in terms of photon flux or counts per second. As described herein, the first and second intensities may also be quantified as relative measurement for a cluster labeled with a single nucleotide as compared to a cluster labeled with two different nucleotides. [0110] In some embodiments, the disclosure provides for systems and methods for DNA sequencing using detection schemes other than fluorescence. For example, some DNA sequencing systems and methods employ voltage detectors instead of light detectors to detect specific nucleotides within a polynucleotide. In some embodiments, these techniques may or may not require amplification of the DNA or RNA sample into a cluster, for example, to be analyzed. In some embodiments, these techniques may or may not require the labelling of the DNA or RNA sample in order to be analyzed. The methods disclosed herein may be applied to voltage-based systems via an analogous process to the fluorescent detection systems. In some embodiments, instead of detecting fluorescent emissions from a labelled nucleotide and determining the nucleotide based on the emission wavelength and intensity, a voltage detection system may detect various parameters of a voltage signal, such as amplitude, frequency, or waveform characteristics, to extract relevant information to determine the presence of a particular nucleotide. In some embodiments, various parameters of a voltage signal, such as amplitude, frequency, or waveform characteristics may be characterized as a channel, with “on” and “off” states. For example, a voltage signal corresponding to a “T” nucleotide may be detected as a voltage signal with a characteristic frequency and at a first amplitude. In some embodiments, a voltage signal corresponding to a “C” nucleotide may be detected at the same characteristic frequency but at a second amplitude. [0111] After detecting the fluorescence emissions from the clusters at step 230, the method 200 moves to a step 240, where the method detects the absence of fluorescent emissions from a fourth labeled nucleotide. For example, at step 240, the method may have excited the clusters at first and second excitation wavelengths and detected the absence of emissions. After detecting the absence fluorescence emissions from the clusters at step 240, the method 200 may move to a step 250, where the method detects fluorescent emissions from a fifth labeled nucleotide at a third wavelength. By way of example, at step 250, fluorescent emissions from a fifth labeled nucleotide may be detected at a third wavelength that is different from either the first or second wavelength. In other examples, at step 250, fluorescent emissions from a fifth labeled nucleotide may be detected at a third wavelength that is the same wavelength as either the first or second emission wavelength. In some examples, the fifth nucleotide may be one or more modified nucleotides. In some embodiments the fifth labeled nucleotide is a modified nucleotide. [0112] Any of the preceding steps may be performed in a different order. One of skill in the art will understand that step 240 may be performed before or after step 250. For example, in some embodiments, the method may have excited the clusters at first, second, and third excitation wavelengths before detecting the absence of emissions. [0113] In some embodiments, one or more modified nucleotides includes a 5- methylcytosine, a N6-methyladenine, and an inosine. In some embodiments, a method may include the step of detecting fluorescent emissions at the third wavelength, and may include detecting the fluorescent emissions at the third wavelength at a first intensity and at a second intensity, wherein the first intensity corresponds to a first modified nucleotide and wherein the second intensity corresponds to a second modified nucleotide. [0114] The method 200 then moves to a decision step 255 to determine if the method 200 should repeat for an additional SBS cycle of reading sequence data. A determination may be made at decision step 255 whether to detect more nucleotides based on, for example, the quality of the signal or after a predetermined number of bases. If more nucleotides are to be detected, then the method 200 may loop back to the step 210 to start a next sequencing cycle. [0115] The method 200 may then move to a step 260 wherein the system may determine the sequence of the polynucleotides with one or more modified nucleotides based on the detected fluorescent emissions. For example, at the step 260 the method may identify clusters that have added nucleotides with emissions at wavelengths and intensities corresponding to the first, second, third, fourth and/or fifth labeled nucleotides. In some embodiments, at the step 260 the method may identify clusters which had no fluorescent emissions following excitation at the first, second, and/or third excitation wavelengths and determine that the cluster corresponds to the fourth labeled nucleotide. [0116] As mentioned above, a cluster may be identified by the lack of any fluorescence after excitation at some or all of the first, second, and third wavelengths. Because the lack of fluorescent emissions is dark or undetectable on an image, the system may track the position of each cluster on a flow cell. Here, the term “undetectable” refers to fluorescent emissions from a nucleotide that are intentionally or unintentionally reduced to an intensity that is not effectively distinguishable from noise by a detection scheme. Once a cluster has been identified at a particular position, the system may then note in subsequence sequencing rounds whether there is a fluorescent emission at the position of the cluster. If no fluorescent emissions are noted at a known position of a cluster, the system may determine that the nucleotide added in the latest sequencing round was the unlabeled nucleotide. For example, in some cases the unlabeled nucleotide may be a “G”. In another example, the unlabeled nucleotide may correspond to no nucleotide being sequenced—from either an empty well or a finished cluster. However, it should be realized that any nucleotide could be chosen as the one which is unlabeled and still be within the scope of the invention. [0117] In some embodiments, the flow cell includes wells configured to bind polynucleotides; further wherein the incorporation of one of the at least four labelled nucleotide conjugates into a well that is detected from at least one signal state. In some embodiments, the presence of an empty well is determined from a dark state. For example, labelled nucleotides may be distributed as clouds in an X-Y scatterplot on a three by three grid, where the X axis corresponds to signal states in a first channel in off, intermediate, and on states, and the Y axis corresponds to signal states in a second channel in off, intermediate, and on states. This three by three grid corresponds to a total of nine possible locations on the X-Y scatterplot, where the four traditional nucleotides and four modified nucleotides could occupy eight clouds displaced from the origin (the off-off state in the first and second channels). The remaining off-off state may be used to distinguish empty wells, because there is no nucleotide corresponding to this dark state. In some embodiments, base calling may be performed by a clustering model where each of the at least four labelled nucleotides are distributed as a cloud in an X-Y scatterplot with an intensity level of at least one signal state such that each labelled nucleotide is more similar to nucleotides of the same label than to those from different labelled nucleotides. In some embodiments, at least four labelled nucleotides are distributed as clouds with an intensity level in at least two signal states. [0118] In some embodiments, prior to the next sequencing cycle, the fluorescent labels attached to each nucleotide may be removed from the incorporated nucleotide analogs, and the reversible 3ƍ blocks may be removed so that another nucleotide analog may be added onto each extending primer-polynucleotide. If a determination is made at the decision step 255 that there are no more additional rounds of sequencing necessary, the method 200 then moves to an end step 270 and terminates the method 200. Real-time analysis typically only processes and stores the information used during the sequencing run. This process may minimize the amount of data generated and stored in real-time. For each nucleotide base determined, a quality score may be determined. After all the fluorescent images are processed, the method 200 may terminate at the step 270. Amplitude Multiplexing [0119] As mentioned above, an aspect of the disclosure is directed to amplitude multiplexing. Expanding the encoding space of current systems by assigning unique identifiers or labels to each sample or data stream is referred to herein as multiplexing, and allows for greater than four bases to be identified in a single sequencing experiment. As an example of amplitude multiplexing, one channel may include an “off” state, an “on” state, and an intermediate state that is “half on, half off” state with approximately 50% intensity of the full “on” state. [0120] For example, an aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flow cell, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting fluorescent emissions from a fourth labeled nucleotide at the first wavelength and a third intensity that is less than the first intensity; detecting fluorescent emissions from a fifth labeled nucleotide at the second wavelength and a fourth intensity that is less than the second intensity; and determining the sequence of the polynucleotides based on the detected fluorescent emission and intensity. [0121] Fig. 3 shows a flowchart of a method 300 of sequencing clusters of labeled polynucleotides bound to a flow cell according to one embodiment. The method may begin at a start step 302, by, for example, gathering DNA samples, and putting the sample into a sequencing system, such as described above. Sequencing systems according to the disclosure may include flow cell sequencers such as those produced by ILLUMINA®, INC. (San Diego, CA). For example, when the method 300 begins at step 302, a flow cell may have been prepared by embedding the flow cell with fragmented polynucleotides (e.g., fragmented single- or double- stranded polynucleotide fragments). Fragmented polynucleotides may be generated from a deoxyribonucleic acid (DNA) sample. DNA samples may be from various sources, for example, a biological sample, a cell sample, an environmental sample, or any combination thereof. The lengths of fragmented polynucleotide fragments may range from, for example, 100 bases to 1000 bases, or more. [0122] After generating clusters within the flow cell, the method 300 may begin a Sequencing by Synthesis process. During each sequencing cycle, four or more types of nucleotide analogs are added and incorporated onto the growing primer-polynucleotides. The four or more types of nucleotide analogs may have different modifications. For example, a first type of nucleotide may be an analog of deoxyguanosine triphosphate (dGTP), which is partially conjugated via a linker with a first type of fluorescent label that, after an excitation, can emit light at a first emission wavelength. In some embodiments, only a fraction of the dGTP nucleotides are labeled so that each cluster which has a dGTP as the next incorporated nucleotide will fluoresce with a reduced intensity as compared to a cluster where every incorporated nucleotide was labeled. For example, only 10, 20, 30, 40, 50, 60, or 70 percent of the nucleotides in a dGTP mixture that is used during SBS may be labeled. Thus, the intensity of the fluorescence coming from a cluster that is labeled with dGTP would be detectibly lower than clusters having every growing strand labeled with a fluorescent dye. This allows the same dye to be used on dGTP nucleotides as on another nucleotide, but still have the dGTP clusters be detectibly different from the other clusters. [0123] Once the clusters are created on the flow cell, the method 300 moves to a step 310 of detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity. For example, step 310 may include exciting all of the clusters of labeled polynucleotides on a flow cell at a first excitation wavelength. A single light source, such as a laser or an LED source, may excite a fluorescent label at the predetermined excitation wavelength. In some embodiments, the single laser or the LED source may be non-tunable. Step 310 may also include detecting any fluorescent emissions from the clusters at a first detection wavelength or range of wavelengths to detect the presence of a first labeled nucleotide. In general, the first detection wavelength will be red-shifted to a longer wavelength relative to the first excitation wavelength. [0124] Image processing yields base calling, as described in further detail below, where the complementary nucleotides added to the molecules in a cluster during a cycle are identified. In some embodiments, the fluorescent images may be processed to determine the sequence of the growing primer-polynucleotides in each cluster in real time. After detecting the fluorescence emissions from the clusters at step 310, the method 300 moves to a step 320, where the method detects fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength. [0125] When the method detects fluorescent emissions from a first wavelength in step 310 different than a second wavelength in step 320, the method may be described as employing at least two channel base calling. A “channel” may be used to describe an emission/detection process for a labeled or unlabeled base that is specific for a particular excitation wavelength and detection wavelength pair. Thus, a channel may include a particular first range of wavelengths of light used to excite a fluorescent dye and a second range of wavelengths of light used to detect the fluorescent emissions from the excited dyes. In the context of one excitation, two channel systems and methods, a first excitation wavelength may excite two types of dyes to emit light in two different wavelengths, where each emission wavelength is referred to as a channel. The disclosure provides for, inter alia, two or more channel base calling for four or more bases. [0126] After detecting the fluorescence emissions from the clusters at step 320, the method 300 moves to a step 330, where the method detects fluorescent emissions from a third labeled nucleotide at the first and second wavelengths. For example, at step 330 fluorescent emissions from a third labeled nucleotide may be detected at the second wavelength at some intensity and at the first wavelength may be detected at the first intensity. In some embodiments, fluorescent emissions from a third labeled nucleotide at the first and second wavelength may be detected at some non-zero intensity other than the first intensity. [0127] After detecting the fluorescence emissions from the clusters at step 330, the method 300 moves to a step 340, where the method detects fluorescent emissions from a fourth labeled nucleotide at the first wavelength and a third intensity that is less than the first intensity. For example, at step 340, the method may detect fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a third intensity that is approximately one half of the first intensity. The intensity of the detected emissions at the first wavelength may be quantified as an absolute measurement in terms of photon flux or counts per second. In some embodiments, the intensity of detected emissions may be normalized such that intensities in a channel are relative to the maximum emission in that channel. As described herein, the intensities may also be quantified as relative measurement for a cluster labeled with a single nucleotide as compared to a cluster labeled with two different nucleotides. In some embodiments, at step 340, the method may detect fluorescent emissions from the fourth labeled nucleotide at the first wavelength and also at a second and/or third wavelength. In some embodiments, at step 340, the method may detect fluorescent emissions from the fourth labeled nucleotide only at the first wavelength. [0128] After detecting the fluorescence emissions from the clusters at step 340, the method 300 moves to a step 350, where the method detects fluorescent emissions from a fifth labeled nucleotide at the second wavelength and a fourth intensity that is less than the second intensity. For example, at step 350, the method may detect fluorescent emissions from the fifth labeled nucleotide at the second wavelength and a fourth intensity that is approximately one half of the second intensity. In some embodiments, the fourth intensity may be approximately equal to the third intensity. In some embodiments, at step 350, the method may detect fluorescent emissions from the fifth labeled nucleotide at the second wavelength and also at the first and/or a third wavelength. In some embodiments, at step 340, the method may detect fluorescent emissions from the fifth labeled nucleotide only at the second wavelength. [0129] The method 300 then moves to a decision step 355 to determine if the method 300 should repeat for an additional SBS cycle of reading sequence data. A determination may be made at decision step 355 whether to detect more nucleotides based on, for example, the quality of the signal or after a predetermined number of bases. If more nucleotides are to be detected, then the method 300 may loop back to the step 310 to start a next sequencing cycle. [0130] The method 300 may then move to a step 360 wherein the system may determine the nucleotide sequence of the polynucleotide based on the detected fluorescent emissions and intensities. For example, at the step 360 the method may identify clusters that have added nucleotides with emissions at wavelengths and intensities corresponding to the first, second, third and/or fourth labeled nucleotides. In some embodiments, at the step 360 the method may identify clusters which had no fluorescent emissions following excitation at the first and/or second excitation wavelength and determine that the cluster corresponds to an empty well or to a short insert that has completed sequencing. [0131] In some embodiments, a cluster may be identified by the lack of any fluorescence after excitation at either the first or second wavelengths. Because the lack of fluorescent emissions is dark or “missing” on an image, the system may track the position of each cluster on a flow cell. Once a cluster has been identified at a particular position, the system may then note in subsequence sequencing rounds whether there is a fluorescent emission at the position of the cluster. If no fluorescent emissions are noted at a known position of a cluster, the system may determine that the nucleotide added in the latest sequencing round was the unlabeled nucleotide. For example, in some cases the unlabeled nucleotide may be a “G”. In another example, the unlabeled nucleotide may correspond to no nucleotide being sequenced—from either an empty well or a finished cluster. However, it should be realized that any nucleotide could be chosen as the one which is unlabeled and still be within the scope of the invention. Using the lack of fluorescence to identify a nucleotide is broadly applicable to other embodiments according to the disclosure, and it is expressly considered that this feature may be combined with other embodiments according to the disclosure. [0132] In some embodiments, prior to the next sequencing cycle, the fluorescent labels attached to each nucleotide may be removed from the incorporated nucleotide analogs, and the reversible 3ƍ blocks may be removed so that another nucleotide analog may be added onto each extending primer-polynucleotide. If a determination is made at the decision step 355 that there are no more additional rounds of sequencing necessary, the method 300 then moves to an end step 370 and terminates the method 300. For each nucleotide base determined, a quality score may be determined. After all the fluorescent images are processed, the method 300 may terminate at the step 370. [0133] In some embodiments, a method may include a further step of detecting fluorescent emissions from a sixth labeled nucleotide at the first wavelength and a fifth intensity that is less than the third intensity, and at the second wavelength and a sixth intensity that is less than the fourth intensity. [0134] An aspect of the disclosure is directed to increasing the encoding space for base calls while using two channels. Fig. 4 shows three panels, where each panel shows a different exemplary illustration of an X-Y axis scatterplot of the fluorescence emissions in a first channel and a second channel. The first panel on the left shows a two-dimensional scatterplot of intensities of a traditional two excitation, two channel sequencing system. The Y axes 401, and X axes 402 for each panel in Fig. 4 correspond to intensities of fluorescent signals detected in a detection channel. In some embodiments, as shown here, the Y axis 401 may correspond to intensities of fluorescence detected from a channel (e.g., a first channel or Channel 1) detecting green wavelengths. The X axis 402 may correspond to intensities of fluorescence detected from a channel (a second channel, or Channel 0) detecting red wavelengths. Note that the intensities of each axis may be normalized, and the total non-normalized intensities may be increased by using a dye with a higher quantum yield of fluorescence, or a larger percentage of a dye with a high quantum yield. [0135] Nucleotide “C” 410 is shown as a cloud in the top left corner in the first panel of Fig.4 and corresponds to detections of fluorescence in the green channel, and no detections (or minimal detections) in the red channel. Nucleotide “A” 420 is shown as a cloud in the top right corner and corresponds to a detection of fluorescence in the green channel and in the red channel. Nucleotide “T” 430 is shown as a cloud in the bottom right corner and corresponds to a detection of fluorescence in the red channel and no detections (or minimal detections) in the green channel. Nucleotide “G” 440 is shown as a cloud in the bottom left corner of Fig. 4 and corresponds to typical dark detection of nucleotide “G”—no detections (or minimal detections) in the green and red channels. [0136] The separation between some of the clouds may depend on the amount of cross- talk between some of the fluorescent labels, and an aspect of the disclosure is related to minimizing this effect and increasing the signal to noise of the sequencing system. The separation between nucleotides, for example nucleotide “A” 420 and nucleotide “C” 410, may depend on several factors like the crosstalk between the dyes labeling nucleotide “A” 420 and nucleotide “C” 410 that would lead to partial detection of channel 0 detections when detecting emissions for nucleotide “C” 410. In some embodiments, the signal to noise may be improved by increasing the brightness of the dye associated with either the green channel or the red channel, which in allows for sufficient resolution between the clouds that an additional cloud may be included in one or more channel. [0137] The top right panel of Fig. 4 shows a two-dimensional scatterplot of a two excitation, two channel sequencing system with at least the four labeled nucleotides from the last panel, and five additional possible clouds corresponding to modified nucleotides or to empty wells or base calls past the read length of an insert. In some embodiments, the techniques described herein relate to a method, wherein additional bases beyond a fourth labeled nucleotide are modified nucleotides. Examples of modified nucleotides are including throughout the disclosure, including the definition section. However, one skilled in the art will understand that new modified nucleotides beyond those currently discovered may be used as one of the modified bases. [0138] Nucleotide “C” 411 is shown as a cloud in the top left corner in the first panel of Fig. 4 and corresponds to maximum detections of fluorescence in the green channel, and no detections (or minimal detections) in the red channel. Nucleotide “A” 421 is shown as a cloud in the top right corner and corresponds to maximum detections of fluorescence in the green channel and in the red channel. In some embodiments, nucleotide “A” 421 may correspond to maximum detections of fluorescence in the green channel and non-maximum detections the red channel, at for example cloud 435. [0139] Nucleotide “T” 431 is shown as a cloud in the bottom right corner and corresponds to a maximum detection of fluorescence in the red channel and no detections (or minimal detections) in the green channel. Nucleotide “G” 441 is shown as a cloud in the bottom left corner of Fig. 4, and corresponds to typical dark detection of nucleotide “G”—no detections (or minimal detections) in the green and red channels. In some embodiments, nucleotide “G” 441 may be labeled and may appear at any of the shaded clouds, including shaded cloud 435. The shaded clouds, including shaded cloud 435, correspond to locations of potential clouds for additional nucleotides or empty well detection. In some embodiments, all five shaded clouds are occupied by modified nucleotides such that there is a total of nine clouds in a scatterplot. In some embodiments, less than five of the five shaded clouds are occupied by modified nucleotides such that there is a total of any of five, six, seven and eight clouds in a scatterplot. In some embodiments, one or more of the clouds associated with the four nucleotides (411, 421, 431, 441) may not be used, and one of the five shaded clouds may be used instead. In some embodiments, less than five of the five shaded clouds are occupied by modified nucleotides and one or more of the clouds associated with the four nucleotides may be replaced by the less than five shaded clouds. [0140] The bottom right panel of Fig. 4 also shows a two-dimensional scatterplot of a two excitation, two channel sequencing system with a total of nine clouds corresponding to encoding space for nucleotide fluorescence detection. The bottom right panel of Fig. 4 shows clouds labeled “1” through “9” and demonstrates an example of amplitude multiplexing where additional clouds are added sequentially. In some embodiments, the clouds may correspond to any order of the four usual nucleotides and any additional modified nucleotides. In some embodiments, less than all nine clouds may be present. [0141] In some embodiments, a method of sequencing polynucleotides bound to a flow cell, may include detecting fluorescent emissions from a first labeled nucleotide at cloud “1” at a first wavelength and a first intensity. The bottom right panel of Fig. 4 illustrates detections of fluorescent emissions from a second labeled nucleotide at cloud “2” at a second wavelength and a second intensity. The bottom right panel of Fig.4 shows that the first wavelength as different from the second wavelength. [0142] The bottom right panel of Fig. 4 illustrates detections of fluorescent emissions from a third labeled nucleotide at cloud “3” at high levels of detected fluorescence in both the first and second wavelengths. Fluorescent emissions from a fourth labeled nucleotide are shown at cloud “4” at the first wavelength and a third intensity that may be less than the first intensity. In some embodiments, fluorescent emissions from the fourth labeled nucleotide may also include some fluorescence in the second wavelength at some non-zero intensity. Fluorescent emissions from a fifth labeled nucleotide are shown at cloud “5” at the second wavelength and a fourth intensity less than the second intensity. [0143] Fluorescent emissions from a sixth labeled nucleotide are shown at cloud “6” at the first wavelength and a fifth intensity less than the third intensity, and also at the second wavelength and a sixth intensity less than the fourth intensity. In some embodiments, the method may include detecting fluorescent emissions from a seventh labeled nucleotide shown at cloud “7” at the first wavelength and a seventh intensity that may be greater than the third intensity, and at the second wavelength and an eighth intensity that may be less than the second intensity. In some embodiments, detecting fluorescent emissions from a third labeled nucleotide at the first wavelength may be greater than the third intensity and at the second wavelength may be greater than the fourth intensity. In some embodiments, the method may include detecting fluorescent emissions from an eighth labeled nucleotide shown at cloud “8” at the first wavelength and a ninth intensity that may be less than the first intensity, and at the second wavelength and tenth intensity that may be greater than the fourth intensity. [0144] In some embodiments, the absence of fluorescent emissions at the first wavelength and at the second wavelength, shown as cloud “9” may indicate the absence of a polynucleotide, or the presence of a labeled nucleotide. In some embodiments, cloud “9” may correspond to a dark base, where the absence of detection of fluorescence may indicate either that a well is empty or the presence of the dark base. [0145] An aspect of the disclosure is directed to providing a sequencing system with fluorescent emissions with different relative intensities as compared to prior systems which may have only utilized an “on” versus “off” intensity. An increase or reduction in brightness may be quantified as an absolute measurement in terms of photon flux or counts per second. The increase in brightness may also be quantified as relative measurement for a cluster labeled with a single nucleotide as compared to a cluster labeled with two different nucleotides. For SNR optimization reasons, some sequencing systems employ a square constraint on a scatterplot of a two-channel detection, wherein a cluster labeled with a single nucleotide is constrained to have a similar intensity as a cluster labeled with two nucleotides (e.g. base C in a corner cloud). Such constraints in intensity/brightness of a cluster may be controlled by diluting labeled nucleotides with a non- fluorescing tag, such that all clusters are emitting at approximately half of the potential brightness. Relaxing this constraint according to this disclosure may improve increase signal to noise ratio (SNR). [0146] It should be realized that on any flow cell, the different clusters may have varying brightness. For example, some clusters can be bright, and some clusters can be dim in comparison to each other. In embodiments, the intensity values vary between base calling cycles and thus the classification of bright and dim may also change between cycles. Some examples of intensity value ratios of emissions between bright and dim clusters include 0.55:0.45, 0.60:0.40, 0.65:0.35, 0.70:0.30, 0.75:0.25, 0.80:0.20, 0.85:0.15, 0.90:0.10, and 0.95:0.05. During each sampling event (e.g., each illumination stage or each image acquisition stage), a detector may image clusters with different intensities or clusters generating different types of signals. [0147] In some embodiments, a method according to this disclosure may allow for each cluster to be labeled with a single dye, and that dye, or faction of that dye, may be chosen to increase the brightness of the cluster. In some embodiments, methods and systems may not require clusters to be labeled with two nucleotides. Some embodiments may result in an increase in the brightness of a cluster relative to an intensity of fluorescent emissions from a labeled nucleotide labeled with two fluorescent dyes, wherein the two fluorescent dyes are detected in two different channels. Some embodiments may result in an increase in the brightness of a cluster relative to an intensity of fluorescent emissions from a nucleotide forming a corner cloud in a two-excitation, two-channel detection method. [0148] Some embodiments may include a first labeled nucleotide that is labeled with one fluorescent dye at a first intensity, wherein the first intensity is at least 10% higher than an intensity of fluorescent emissions from, for example, a fourth labeled nucleotide at the second intensity. In some embodiments, the first intensity may be at least 20% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 30% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 40% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 50% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 100% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 110% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 120% higher relative to a fourth labeled nucleotide. [0149] The brightness of any cluster may also be affected by fragment length distribution of the sample. The varying brightness of the cluster population can have the effect of elongating the ‘on’ populations in the base calling scatterplot. By way of example, without normalizing the level of amplification before trying to increase the brightness of all the clusters, some over-amplified AT rich sequences may be much brighter than similar GC rich sequences, and become even more ‘over amplified’ than the GC rich clusters. In some embodiments, it may be advantageous to normalize each cluster’s intensity by its mean intensity in the first 10 cycles to reduce population intensity variation. For example, in the first ten cycles, for every non- guanine(G) base call, two radii can be calculated: the distance of the population intensity from the origin, and the distance of the corresponding Gaussian mean from the origin. Cluster scaling can include normalizing to the mean of the ratio of these two radii averaged over, for example, the first 10 cycles. All cluster intensities can be normalized by this scaling factor before phase correction and base calling are performed. Cluster scaling can advantageously increase throughput and decrease error rates, for example, for samples with large fragment length distributions. [0150] It is also possible to improve the brightness of all clusters, for example, by carrying out a higher number of amplification cycles, or by changing the chemistry/fraction of the dyes, or by changing the detection scheme (two to three-channel detection as described herein). In some embodiments, the methods and systems may be employed in a two-channel system, however, one of skill in the art will understand that the labeling in one channel with different/same dyes for two nucleotides may be applied to, for example, a four-channel system. Base calling with five bases [0151] An aspect of the disclosure is directed to channel multiplexing. Channel based multiplexing may expand the number of possible states for encoding nucleotides by using additional color channels. In some embodiments, channel multiplexing may be used in combination with amplitude multiplexing, where additional states are encoded into existing channels by changing amplitudes of the fluorescence emissions in the existing channels. In some embodiments, specifically in the context of greater than four base sequencing, a third color channel may encode an additional base (a fifth base) or more bases as required. [0152] Fig. 5 shows a flowchart of a method 500 of sequencing clusters of labeled polynucleotides bound to a flow cell according to one embodiment. The method may begin at a start step 502, by, for example, gathering DNA samples, and putting the sample into a sequencing system, such as described above. Sequencing systems according to the disclosure may include flow cell sequencers such as those produced by ILLUMINA®, INC. (San Diego, CA). For example, when the method 500 begins at step 502, a flow cell may have been prepared by embedding the flow cell with fragmented polynucleotides (e.g., fragmented single- or double- stranded polynucleotide fragments). Fragmented polynucleotides may be generated from a deoxyribonucleic acid (DNA) sample. DNA samples may be from various sources, for example, a biological sample, a cell sample, an environmental sample, or any combination thereof. The lengths of fragmented polynucleotide fragments may range from, for example, 100 bases to 1000 bases, or more. [0153] After generating clusters within the flow cell, the method 500 may begin a Sequencing by Synthesis process. During each sequencing cycle, four or more types of nucleotide analogs are added and incorporated onto the growing primer-polynucleotides. The four or more types of nucleotide analogs may have different modifications. For example, a first type of nucleotide may be an analog of deoxyguanosine triphosphate (dGTP), which is partially conjugated via a linker with a first type of fluorescent label that, after an excitation, can emit light at a first emission wavelength. In some embodiments, only a fraction of the dGTP nucleotides are labeled so that each cluster which has a dGTP as the next incorporated nucleotide will fluoresce with a reduced intensity as compared to a cluster where every incorporated nucleotide was labeled. For example, only 10, 20, 30, 40, 50, 60, or 70 percent of the nucleotides in a dGTP mixture that is used during SBS may be labeled. Thus, the intensity of the fluorescence coming from a cluster that is labeled with dGTP would be detectibly lower than clusters having every growing strand labeled with a fluorescent dye. This allows the same dye to be used on dGTP nucleotides as on another nucleotide, but still have the dGTP clusters be detectibly different from the other clusters. [0154] Once the clusters are created on the flow cell, the method 500 moves to a step 510 of detecting fluorescent emissions from a first labeled nucleotide at a first wavelength. For example, step 510 may include exciting all of the clusters of labeled polynucleotides on a flow cell at a first excitation wavelength. A single light source such as a laser or an LED source may excite a fluorescent label at the predetermined excitation wavelength. In some embodiments, the single laser or the LED source may be non-tunable. Step 510 may also include detecting any fluorescent emissions from the clusters at a first detection wavelength or range of wavelengths to detect the presence of a first labeled nucleotide. In general, the first detection wavelength will be red-shifted to a longer wavelength relative to the first excitation wavelength. [0155] After detecting the fluorescence emissions from the clusters at step 510, the method 500 moves to a step 520, where the method detects fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength. [0156] When the method detects fluorescent emissions from a first wavelength in step 510 different than a second wavelength in step 520, the method may be described as employing at least two channel base calling. A “channel” may be used to describe an emission/detection process for a labeled or unlabeled base that is specific for a particular excitation wavelength and detection wavelength pair. Thus, a channel may include a particular first range of wavelengths of light used to excite a fluorescent dye and a second range of wavelengths of light used to detect the fluorescent emissions from the excited dyes. In the context of one excitation, two channel systems and methods, a first excitation wavelength may excite two types of dyes to emit light in two different wavelengths, where each emission wavelength is referred to as a channel. The disclosure provides for, inter alia, two or more channel base calling for four or more bases. [0157] After detecting the fluorescence emissions from the clusters at step 520, the method 500 moves to a step 530, where the method detects fluorescent emissions from a third labeled nucleotide at the first and second wavelengths. For example, at step 530 fluorescent emissions from a third labeled nucleotide may be detected at the second wavelength at some intensity and at the first wavelength may be detected at an intensity. In some embodiments, fluorescent emissions from a third labeled nucleotide at the first and second wavelength may be detected at some non-zero intensity. The intensity of the detected emissions at the first wavelength may be quantified as an absolute measurement in terms of photon flux or counts per second. In some embodiments, the intensity of detected emissions may be normalized such that intensities in a channel are relative to the maximum emission in that channel. As described herein, the intensities may also be quantified as relative measurement for a cluster labeled with a single nucleotide as compared to a cluster labeled with two different nucleotides. [0158] After detecting the fluorescence emissions from the clusters at step 530, the method 500 moves to a step 540, where the method detects the absence of fluorescent emissions from the fourth labeled nucleotide. For example, at step 540, the method may detect fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a third intensity that is approximately one half of the first intensity. In some embodiments, at step 540, the method may detect fluorescent emissions from the fourth labeled nucleotide at the first wavelength and also at a second and/or third wavelength. In some embodiments, at step 540, the method may detect fluorescent emissions from the fourth labeled nucleotide only at the first wavelength. For example, a cluster may be identified by the lack of any fluorescence after excitation at either the first or second wavelengths. Because the lack of fluorescent emissions is dark or “missing” on an image, the system may track the position of each cluster on a flow cell. Once a cluster has been identified at a particular position, the system may then note in subsequence sequencing rounds whether there is a fluorescent emission at the position of the cluster. If no fluorescent emissions are noted at a known position of a cluster, the system may determine that the nucleotide added in the latest sequencing round was the unlabeled nucleotide. For example, in some cases the unlabeled nucleotide may be a “G”. In another example, the unlabeled nucleotide may correspond to no nucleotide being sequenced—from either an empty well or a finished cluster. However, it should be realized that any nucleotide could be chosen as the one which is unlabeled and still be within the scope of the invention. [0159] After detecting fluorescence emissions, or lack thereof, from the clusters at step 540, the method 500 moves to a step 550, where the method detects fluorescent emissions from a fifth nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths. In a variation of step 550 some embodiments may include the third wavelength as the same as one of the first and second wavelengths. For example, at step 550, the method may detect fluorescent emissions from the fifth labeled nucleotide at the second wavelength and also at the first and/or a third wavelength. In some embodiments, at step 550, the method may detect fluorescent emissions from the fifth labeled nucleotide only at the second wavelength. [0160] The method 500 then moves to a decision step 555 to determine if the method 500 should repeat for an additional SBS cycle of reading sequence data. A determination may be made at decision step 555 whether to detect more nucleotides based on, for example, the quality of the signal or after a predetermined number of bases. If more nucleotides are to be detected, then the method 500 may loop back to the step 510 to start a next sequencing cycle. [0161] The method 500 may then move to a step 560 wherein the system may determine the nucleotide sequence of the polynucleotide based on the detected fluorescent emissions. For example, at the step 560 the method may identify clusters that have added nucleotides with emissions at wavelengths and intensities corresponding to the first, second, third fourth and/or fifth labeled nucleotides. In some embodiments, at the step 560 the method may identify clusters which had no fluorescent emissions following excitation at the first and/or second excitation wavelength and determine that the cluster corresponds to an empty well or to a short insert that has completed sequencing. [0162] In some embodiments, prior to the next sequencing cycle, the fluorescent labels attached to each nucleotide may be removed from the incorporated nucleotide analogs, and the reversible 3ƍ blocks may be removed so that another nucleotide analog may be added onto each extending primer-polynucleotide. If a determination is made at the decision step 555 that there are no more additional rounds of sequencing necessary, the method 500 then moves to an end step 570 and terminates the method 500. For each nucleotide base determined, a quality score may be determined. After all the fluorescent images are processed, the method 500 may terminate at the step 570. [0163] Fig. 6 illustrates an example of the results from labeling nucleotides and doing base calling for five bases within embodiments of the invention. The left panel of Fig.6 shows an X-Y axis scatterplot of the fluorescence emissions in a first channel (channel 0) and a second channel (channel 1). The Y axis 601and X axis 602 correspond to intensities of fluorescent signals detected in a detection channel. In some embodiments, the Y axis 601 may correspond to intensities of fluorescence detected from a channel (for example, a first channel or Channel 0) detecting green wavelengths. The X axis 602 may correspond to intensities of fluorescence detected from a channel (for example, a second channel, or Channel 1) detecting red wavelengths. [0164] Nucleotide “C” 610 is shown as a cloud in the top left corner in the left panel of Fig.6 and corresponds to detections of fluorescence in the green channel, and no detections (or minimal detections) in the red channel. Nucleotide “A” 620 is shown as a cloud in the top right corner and corresponds to detection of fluorescence in the green channel and in the red channel. Nucleotide “T” 630 is shown as a cloud in the bottom right corner and corresponds to detection of fluorescence in the red channel and no detections (or minimal detections) in the green channel. Nucleotide “G” 640 is shown as a cloud in the bottom left corner of Fig. 6 and corresponds to typical “dark detection” of nucleotide “G”—no detections (or minimal detections) in the green and red channels. A fifth nucleotide “X” 645 is shown as a small circle at the same general position as nucleotide “G” 640 and corresponds to no detection (or minimal detections) in the green and red channels, but detection in a third channel (for example, channel two) that detects fluorescent emissions at a third wavelength. In some embodiments, the third channel may detect fluorescent emissions at orange wavelengths. In some embodiments, the relative sizes of the clouds for nucleotide “G” 640 and the fifth nucleotide “X” 645 do not represent any difference in signal to noise or the range of values for the cloud in the X-Y scatterplot. In other embodiments, either Nucleotide “G” 640 or the fifth nucleotide “X” 645 may have a smaller cloud in the X-Y scatterplot because the unlabeled dyes or dyes emitting in the third channel contribute to less crosstalk in the first and second channels. The fifth nucleotide “X” 645 is shown as encoded using the “on-state” of the third channel. In some embodiments, the “on-state” of the third channel may refer to a dye that emits in a third channel. In some embodiments, the “on-state” of the third channel may refer to a dye that emits in either the first or second channel after a chemical processing step. The four naturally occurring DNA bases are shown represented using a standard two-channel base calling encoding scheme. [0165] The right panel of Fig. 6 shows an X-Y axis graph of the probability density distribution for detecting fluorescent emissions over channel three for the five nucleotides in the left panel. The Y axis 603 corresponds to the probabilistic frequency of detections of fluorescent emissions. The X-axis 604 corresponds to intensities of fluorescence detected from a third channel (here, Channel 3). As shown in the left panel of Fig. 6, the third channel only encodes one additional base, such that there are only two options for intensities in the third channel—on and off. In two channel base calling systems, the usual four DNA bases are encoded using two bits of information from each color channel. Software methods may proceed through a series of intensity extraction, normalization, signal correction steps before base calling. During base calling, a probabilistic model may be built to jointly map a probability density distribution over the two- color channels. Once this probabilistic model is determined, the base calls may then be assigned based on the inference outcome of the probabilistic model (for example, at maximum likelihood). [0166] The graph in the right panel of Fig. 6 shows two peaks in probabilistic frequency. The first peak corresponds to the probabilistic frequency of detecting the off state (at lower values on the X axis) and the second peak corresponds to the probabilistic frequency of detecting the on state. The on state (at higher values on the X axis) is shown to have a lower probabilistic frequency because the other four nucleotides will not emit in the third channel, and the nucleotide assigned to emit in the third channel may not be present in an equal proportion in the polynucleotide to be sequenced. In some embodiments using more than five bases, additional multiplexing may be implemented by jointly combining the information from three channels together into a 3-dimensional scatter plot system. [0167] Fig.7 shows a three-dimensional X-Y-Z scatterplot of the detected fluorescence emissions in a first channel, a second channel, and a third channel. The axes correspond to intensities of fluorescent signals detected in a detection channel. The X-axis 701 corresponds to intensities of fluorescence detected from a first channel (here, Channel 0).The Y axis 702 corresponds to intensities of fluorescence detected from a second channel (here, Channel 1). The Z axis 703 corresponds to intensities of fluorescence detected from a second channel (here, Channel 2). [0168] In some embodiments, the Y axis 702 may correspond to intensities of fluorescence detected from a channel detecting green wavelengths. The X axis 701 may correspond to intensities of fluorescence detected from a channel detecting red wavelengths. The Z axis may correspond to intensities of fluorescence detected from a channel detecting orange wavelengths. Note that the intensities of each axis may be normalized, and the total non- normalized intensities may be increased by using a dye with a higher quantum yield of fluorescence, or a larger percentage of a dye with a high quantum yield. [0169] Nucleotide “C” 710 is shown as a cloud in the top left corner in Fig. 7 and corresponds to detections of fluorescence in the green channel, and no detections (or minimal detections) in the red and orange channels. Nucleotide “A” 720 is shown as a cloud in the top right corner and corresponds to a detection of fluorescence in the green channel and in the red channel, and no detection in the orange channel. Nucleotide “T” 730 is shown as a cloud in the middle right side and corresponds to a detection of fluorescence in the red channel and no detections (or minimal detection) in the green channel or the orange channel. Nucleotide “G” 740 is shown as a cloud near the origin of the scatterplot of Fig. 7 and corresponds to typical dark detection of nucleotide “G”—no detection (or minimal detection) in the green, red, and orange channels. Nucleotide “X” 750 is shown as a cloud in the bottom left corner and corresponds to a detection of fluorescence in the orange channel and no detections (or minimal detections) in the green channel or the red channel. In some embodiments, Nucleotide “X” 750 may be equivalent to nucleotide 645 in Fig. 6. Nucleotide “Y” 760 is shown as a cloud in the bottom right corner and corresponds to a detection of fluorescence in the red channel and the orange channel and no detections (or minimal detections) in the green channel. [0170] While not shown, Fig. 7 suggests that a three-channel system may encode at least two additional nucleotides. For example, a nucleotide may emit in a cloud (not shown) corresponding to emissions in both the orange and green channels. In some embodiments, a nucleotide may emit in a cloud (not shown) corresponding to emissions in the green, red and orange channels. In some embodiments, the nucleotide corresponding to emissions in the green red and orange channels is omitted in order to reduce any fluorescence intensity limits imposed by labeling a nucleotide with three different dyes (where the emission intensity in each channel may be reduced to a third of maximum intensity. [0171] As Fig. 7 illustrates, systems and methods disclosed herein may encode four additional states (only “X” and “Y” are shown to improve clarity) using three channels. Table 1 below summarizes the options for two level encoding for three channels. Table 1 Base Channel 0 Channel 1 Channel 2 A 1 1 0 C 1 0 0 G 0 0 0 T 0 1 0 X 1 1 1 Y 1 0 1 Z 0 0 1 W 0 1 1 [0172] An aspect of the disclosure is directed to base calling systems and methods that address issues that may arise from low nucleotide diversity. In general, base calls with the highest accuracy are determined when a polynucleotide sequence has a roughly equal representation of all DNA bases. The non-naturally occurring bases are rarer and will generally not satisfy the condition of equal representation of all DNA bases. In some embodiments, raw data from the polynucleotide sequence may be supplemented with an excess of the rare bases in order to improve base call accuracy. [0173] As another example, the method outlined in Fig. 6 will have less stringent requirements on base diversity and provides highly accurate base calls even if the fifth base is not commonly occurring. Accordingly, in some embodiments, a fifth base with low diversity may be determined as the fifth base in the method outlined in Fig. 6, and be determined by a single “on” state in the third channel. In another embodiment, a low diversity base may be paired for detection in another channel (or set of channels) that also detects another low diversity base. For example, modified nucleotides “X” and “Y” may be paired in the third channel as shown in Fig.7. Embodiments with 4 channels and a dark state [0174] As mentioned above, an aspect of the disclosure is directed to four channel sequencing systems and methods that employ four channels and a dark state to identify five nucleotides. For example, an aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths; detecting fluorescent emissions from a fourth labeled nucleotide at a fourth wavelength, wherein the fourth wavelength is different from the first, second, and third wavelengths; detecting the absence of fluorescent emissions from a fifth labeled nucleotide; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions. [0175] Figure 8 shows a flowchart of a method 800 of sequencing clusters of labeled polynucleotides bound to a flow cell according to one embodiment. The method begins at a start step 802, by, for example, gathering DNA samples, and putting the sample into a sequencing system, such as described above. Sequencing systems according to the disclosure may include flow cell sequencers such as those produced by ILLUMINA®, INC. (San Diego, CA). For example, when the method 800 begins at step 802, a flow cell may have been prepared by embedding the flow cell with fragmented polynucleotides (e.g., fragmented single- or double- stranded polynucleotide fragments). Fragmented polynucleotides may be generated from a deoxyribonucleic acid (DNA) sample. DNA samples may be from various sources, for example, a biological sample, a cell sample, an environmental sample, or any combination thereof. The lengths of fragmented polynucleotide fragments may range from, for example, 100 bases to 1000 bases, or more. [0176] After generating clusters within the flow cell, the method 800 may begin a Sequencing by Synthesis process. During each sequencing cycle, four or more types of nucleotide analogs are added and incorporated onto the growing primer-polynucleotides. The four or more types of nucleotide analogs may have different modifications. For example, a first type of nucleotide may be an analog of deoxyguanosine triphosphate (dGTP), which is partially conjugated via a linker with a first type of fluorescent label that, after an excitation, can emit light at a first emission wavelength. In some embodiments, only a fraction of the dGTP nucleotides are labeled so that each cluster which has a dGTP as the next incorporated nucleotide will fluoresce with a reduced intensity as compared to a cluster where every incorporated nucleotide was labeled. For example, only 10, 20, 30, 40, 50, 60, or 70 percent of the nucleotides in a dGTP mixture that is used during SBS may be labeled. Thus, the intensity of the fluorescence coming from a cluster that is labeled with dGTP would be detectibly lower than clusters having every growing strand labeled with a fluorescent dye. This allows the same dye to be used on dGTP nucleotides as on another nucleotide, but still have the dGTP clusters be detectibly different from the other clusters. [0177] Once the clusters are created on the flow cell, the method 800 moves to a step 810 of detecting fluorescent emissions from a first labeled nucleotide at a first wavelength. For example, step 810 may include exciting all of the clusters of labeled polynucleotides on a flow cell at a first excitation wavelength. A single light source such as a laser or an LED source may excite a fluorescent label at the predetermined excitation wavelength. In some embodiments, the single laser or the LED source may be non-tunable. Step 810 may also include detecting any fluorescent emissions from the clusters at a first detection wavelength or range of wavelengths to detect the presence of a first labeled nucleotide. In general, the first detection wavelength will be red-shifted to a longer wavelength relative to the first excitation wavelength. [0178] After detecting the fluorescence emissions from the clusters at step 810, the method 800 moves to a step 820, where the method detects fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength. [0179] When the method detects fluorescent emissions from a first wavelength in step 810 different than a second wavelength in step 820, the method may be described as employing at least two channel base calling. A “channel” may be used to describe an emission/detection process for a labeled or unlabeled base that is specific for a particular excitation wavelength and detection wavelength pair. Thus, a channel may include a particular first range of wavelengths of light used to excite a fluorescent dye and a second range of wavelengths of light used to detect the fluorescent emissions from the excited dyes. In the context of one excitation, two channel systems and methods, a first excitation wavelength may excite two types of dyes to emit light in two different wavelengths, where each emission wavelength is referred to as a channel. The disclosure provides for, inter alia, four channel base calling for four or more bases. [0180] After detecting the fluorescence emissions from the clusters at step 820, the method 800 moves to a step 830, where the method detects fluorescent emissions from a third labeled nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths. For example, at step 830 fluorescent emissions from a third labeled nucleotide may be detected at a third wavelength at some non-zero intensity. In some embodiments, fluorescent emissions from a third labeled nucleotide may also be detected at one of the first and second wavelength may be detected at some non-zero intensity. The intensity of the detected emissions at the first wavelength may be quantified as an absolute measurement in terms of photon flux or counts per second. In some embodiments, the intensity of detected emissions may be normalized such that intensities in a channel are relative to the maximum emission in that channel. As described herein, the intensities may also be quantified as relative measurement for a cluster labeled with a single nucleotide as compared to a cluster labeled with two different nucleotides. [0181] After detecting the fluorescence emissions from the clusters at step 830, the method 800 moves to a step 840, where the method detects fluorescent emissions from a fourth labeled nucleotide at a fourth wavelength, wherein the fourth wavelength is different from the first, second, and third wavelengths. For example, through step 840, the method 800 may detect fluorescent emissions in a blue channel, a green channel, an orange channel and a red channel. [0182] In some embodiments, at step 840, the method may detect fluorescent emissions from the fourth labeled nucleotide at the fourth wavelength and also at one or more of the first, second and third wavelengths. In some embodiments, additional bases may be detected by detecting fluorescent emissions in more than one channel. In some embodiments, at step 840, the method may detect fluorescent emissions from the fourth labeled nucleotide only at the fourth wavelength. [0183] After detecting the fluorescence emissions from the clusters at step 830, the method 800 moves to a step 850, where the method detects the absence of fluorescent emissions from a fifth labeled nucleotide. For example, a cluster may be identified by the lack of any fluorescence at any of the first, second, third and fourth wavelengths after excitation. Because the lack of fluorescent emissions is dark or “missing” on an image, the system may track the position of each cluster on a flow cell. Once a cluster has been identified at a particular position, the system may then note in subsequence sequencing rounds whether there is a fluorescent emission at the position of the cluster. If no fluorescent emissions are noted at a known position of a cluster, the system may determine that the nucleotide added in the latest sequencing round was the unlabeled nucleotide. For example, in some cases the unlabeled nucleotide may be a “G”. In another example, the unlabeled nucleotide may correspond to no nucleotide being sequenced—from either an empty well or a finished cluster. However, it should be realized that any nucleotide could be chosen as the one which is unlabeled and still be within the scope of the invention. [0184] After detecting fluorescence emissions, or lack thereof, from the clusters at step 850, the method 800 then moves to a decision step 855 to determine if the method 800 should repeat for an additional SBS cycle of reading sequence data. A determination may be made at decision step 855 whether to detect more nucleotides based on, for example, the quality of the signal or after a predetermined number of bases. If more nucleotides are to be detected, then the method 800 may loop back to the step 810 to start a next sequencing cycle. [0185] The method 800 may then move to a step 860 wherein the system may determine the nucleotide sequence of the polynucleotide based on the detected fluorescent emissions. For example, at the step 860 the method may identify clusters that have added nucleotides with emissions at wavelengths and intensities corresponding to the first, second, third fourth and/or fifth labeled nucleotides. In some embodiments, at the step 860 the method may identify clusters which had no fluorescent emissions following excitation at the first and/or second excitation wavelength and determine that the cluster corresponds to an empty well or to a short insert that has completed sequencing. [0186] In some embodiments, prior to the next sequencing cycle, the fluorescent labels attached to each nucleotide may be removed from the incorporated nucleotide analogs, and the reversible 3ƍ blocks may be removed so that another nucleotide analog may be added onto each extending primer-polynucleotide. If a determination is made at the decision step 855 that there are no more additional rounds of sequencing necessary, the method 800 then moves to an end step 870 and terminates the method 800. For each nucleotide base determined, a quality score may be determined. After all the fluorescent images are processed, the method 800 may terminate at the step 870. [0187] An aspect of the disclosure is directed to four channel base calling extended for additional bases. For example, one of the color channels may be paired with “dark” base encoding, thereby encoding five total bases which may include a modified nucleotide. Fig.9 illustrates three examples of base calling in a system configured to detect five bases with four channels, where each panel of Fig. 9 summarizes a different base calling scenario through a X-Y histogram of emissions intensities in four channels. In each panel, the Y-axes correspond to corrected or normalized amplitude of fluorescent emissions, with a dashed line indicating a minimum intensity of fluorescent emissions for each channel to be in an “on” state. The X-axes of the histograms are a number line that has been split into four bins. For each of the four bins, a bar is drawn where the height of the bar represents the detected fluorescent intensity in a channel, where each channel is assigned to a nucleotide. The X-axes include bins corresponding to a first channel assigned to an “A” nucleotide, a second channel assigned to an “C” nucleotide, a third channel assigned to an “X” nucleotide, and a fourth channel assigned to an “T” nucleotide. [0188] The first panel on the left of Fig. 9 shows a two-dimensional histogram of fluorescent emission intensities where “T” is correctly base called via a detection in the “T” channel. The intensities of the “A”, “C”, and “X” channels are in the “off” state and are shown to have random amounts of minimal but non-zero emission detections. The second panel of Fig. 9 shows a two-dimensional histogram of fluorescent emission intensities where “A” is correctly base called via a detection in the “A” channel. Similarly, correct base calls for nucleotides “X” and “T” would correspond to detections in the “X” and “T” channels, respectively (not shown). The third and final panel of Fig. 9 shows a two-dimensional histogram of fluorescent emission intensities where “G” is correctly base called via the absence of detections in all four channels. [0189] Fig. 9 illustrates a four channel system for detecting more than four bases through a "maximum amplitude" strategy. Here, four of the five possible fluorescently labeled DNA bases may be correctly base called with an “on” state that may detect a bright dye for each channel. In some embodiments, wells that are temporarily dark when incorporating the unlabeled nucleotide may be distinguished from truly dark wells by using an adapted empty detection algorithm used for two-channel base calling, where clusters are labeled as "unoccupied" based on observing the clusters’ behavior over multiple cycles of sequencing. [0190] As described above, an aspect of the disclosure is directed to base calling systems and methods that address issues that may arise from low nucleotide diversity. One advantage of using the four-channel base calling scheme illustrated in Fig. 9 is efficient handling of low diversity nucleotides. This model may operate without constructing a probabilistic model that would require joint distribution information to be available in a sufficient quantity in all channels. [0191] Fig. 10 shows an illustration of two X-Y axis scatterplots of the fluorescence emissions of eight nucleotides in a total of four channels. The left two-dimensional scatterplot replicates the intensities of fluorescence emissions from a traditional two excitation, two channel sequencing system. The X and Y axes of the left two-dimensional scatterplot correspond to intensities of fluorescent signals detected in a detection channel, channel 0 and channel 1, respectively. The right two-dimensional scatterplot also replicates the fluorescence emissions from a traditional two excitation, two channel sequencing system, but instead of channel 0 and channel 1, the fluorescence emissions are detected in channel 2 and channel 3. The X and Y axes of the left two-dimensional scatterplot correspond to intensities of fluorescent signals detected in a detection channel, channel 2 and channel 3, respectively. Note that the intensities across each channel may be normalized, and the total non-normalized intensities may be increased by using a dye with a higher quantum yield of fluorescence, or a larger percentage of a dye with a high quantum yield. Also note that the nucleotide G and nucleotide Y are designated as dark bases in each scatterplot but will not necessarily be dark bases in the other corresponding scatterplot. In some embodiments, the dark base may be the same in both scatterplots where seven, instead of eight, bases are determined. In some embodiments, the eight bases may be arranged as in Fig. 7, where instead of the two dually labeled bases in Fig. 10, there would be two dually labeled bases and a triply labeled base. However, in general the scatterplots of Fig. 10 illustrate that a channel multiplexing system may employ base calling methods that reduce a complicated four-dimensional encoding space into two sets of two dimensional encoding space. [0192] The right two-dimensional scatterplot shows clouds of fluorescent emissions for modified nucleotides W, X, Y and Z. These modified nucleotides may all have low base diversity as compared to the natural bases (A, C, T, G nucleotides). In some embodiments, the four natural bases may be at any position in either the left or right scatterplots and may be swapped with the modified bases shown in Fig. 10. In some embodiments, the four natural bases and the modified bases are intentionally grouped together as shown in Fig. 10. In some embodiments, greater than five bases base calling may jointly encode two channel states together to represent the additional nucleotides. In some embodiments, an independence assumption may be employed to limit the number of independent variables to just two. This assumption may simplify a probabilistic model and provide for higher accuracy base calling models with minimal training data. [0193] In some embodiments using joint distribution estimations, the base diversity assumption may be used where the additional nucleotide bases are roughly in equal proportion to the naturally occurring “ACGT”. In some embodiments, two sets of probabilistic models may be used by breaking existing 4 channel (R/G) into two sets of two channels. In some embodiments, additional blue lasers and dyes may be used for the second group of two channels. [0194] As described above, an aspect of the disclosure is directed to base calling systems and methods that address issues that may arise from low nucleotide diversity. One advantage of using the four-channel base calling scheme illustrated in Fig.10 is efficient handling of low diversity nucleotides. In some embodiments this model may operate by constructing a probabilistic model that assumes joint distribution information to be available in a sufficient, but not necessarily equal, quantity in all channels. [0195] In general, while sequencing libraries with low initial sequence diversity typically produces low quality data due to the limitations of cluster calling algorithms, systems and methods of this disclosure may sequence more than four bases using adapted two or four channel systems, while retaining high signal to noise and producing high base call accuracy. [0196] When template generation occurs during the sequencing cycle, a rapid generation rate is highly desired. In a typical protocol, only images from the first four sequencing cycles are used to generate templates (which are then used for cluster calling) in order to increase efficiency and reduce computational cost. The first four iterations might be chosen for template generation on the premise that those images are the highest quality. However, when a sample includes modified bases, the library is likely to lack initial sequence diversity, such that cluster recognition may be difficult in the initial cycles. Accordingly, methods of the disclosure may perform template generation in the middle of a sequencing read. [0197] In some embodiments, an accurate template may be generated for a library that otherwise has low base diversity by delaying template generation for a number of cycles. In some embodiments, an accurate template may be generated for a library that otherwise has low base diversity by mixing low diversity samples with high-diversity samples. In some embodiments, an accurate template may be generated for a library that otherwise has low base diversity by lowering cluster density. Accordingly, the signal to noise ratio of all images on the flow cell may be improved. In some embodiments, an accurate template may be generated for a library that otherwise has low base diversity by image analysis from previously saved images that already have a template generated for modified bases. [0198] An aspect of the disclosure is directed to methods that may encode less than the maximum number of states to improve signal to noise ratio and minimize base call error. The general relationship of encoding levels and channels to represent different DNA bases follows a simple relationship: S=L^C where S is the number of states, L is the number of levels encoded in each channel, and C is the number of channels. In some embodiments, the disclosure provides methods to address base diversity challenges by introducing known sequences including multiples of the less abundant fifth or 6th base to train RTA to achieve higher accuracy of calling fifth or sixth bases. [0199] Some embodiments according to the disclosure are summarized in Table 2 below: Table 2 Method Channels Levels Max. Used Bases Bases 2-channel 2 2 4 4 4-channel 4 1 4 4 2-channel with Amp. 2 3 9 5-9 Multiplexing (See also FIG.4) 3 channel option 1 3 2 8 5 (See also FIG.6) 3 channel option 2 3 2 8 5-8 (See also FIG.7) 4 channel option 1 4 1 5 5 (See also FIG.9) 4 channel option 2 4 2 64 8 (See also FIG.10) Systems according to the disclosure [0200] Embodiments of the present disclosure also include a system for analyzing and assembling sequences of polynucleotides. Fig. 11 is a block diagram of an exemplary computing system 1100 that may be used in connection with an illustrative sequencing system. The computing system 1100 may be configured to determine a DNA sequence by using the sequencing and assembly methods disclosed herein. The general architecture of the computing system 1100 depicted in Fig.1A includes an arrangement of computer hardware and software components. The computing system 1100 may include many more (or fewer) elements than those shown in Fig.1A. It is not necessary, however, that all of these generally conventional elements be shown in order to provide an enabling disclosure. [0201] As illustrated, the computing system 1100 includes a processing unit 1110, a network interface 1120, a computer-readable medium drive 1130, an input/output device interface 1140, a display 1150, and an input device 1160, all of which may communicate with one another by way of a communication bus. The network interface 1170 may provide connectivity to one or more networks or computing systems. The processing unit 1110 may thus receive information and instructions from other computing systems or services via a network. The processing unit 1110 may also communicate to and from memory 1170 and further provide output information for an optional display 1150 via the input/output device interface 1140. The input/output device interface 1140 may also accept input from the optional input device 1160, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device. [0202] The memory 1170 may contain computer program instructions (grouped as modules or components in some embodiments) that the processing unit 1110 executes in order to implement one or more embodiments. The memory 1170 generally includes RAM, ROM and/or other persistent, auxiliary or non-transitory computer-readable media. The memory 1170 may store an operating system 1172 that provides computer program instructions for use by the processing unit 1110 in the general administration and operation of the computing device 1100. The memory 1170 may further include computer program instructions and other information for implementing aspects of the present disclosure. [0203] For example, in one embodiment, the memory 1170 includes a two-channel sequencing module 1174 for analyzing and assembling sequences of polynucleotides. The two- channel sequencing module 1174 can perform the methods disclosed herein, including the method described with respect to the flow diagrams of, for example, Figs. 2, 3, 5 and 8. In addition, memory 1170 may include or communicate with the data store 1190 and/or one or more other data stores that store one or more inputs, one or more outputs, and/or one or more results (including intermediate results) of determining a DNA sequence and providing an assembly process according to the present disclosure. [0204] Particular embodiments of the method of sequencing may utilize a two-channel detection system (also known as 2Ex-2Ch), a three excitation, three channel detection system (also known as 3Ex-4Ch) or a four channel detection system (with two, three, four or more excitation sources). Detailed disclosures are provided in WO 2018/165099 and U.S. Ser. No. 17/338590, each of which is incorporated by reference in its entirety. However, the number of channels in a system does not change whether aspects of the configurations are mutually exclusive, and these configurations can be used in various combinations. For example, some dyes used in 1Ex-2Ch may be used in 2Ex-2Ch configurations. [0205] In some embodiments, methods according to the disclosure may be performed on an automated sequencing instrument, and wherein the automatic sequencing instrument may comprise two light sources operating at different wavelengths (e.g., at 350-360 nm (blue), 520- 530 nm (green), 630 nm-670 nm (red)). The incorporation of the first type of the nucleotide conjugates is determined by a signal state in the first imaging event and a dark state in the second imaging event. The incorporation of the second type of the nucleotide conjugates is determined by a dark state in the first imaging event and a signal state in the second imaging event. The incorporation of the third type of the nucleotide conjugates is determined by a signal state in both the first imaging event and the second imaging event. The incorporation of the fourth type of the nucleotide conjugates is determined by a dark state in the first imaging event and a partial signal state in the second imaging event. The incorporation of the fifth type of the nucleotide conjugates is determined by a dark state in both the first imaging event and the second imaging event. [0206] In some embodiments, the incorporation of the first type of the nucleotide conjugates is determined by a signal state in the first imaging event and a dark state in the second imaging event. The incorporation of a fifth type of the nucleotide conjugates is determined by a reduced intensity signal state in the first imaging event and a dark state in the second imaging event. The incorporation of the second type of the nucleotide conjugates is determined by a dark state in the first imaging event and a signal state in the second imaging event. The incorporation of the third type of the nucleotide conjugates is determined by a signal state in both the first imaging event and the second imaging event. The incorporation of the fourth type of the nucleotide conjugates is determined by a dark state in the first imaging event and a partial signal state in the second imaging event. The incorporation of a sixth type of the nucleotide conjugates is determined by a dark state in both the first imaging event and the second imaging event. [0207] In some embodiments, the automatic sequencing instrument may comprise three or more light sources, and an automated sequencing instrument may perform three or more imaging steps. In some embodiments, the incorporation of the first type of the nucleotide conjugates is determined by a signal state in the first imaging event and a dark state in the second and third imaging events. The incorporation of a fifth type of the nucleotide conjugates is determined by a reduced intensity signal state in the first imaging event and a dark state in the second and third imaging events. The incorporation of the second type of the nucleotide conjugates is determined by a dark state in the first and third imaging events and a signal state in the second imaging event. The incorporation of the third type of the nucleotide conjugates is determined by a signal state in both the first imaging event and the second imaging event, and a dark state in the third imaging event. The incorporation of the fourth type of the nucleotide conjugates is determined by a dark state in the first and third imaging events and a partial signal state in the second imaging event. The incorporation of a sixth type of the nucleotide conjugates is determined by a signal state in the third imaging event, and a dark state in both the first imaging event and the second imaging event. [0208] In some embodiments, the automatic sequencing instrument may comprise a single light source operating with a blue laser at about 350 nm to about 360 nm. The incorporation of the first type of the nucleotide may be determined by detection in the one of the blue or green channel/region (e.g., at a blue region with a wavelength ranging from about 372 to about 520 nm, or at a green region with a wavelength ranging from about 540 nm to about 640nm). The incorporation of the second type of nucleotide is determined by detection in the other one of the blue or green detection channel/region. The incorporation of the third type of nucleotide is determined by detection in both the blue and green channels/regions. The incorporation of the fourth type of nucleotide is determined by a partial detection in the blue channel/region but no detection green channel/region. The incorporation of the fifth type of nucleotide is determined by no detection in either the blue or detection green channels/regions. [0209] In some embodiments, the disclosed systems and methods may involve approaches for shifting or distributing certain sequence data analysis features and sequence data storage to a cloud computing environment or cloud-based network. User interaction with sequencing data, genome data, or other types of biological data may be mediated via a central hub that stores and controls access to various interactions with the data. In some embodiments, the cloud computing environment may also provide sharing of protocols, analysis methods, libraries, sequence data as well as distributed processing for sequencing, analysis, and reporting. In some embodiments, the cloud computing environment facilitates modification or annotation of sequence data by users. In some embodiments, the systems and methods may be implemented in a computer browser, on-demand or on-line. [0210] In some embodiments, software written to perform the methods as described herein is stored in some form of computer readable medium, such as memory, CD-ROM, DVD- ROM, memory stick, flash drive, hard drive, SSD hard drive, server, mainframe storage system and the like. [0211] In some embodiments, computational methods as described herein are carried out on a collection of inter- or intra-connected computer systems (i.e., grid technology) which may run a variety of operating systems in a coordinated manner. For example, the CONDOR framework (University of Wisconsin-Madison) and systems available through United Devices are exemplary of the coordination of multiple stand-alone computer systems for the purpose of dealing with large amounts of data. These systems may offer Perl interfaces to submit, monitor and manage large sequence analysis jobs on a cluster in serial or parallel configurations. One aspect of the disclosure is directed to a workflow module that may be integrated into existing workflows. In some embodiments, a workflow module may be a two-channel sequencing module and may be integrated into a NGS sequence analysis platform, for example the DRAGEN™ Bio-ID platform from Illumina. Samples [0212] In some embodiments, the sample comprises or consists of a purified or isolated polynucleotide derived from a tissue sample, a biological fluid sample, a cell sample, and the like. Suitable biological fluid samples include, but are not limited to blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginal flow, trans-cervical lavage, brain fluid, ascites, milk, secretions of the respiratory, intestinal and genitourinary tracts, amniotic fluid, milk, and leukophoresis samples. In some embodiments, the sample is a sample that is easily obtainable by non-invasive procedures, e.g., blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, saliva or feces. In certain embodiments the sample is a peripheral blood sample, or the plasma and/or serum fractions of a peripheral blood sample. In other embodiments, the biological sample is a swab or smear, a biopsy specimen, or a cell culture. In another embodiment, the sample is a mixture of two or more biological samples, e.g., a biological sample can comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample. As used herein, the terms “blood,” “plasma” and “serum” expressly encompass fractions or processed portions thereof. Similarly, where a sample is taken from a biopsy, swab, smear, etc., the “sample” encompasses a processed fraction or portion derived from the biopsy, swab, smear, etc. [0213] In certain embodiments, samples can be obtained from sources, including, but not limited to, samples from different individuals, samples from different developmental stages of the same or different individuals, samples from different diseased individuals (e.g., individuals with cancer or suspected of having a genetic disorder), normal individuals, samples obtained at different stages of a disease in an individual, samples obtained from an individual subjected to different treatments for a disease, samples from individuals subjected to different environmental factors, samples from individuals with predisposition to a pathology, samples individuals with exposure to an infectious disease agent, and the like. [0214] In one illustrative, but non-limiting embodiment, the sample is a maternal sample that is obtained from a pregnant female, for example a pregnant woman. The maternal sample can be a tissue sample, a biological fluid sample, or a cell sample. In another illustrative, but non-limiting embodiment, the maternal sample is a mixture of two or more biological samples, e.g., the biological sample can comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample. [0215] In certain embodiments samples can also be obtained from in vitro cultured tissues, cells, or other polynucleotide-containing sources. The cultured samples can be taken from sources including, but not limited to, cultures (e.g., tissue or cells) maintained in different media and conditions (e.g., pH, pressure, or temperature), cultures (e.g., tissue or cells) maintained for different periods of length, cultures (e.g., tissue or cells) treated with different factors or reagents (e.g., a drug candidate, or a modulator), or cultures of different types of tissue and/or cells. [0216] In some embodiments, the use of the disclosed sequencing technology does not involve the preparation of sequencing libraries. In other embodiments, the sequencing technology contemplated herein involves the preparation of sequencing libraries. In one illustrative approach, sequencing library preparation involves the production of a random collection of adapter-modified DNA fragments (e.g., polynucleotides) that are ready to be sequenced. [0217] Sequencing libraries of polynucleotides can be prepared from DNA or RNA, including equivalents, analogs of either DNA or cDNA, for example, DNA or cDNA that is complementary or copy DNA produced from an RNA template, by the action of reverse transcriptase. The polynucleotides may originate in double-stranded form (e.g., dsDNA such as genomic DNA fragments, cDNA, PCR amplification products, and the like) or, in certain embodiments, the polynucleotides may originate in single-stranded form (e.g., ssDNA, RNA, etc.) and have been converted to dsDNA form. By way of illustration, in certain embodiments, single stranded mRNA molecules may be copied into double-stranded cDNAs suitable for use in preparing a sequencing library. The precise sequence of the primary polynucleotide molecules is generally not material to the method of library preparation, and may be known or unknown. In one embodiment, the polynucleotide molecules are DNA molecules. More particularly, in certain embodiments, the polynucleotide molecules represent the entire genetic complement of an organism or substantially the entire genetic complement of an organism, and are genomic DNA molecules (e.g., cellular DNA, cell free DNA (cfDNA), etc.), that typically include both intron sequence and exon sequence (coding sequence), as well as non-coding regulatory sequences such as promoter and enhancer sequences. In certain embodiments, the primary polynucleotide molecules comprise human genomic DNA molecules, e.g., cfDNA molecules present in peripheral blood of a pregnant subject. [0218] Methods of isolating nucleic acids from biological sources may differ depending upon the nature of the source. One of skill in the art can readily isolate nucleic acids from a source as needed for the method described herein. In some instances, it can be advantageous to fragment large nucleic acid molecules (e.g., cellular genomic DNA) in the nucleic acid sample to obtain polynucleotides in the desired size range. Fragmentation can be random, or it can be specific, as achieved, for example, using restriction endonuclease digestion. Methods for random fragmentation may include, for example, limited DNase digestion, alkali treatment and physical shearing. Fragmentation can also be achieved by any of a number of methods known to those of skill in the art. For example, fragmentation can be achieved by mechanical means including, but not limited to nebulization, sonication and hydroshear. [0219] In some embodiments, sample nucleic acids are obtained from cfDNA, which is not subjected to fragmentation. For example, cfDNA typically exists as fragments of less than about 300 base pairs and consequently, fragmentation is not typically necessary for generating a sequencing library using cfDNA samples. [0220] Typically, whether polynucleotides are forcibly fragmented (e.g., fragmented in vitro), or naturally exist as fragments, they are converted to blunt-ended DNA having 5’- phosphates and 3’-hydroxyl. Standard protocols, e.g., protocols for sequencing using, for example, the Illumina platform, instruct users to end-repair sample DNA, to purify the end-repaired products prior to dA-tailing, and to purify the dA-tailing products prior to the adaptor-ligating steps of the library preparation. [0221] In various embodiments, verification of the integrity of the samples and sample tracking can be accomplished by sequencing mixtures of sample genomic nucleic acids, e.g., cfDNA, and accompanying marker nucleic acids that have been introduced into the samples, e.g., prior to processing. Sequencing Techniques [0222] The disclosed sequencing systems and methods may be compatible with any sequencing techniques based on optical detection, for example, next-generation sequencing (NGS), fluorescent in situ sequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS). In one embodiment, the disclosed systems and methods may be compatible with NGS technologies that allow multiple samples to be sequenced individually as genomic molecules (i.e., singleplex sequencing) or as pooled samples comprising indexed genomic molecules (e.g., multiplex sequencing) on a single sequencing run. These methods can generate up to several hundred million reads of DNA sequences. [0223] The disclosed technology may implement sequencing reactions such as those incorporating sequencing-by-synthesis methods described in U.S. Patent Application Publication Numbers 2007/0166705, 2006/0188901, 2006/0240439, 2006/0281109, 2005/0100900, U.S. Patent Number 7,057,026, PCT Application Publication Numbers WO 2005/065814, WO 2006/064199, and WO 2007/010251, the disclosures of which are incorporated herein by reference in their entireties. In some embodiments, the sequencers may implement sequencing-by-synthesis methods similar to those used in the HiSeq, MiSeq, or HiScanSQ systems from Illumina (San Diego, Calif.). [0224] Alternatively, sequencing by ligation techniques may be used in the disclosed technology, such as described in U.S. Patent Numbers 6,969,488, 6,172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entireties. Sequencing by ligation techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. [0225] The disclosed technology may be implemented in some sequencing techniques which are available commercially, such as the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, CA) and the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, CT) and Helicos Biosciences (Cambridge, MA), the sequencing-by-ligation platform from Applied Biosystems (Foster City, CA), or the SMRT technology of Pacific Biosciences. [0226] In one illustrative, but non-limiting, embodiment, the methods described herein comprise obtaining sequence information for the nucleic acids in a sample using Illumina’s sequencing-by-synthesis and reversible terminator-based sequencing chemistry (e.g., as described in Bentley et al., Nature 6:53-59 [2009]). Illumina’s sequencing technology may include the attachment of fragmented genomic DNA to a planar, optically transparent surface on which oligonucleotide anchors are bound. For example, template DNA is end-repaired to generate 5’- phosphorylated blunt ends, and the polymerase activity of Klenow fragment is used to add a single A base to the 3’ end of the blunt phosphorylated DNA fragments. This addition prepares the DNA fragments for ligation to oligonucleotide adapters, which have an overhang of a single T base at their 3’ end to increase ligation efficiency. The adapter oligonucleotides are complementary to the flow cell anchor oligos. Under limiting-dilution conditions, adapter-modified, single-stranded template DNA is added to the flow cell and immobilized by hybridization to the anchor oligos. Attached DNA fragments are extended and bridge amplified to create an ultra-high density sequencing flow cell with hundreds of millions of clusters, each containing about 1,000 copies of the same template. In one embodiment, the randomly fragmented genomic DNA is amplified using PCR before it is subjected to cluster amplification. Alternatively, an amplification-free (e.g., PCR free) genomic library preparation is used, and the randomly fragmented genomic DNA is enriched using the cluster amplification alone (Kozarewa et al., Nature Methods 6:291-295 [2009]). The sequencing-by-synthesis reaction may employ reversible terminators with removable fluorescent dyes. Short sequence reads of about tens to a few hundred base pairs are aligned against a reference genome and unique mapping of the short sequence reads to the reference genome are identified. After completion of the first read, the templates can be regenerated in situ to enable a second read from the opposite end of the fragments. Thus, either single-end or paired end sequencing of the DNA fragments can be used. Detailed information about paired end sequencing can be found in US Patent No. 7601499 and US Patent Publication No. 2012/0,053,063, which are incorporated by reference. [0227] In some embodiments, the sequencing by synthesis platform by Illumina involves clustering fragments. Clustering is a process in which each fragment molecule is isothermally amplified. In some embodiments, the fragment has two different adaptors attached to the two ends of the fragment, the adaptors allowing the fragment to hybridize with the two different oligos on the surface of a flow cell lane. The fragment further includes or is connected to two index sequences at two ends of the fragment, where index sequences provide labels to identify different samples in multiplex sequencing. [0228] In some implementation, a flow cell for clustering in the Illumina platform is a glass slide with lanes. Each lane is a glass channel coated with a lawn of two types of oligos. Hybridization is enabled by the first of the two types of oligos on the surface. This oligo is complementary to a first adapter on one end of the fragment. A polymerase creates a compliment strand of the hybridized fragment. The double-stranded molecule is denatured, and the original template strand is washed away. The remaining strand, in parallel with many other remaining strands, is clonally amplified through bridge application. [0229] In bridge amplification, a strand folds over, and a second adapter region on a second end of the strand hybridizes with the second type of oligos on the flow cell surface. A polymerase generates a complimentary strand, forming a double-stranded bridge molecule. This double-stranded molecule is denatured resulting in two single-stranded molecules tethered to the flow cell through two different oligos. The process is then repeated over and over, and occurs simultaneously for millions of clusters resulting in clonal amplification of all the fragments. After bridge amplification, the reverse strands are cleaved and washed off, leaving only the forward strands. The 3’ ends are blocked to prevent unwanted priming. [0230] After clustering, sequencing starts with extending a first sequencing primer to generate the first read. With each cycle, fluorescently tagged nucleotides compete for addition to the growing chain. Only one is incorporated based on the sequence of the template. After the addition of each nucleotide, the cluster is excited by a light source, and a characteristic fluorescent signal is emitted. The number of cycles determines the length of the read. The emission wavelength and the signal intensity determine the base call. For a given cluster all identical strands are read simultaneously. Hundreds of millions of clusters, or thousands to tens of thousands of millions of clusters, are sequenced in a massively parallel manner. At the completion of the first read, the read product is washed away. [0231] In processes involving two index primers, an index 1 primer is introduced and hybridized to an index 1 region on the template. Index regions provide identification of fragments, which is useful for de-multiplexing samples in a multiplex sequencing process. The index 1 read is generated similar to the first read. After completion of the index 1 read, the read product is washed away and the 3’ end of the strand is de-protected. The template strand then folds over and binds to a second oligo on the flow cell. An index 2 sequence is read in the same manner as index 1. Then an index 2 read product is washed off at the completion of the step. [0232] After reading two indices, read 2 initiates by using polymerases to extend the second flow cell oligos, forming a double-stranded bridge. This double-stranded DNA is denatured, and the 3’ end is blocked. The original forward strand is cleaved off and washed away, leaving the reverse strand. Read 2 begins with the introduction of a read 2 sequencing primer. As with read 1, the sequencing steps are repeated until the desired length is achieved. The read 2 product is washed away. This entire process generates millions of reads, representing all the fragments. Sequences from pooled sample libraries are separated based on the unique indices introduced during sample preparation. For each sample, reads of similar stretches of base calls are locally clustered. Forward and reversed reads are paired creating contiguous sequences. These contiguous sequences are aligned to the reference genome for variant identification. Systems and Instruments [0233] In some embodiments, the methods may be written in any of various suitable programming languages, for example compiled languages such as C, C#, C++, Fortran, and Java. Other programming languages could be script languages, such as Perl, MatLab, SAS, SPSS, Python, Ruby, Pascal, Delphi, R and PHP. In some embodiments, the methods are written in C, C#, C++, Fortran, Java, Perl, R, Java or Python. In some embodiments, the method may be an independent application with data input and data display modules. Alternatively, the method may be a computer software product and may include classes wherein distributed objects comprise applications including computational methods as described herein. [0234] In some embodiments, the methods may be incorporated into pre-existing data analysis software, such as that found on sequencing instruments. Software comprising computer implemented methods as described herein are installed either onto a computer system directly, or are indirectly held on a computer readable medium and loaded as needed onto a computer system. Further, the methods may be located on computers that are remote to where the data is being produced, such as software found on servers and the like that are maintained in another location relative to where the data is being produced, such as that provided by a third party service provider. [0235] An assay instrument, desktop computer, laptop computer, or server which may contain a processor in operational communication with accessible memory comprising instructions for implementation of systems and methods. In some embodiments, a desktop computer or a laptop computer is in operational communication with one or more computer readable storage media or devices and/or outputting devices. An assay instrument, desktop computer and a laptop computer may operate under a number of different computer based operational languages, such as those utilized by Apple based computer systems or PC based computer systems. An assay instrument, desktop and/or laptop computers and/or server system may further provide a computer interface for creating or modifying experimental definitions and/or conditions, viewing data results and monitoring experimental progress. In some embodiments, an outputting device may be a graphic user interface such as a computer monitor or a computer screen, a printer, a hand-held device such as a personal digital assistant (i.e., PDA, Blackberry, iPhone), a tablet computer (for example, iPAD), a hard drive, a server, a memory stick, a flash drive and the like. [0236] A computer readable storage device or medium may be any device such as a server, a mainframe, a supercomputer, a magnetic tape system and the like. In some embodiments, a storage device may be located onsite in a location proximate to the assay instrument, for example adjacent to or in close proximity to, an assay instrument. For example, a storage device may be located in the same room, in the same building, in an adjacent building, on the same floor in a building, on different floors in a building, etc. in relation to the assay instrument. In some embodiments, a storage device may be located off-site, or distal, to the assay instrument. For example, a storage device may be located in a different part of a city, in a different city, in a different state, in a different country, etc. relative to the assay instrument. In embodiments where a storage device is located distal to the assay instrument, communication between the assay instrument and one or more of a desktop, laptop, or server is typically via Internet connection, either wireless or by a network cable through an access point. In some embodiments, a storage device may be maintained and managed by the individual or entity directly associated with an assay instrument, whereas in other embodiments a storage device may be maintained and managed by a third party, typically at a distal location to the individual or entity associated with an assay instrument. In embodiments as described herein, an outputting device may be any device for visualizing data. [0237] An assay instrument, desktop, laptop and/or server system may be used itself to store and/or retrieve computer implemented software programs incorporating computer code for performing and implementing computational methods as described herein, data for use in the implementation of the computational methods, and the like. One or more of an assay instrument, desktop, laptop and/or server may comprise one or more computer readable storage media for storing and/or retrieving software programs incorporating computer code for performing and implementing computational methods as described herein, data for use in the implementation of the computational methods, and the like. Computer readable storage media may include, but is not limited to, one or more of a hard drive, a SSD hard drive, a CD-ROM drive, a DVD-ROM drive, a floppy disk, a tape, a flash memory stick or card, and the like. Further, a network including the Internet may be the computer readable storage media. In some embodiments, computer readable storage media refers to computational resource storage accessible by a computer network via the Internet or a company network offered by a service provider rather than, for example, from a local desktop or laptop computer at a distal location to the assay instrument. [0238] In some embodiments, computer readable storage media for storing and/or retrieving computer implemented software programs incorporating computer code for performing and implementing computational methods as described herein, data for use in the implementation of the computational methods, and the like, is operated and maintained by a service provider in operational communication with an assay instrument, desktop, laptop and/or server system via an Internet connection or network connection. [0239] In some embodiments, a hardware platform for providing a computational environment comprises a processor (i.e., CPU) wherein processor time and memory layout such as random access memory (i.e., RAM) are systems considerations. For example, smaller computer systems offer inexpensive, fast processors and large memory and storage capabilities. In some embodiments, graphics processing units (GPUs) can be used. In some embodiments, hardware platforms for performing computational methods as described herein comprise one or more computer systems with one or more processors. In some embodiments, smaller computer are clustered together to yield a supercomputer network. [0240] In some embodiments, computational methods as described herein are carried out on a collection of inter- or intra-connected computer systems (i.e., grid technology) which may run a variety of operating systems in a coordinated manner. For example, the CONDOR framework (University of Wisconsin-Madison) and systems available through United Devices are exemplary of the coordination of multiple stand-alone computer systems for the purpose dealing with large amounts of data. These systems may offer Perl interfaces to submit, monitor and manage large sequence analysis jobs on a cluster in serial or parallel configurations. One aspect of the disclosure is directed to a workflow module that may be integrated into existing workflows. In some embodiments, a workflow module may be a two-channel sequencing module and may be integrated into a NGS sequence analysis platform, for example the DRAGEN™ Bio-ID platform from Illumina, Inc. (San Diego, CA).

Claims

WHAT IS CLAIMED IS: 1. A method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, comprising: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting the absence of fluorescent emissions from a fourth labeled nucleotide; detecting fluorescent emissions from a fifth labeled nucleotide at a third wavelength; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions, wherein at least one of the labeled nucleotides is a modified nucleotide.

2. The method of claim 1, wherein one or more modified nucleotides is selected from the group comprising 5-methylcytosine, N6-methyladenine, and inosine.

3. The method of claim 1, wherein detecting the fluorescent emissions at the third wavelength comprises detecting the fluorescent emissions at a first intensity and at a second intensity, wherein the first intensity corresponds to a first modified nucleotide and wherein the second intensity corresponds to a second modified nucleotide, wherein the third wavelength is different than the first wavelength and the second wavelength.

4. The method of claim 1, wherein the flow cell comprises wells configured to bind polynucleotides; further wherein the incorporation of one of the at least four labelled nucleotide conjugates into a well is detected from at least one signal state.

5. The method of claim 4, wherein the presence of an empty well is determined from a dark state.

6. The method of claims 4 or 5, wherein each of the at least four labelled nucleotides are distributed as a cloud with an intensity level of at least one signal state such that each labelled nucleotide is more similar to nucleotides of the same label than to those from different labelled nucleotides.

7. The method of claim 6, wherein the at least four labelled nucleotides are distributed as a cloud with an intensity level of at least two signal states.

8. The method of claim 6, wherein empty wells are distributed as a cloud with an intensity of at least one signal state such that each empty well is more similar to empty wells than to those from the different detectable nucleotide conjugate types, and there are at least five clouds.

9. The method of claim 1, further comprising determining the presence of an empty well based on the detection pattern of the at least four labelled nucleotides into the polynucleotide thereby determining the sequence of a polynucleotide, wherein determining the presence of an empty well comprises using four clouds, and the empty well is labelled as one of the at least four different types of detectable nucleotide conjugates.

10. The method of claim 1, further comprising determining the presence of an empty well based on the detection pattern of the at least four labelled nucleotides into the polynucleotide thereby determining the sequence of a polynucleotide, wherein determining the presence of an empty well comprises using five clouds, and the empty well is labelled as a separate cloud.

11. The method of any of claims 1 through 10, wherein a labelling of empty wells is performed by analyzing if a dark state exists for the first ten cycles of a sequencing run.

12. The method of claim 11, wherein a labelling of empty wells is performed by analyzing if a series of a single nucleotide exists in the first ten cycles of a sequencing, and relabeling subsequent nucleotides as an empty well.

13. The method of any of claims 1 through 12, wherein a population of a cloud that comprises a nucleotide and an empty well are segmented into two populations.

14. The method of any of claims 1 through 13, wherein the population of the cloud is segmented by Otsu’s method.

15. The method of any of claims 1 through 14, wherein the population of the cloud is segmented by a k-means algorithm.

16. The method of any of claims 1 through 15, wherein the population of the cloud is segmented by an expectation maximization algorithm.

17. The method of any of claims 1 through 16, wherein the determining step is performed for at least one of a single cycle, a few early cycles, or every cycle in a sequencing run.

18. The method of any of claims 1 through 17, wherein the determining step labels a fifth cloud with a placeholder label until the cloud is confirmed to correspond to an empty well after the first 20 cycles in a sequencing run.

19. The method of any of claims 1 through 18, wherein the determining step is present for every cycle in a sequencing run, and the determining step is further used to detect that an insert was completely sequenced.

20. A non-transitory computer-readable medium storing a polynucleotide sequencing program including instructions that, when executed by a processor, causes a polynucleotide sequencing apparatus, to: detect fluorescent emissions from a first labeled nucleotide at a first wavelength; detect fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detect fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detect the absence of fluorescent emissions from the fourth labeled nucleotide; detect fluorescent emissions from one or more modified nucleotides at a third wavelength; and determine the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions.

21. A system for sequencing polynucleotides bound to a flow cell, comprising: a machine-readable memory; and a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting the absence of fluorescent emissions from the fourth labeled nucleotide; detecting fluorescent emissions from one or more modified nucleotides at a third wavelength; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions.

22. A method of sequencing polynucleotides bound to a flow cell, comprising: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting fluorescent emissions from a fourth labeled nucleotide at the first wavelength and a third intensity that is less than the first intensity; detecting fluorescent emissions from a fifth labeled nucleotide at the second wavelength and a fourth intensity that is less than the second intensity; and determining the sequence of the polynucleotides based on the detected fluorescent emission and intensity.

23. The method of claim 22, further comprising: detecting fluorescent emissions from a sixth labeled nucleotide at the first wavelength and a fifth intensity that is less than the third intensity, and at the second wavelength and a sixth intensity that is less than the fourth intensity.

24. The method of claims 22 or 23, further comprising: detecting fluorescent emissions from a seventh labeled nucleotide at the first wavelength and a seventh intensity that is greater than the third intensity, and at the second wavelength and an eighth intensity that is less than the second intensity; wherein detecting fluorescent emissions from a third labeled nucleotide at the first wavelength is greater than the third intensity and at the second wavelength is greater than the fourth intensity.

25. The method of claim 24, further comprising: detecting fluorescent emissions from an eighth labeled nucleotide at the first wavelength and a ninth intensity that is less than the first intensity, and at the second wavelength and tenth intensity that is greater than the fourth intensity.

26. The method of any preceding claim, further comprising detecting the absence of fluorescent emissions at the first wavelength and at the second wavelength and determining the absence of a polynucleotide.

27. The method of any preceding claim, further comprising detecting the absence of fluorescent emissions at the first wavelength and at the second wavelength and determining the absence of a ninth labeled polynucleotide.

28. The method of any preceding claim, wherein the intensity of fluorescent emissions at a wavelength for a polynucleotide is reduced, relative to other fluorescent emissions at the wavelength for a different polynucleotide, by using smaller stoichiometries of a fluorophore dye for the nucleotide detected at a lower intensity.

29. The method of any preceding claim, wherein the intensity of fluorescent emissions at a wavelength for a polynucleotide is reduced, relative to other fluorescent emissions at the wavelength for a different polynucleotide, by using a different fluorophore dye with lower emission at the wavelength for the nucleotide detected at a lower intensity.

30. A method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, comprising: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting the absence of fluorescent emissions from the fourth labeled nucleotide; detecting fluorescent emissions from a fifth nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions.

31. The method of claim 30, wherein detecting fluorescent emissions from a fifth nucleotide at a third wavelength occurs after a chemical processing step.

32. A method of sequencing polynucleotides bound to a flow cell and having one or more modified nucleotides, comprising: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at a third wavelength, wherein the third wavelength is different from the first and second wavelengths; detecting fluorescent emissions from a fourth labeled nucleotide at a fourth wavelength, wherein the fourth wavelength is different from the first, second, and third wavelengths; detecting the absence of fluorescent emissions from a fifth labeled nucleotide; and determining the sequence of the polynucleotides and the one or more modified nucleotides based on the detected fluorescent emissions.