技术领域Technical Field
本发明涉及基因测序技术领域，尤其涉及一种高通量测序文库定量用标准品及其制备方法和应用。The present invention relates to the field of gene sequencing technology, and in particular to a standard product for high-throughput sequencing library quantification, and a preparation method and application thereof.
背景技术Background technique
高通量测序技术能够同时检测多基因、多靶点，现已广泛用于人类及各类其他动植物、微生物基因序列的检测。但是由于高通量测序的成本较高，在上机测序前，需要对待检文库样本进行定量。准确的文库定量分析对获得高质量测序数据十分关键。如果文库定量浓度偏高，则可能导致测序数据产量不足；而定量浓度偏低则可能导致产生低质量的数据，甚至导致测序失败；另外，随着测序仪测序通量的提升，往往会将多个文库合并到一起进行测序，需要按照测序数据量的要求，根据定量结果混合相应浓度的文库，以稳定产出各待检文库样本数据量。因此，文库浓度的精确定量在高通量测序技术中是必不可缺的。High-throughput sequencing technology can detect multiple genes and multiple targets at the same time, and is now widely used in the detection of human and various other animal, plant, and microbial gene sequences. However, due to the high cost of high-throughput sequencing, the sample of the library to be tested needs to be quantified before sequencing. Accurate library quantitative analysis is critical to obtaining high-quality sequencing data. If the library quantitative concentration is too high, it may lead to insufficient sequencing data output; while a low quantitative concentration may result in low-quality data or even sequencing failure; in addition, with the increase in sequencing throughput of sequencers, multiple libraries are often merged together for sequencing. It is necessary to mix libraries of corresponding concentrations according to the quantitative results according to the requirements of the sequencing data volume to stabilize the output of each library sample data volume to be tested. Therefore, accurate quantification of library concentration is indispensable in high-throughput sequencing technology.
目前可通过荧光计、实时荧光定量PCR等方法进行高通量测序待检文库样本的定量。但荧光计在针对双链DNA进行定量时，无法区分无效文库（即无法进行后续高通量测序的双链DNA片段）、有效文库（即含有文库的完整序列结构、可进行后续高通量测序的文库）。而实时荧光定量PCR可以针对完整结构文库两端的接头序列设计引物和探针，通过探针水解进行实时荧光定量PCR的检测，或直接通过接头设计引物结合荧光染料来进行实时荧光定量PCR的检测。这样就可排除单端或双端未连接接头的不可测序无效文库的干扰，因此实时荧光定量PCR法作为文库定量的金标准，准确性最高。Currently, quantification of samples of libraries to be tested for high-throughput sequencing can be performed by fluorometers, real-time fluorescence quantitative PCR and other methods. However, when quantifying double-stranded DNA, the fluorometer cannot distinguish between invalid libraries (i.e., double-stranded DNA fragments that cannot be subsequently sequenced with high throughput sequencing) and valid libraries (i.e., libraries containing the complete sequence structure of the library and can be subsequently sequenced with high throughput sequencing). Real-time fluorescence quantitative PCR can design primers and probes for the adapter sequences at both ends of the complete structure library, perform real-time fluorescence quantitative PCR detection by probe hydrolysis, or directly design primers through adapters and combine them with fluorescent dyes for real-time fluorescence quantitative PCR detection. In this way, interference from invalid libraries that cannot be sequenced without adapters at one or both ends can be eliminated. Therefore, the real-time fluorescence quantitative PCR method is the gold standard for library quantification and has the highest accuracy.
在实时荧光定量PCR检测待检文库样本浓度时，需要一组已知浓度的标准品用来制作标准曲线，X轴、Y轴分别是标准品浓度（或质量）的对数值、标准品在荧光定量PCR中的Ct值，Ct值与模板浓度（或质量）的对数呈现负线性关系，可通过直线拟合得到标准曲线公式（Y=kX+b，其中k、b分别为标准曲线的斜率与截距）。将待检文库样本的Ct值代入线性标准曲线中，即可换算得到待检文库样本的浓度（或质量，单位与标准品的单位一致）。由此可见，标准品的扩增效率与稳定性对于待检文库样本的定量结果准确性是至关重要的。目前在高通量测序领域，对待检文库样本采用实时荧光定量PCR进行定量时使用的标准品，常采用既有文库样本，首先通过PCR扩增先得到较高浓度的文库，有时还会进行片段筛选；然后通过吸光度法或荧光计进行定量；随后稀释得到一系列浓度梯度的文库标准品。这一方法的主要问题是：吸光度法或荧光计无法区分无效文库、有效文库，进而导致定量结果的不准确；既有文库样本是混合物，其中含有的DNA序列组成复杂（由大量不同碱基序列、不同长度的DNA片段组成）、进而导致不同批次间的标准品在序列和长度等方面相差极大、标准品扩增效率也难以实现批间一致性；另外，长期保存时文库标准品可能降解，导致定量结果因文库标准品存放质量下降而带来结果的偏差。以上这些问题最终可导致高通量测序数据量产出与预期相差大、以及不同待检文库的测序数据量之间均匀性差等问题。鉴于此，亟需一种能够快速、稳定、准确制备出高通量测序文库定量用标准品的方法。When real-time fluorescence quantitative PCR is used to detect the concentration of the sample of the library to be tested, a set of standards of known concentration is needed to make a standard curve. The X-axis and Y-axis are the logarithm of the concentration (or mass) of the standard and the Ct value of the standard in fluorescence quantitative PCR, respectively. The Ct value and the logarithm of the template concentration (or mass) show a negative linear relationship, and the standard curve formula can be obtained by linear fitting (Y=kX+b, where k and b are the slope and intercept of the standard curve, respectively). Substituting the Ct value of the sample of the library to be tested into the linear standard curve, the concentration (or mass, the unit is consistent with the unit of the standard) of the sample of the library to be tested can be converted. It can be seen that the amplification efficiency and stability of the standard are crucial to the accuracy of the quantitative results of the sample of the library to be tested. At present, in the field of high-throughput sequencing, the standard used for quantitative analysis of the sample of the library to be tested by real-time fluorescence quantitative PCR often uses existing library samples. First, a high concentration library is obtained by PCR amplification, and sometimes fragment screening is performed; then quantitative analysis is performed by absorbance or fluorescence meter; and then a series of library standards with concentration gradients are obtained by dilution. The main problems of this method are: the absorbance method or fluorometer cannot distinguish between invalid libraries and valid libraries, which leads to inaccurate quantitative results; the existing library samples are mixtures, and the DNA sequences contained in them are complex (composed of a large number of different base sequences and DNA fragments of different lengths), which leads to great differences in sequence and length between different batches of standards, and it is difficult to achieve batch consistency in the amplification efficiency of standards; in addition, library standards may degrade during long-term storage, resulting in deviations in quantitative results due to the decline in the storage quality of library standards. The above problems can ultimately lead to large differences between the output of high-throughput sequencing data and expectations, and poor uniformity between the sequencing data volumes of different libraries to be tested. In view of this, there is an urgent need for a method that can quickly, stably and accurately prepare standards for high-throughput sequencing library quantification.
发明内容Summary of the invention
为解决上述技术问题，本发明提供了一种高通量测序文库定量用标准品及其制备方法和应用，有效降低待测序文库定量不准确带来的风险。In order to solve the above technical problems, the present invention provides a standard for high-throughput sequencing library quantification and a preparation method and application thereof, which effectively reduce the risk caused by inaccurate quantification of the library to be sequenced.
具体的，本发明的技术方案如下：Specifically, the technical solution of the present invention is as follows:
第一方面，本发明提供了一种高通量测序文库定量用标准品，由包括序列插入片段的模板DNA经PCR扩增得到，所述序列插入片段的核苷酸序列如SEQ ID NO.1所示。In a first aspect, the present invention provides a standard for high-throughput sequencing library quantification, which is obtained by PCR amplification of a template DNA including a sequence insert fragment, wherein the nucleotide sequence of the sequence insert fragment is shown in SEQ ID NO.1.
序列插入片段：Sequence insert:
ACGGTAGTCACGCCTCGTATCTACGTCAGGATGAGACTAGTACAGATATACCTGCTGTCGGCAGAAGTGTCATAGGAGTCACTGAATTCGTATAGCCTAATCACATCCGTCTCCGTGCGGGACAGTCACCGACGGCGACTCATAGTCGCCTAGTCGATCAGATTGGTCTAAGTAGCTTATGAGAGTACACTCTGCGATCATTCAGCAGTGATCACTGATGCTCAGTGCAGCTAGCTATGCGGATCGTCACAGATACTAGCCAGCTGATCTCAGCTAGTCTAGTTGACGATCTAGACGTGCACGGTAGTCACGCCTCGTATCTACGTCAGGATGAGACTAGTACAGATATACCTGCTGTCGGCAGAAGTGTCATAGGAGTCACTGAATTCGTATAGCCTAATCACATCCGTCTCCGTGCGGGACAGTCACCGACGGCGACTCATAGTCGCCTAGTCGATCAGATTGGTCTAAGTAGCTTATGAGAGTACACTCTGCGATCATTCAGCAGTGATCACTGATGCTCAGTGCAGCTAGCTATGCGGATCGTCACAGATACTAGCCAGCTGATCTCAGCTAGTCTAGTTGACGATCTAGACGTGC
上述序列插入片段的长度为300bp，且该序列插入片段的第1-60bp、第61-120bp、第121-180bp、第181-240bp、第241-300bp各分区段，以及第1-300bp整个区段的四种碱基（A/C/T/G）占比均为各25%。同时该序列插入片段及其扩增得到的测序文库定量用标准品均无法比对到人、常见动植物和微生物，可有效避免多种原因可能导致的测序文库定量用标准品相关序列的污染，提高待检文库样本测序数据结果分析的准确率。The length of the sequence insert is 300bp, and the four bases (A/C/T/G) in the 1-60bp, 61-120bp, 121-180bp, 181-240bp, 241-300bp segments of the sequence insert, as well as the entire 1-300bp segment, account for 25% each. At the same time, the sequence insert and the sequencing library quantification standard obtained by amplification cannot be compared to humans, common animals, plants and microorganisms, which can effectively avoid the contamination of the sequencing library quantification standard related sequences that may be caused by various reasons, and improve the accuracy of the sequencing data result analysis of the library sample to be tested.
测序文库定量用标准品长度会由于不同测序平台接头长度不同而不同。举例说明本发明涉及的高通量测序文库定量用标准品组成结构，包含但不限于下述结构序列：The length of the standard for sequencing library quantification may vary due to the different lengths of the adapters of different sequencing platforms. The composition structure of the standard for high-throughput sequencing library quantification according to the present invention is illustrated by way of example, including but not limited to the following structural sequences:
illumina测序平台测序文库定量用标准品结构序列：Illumina sequencing platform sequencing library quantification standard structure sequence:
5'-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTT5'-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTT
-序列插入片段（SEQ ID NO.1）-Sequence insert (SEQ ID NO.1)
-AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTCGTATGCCGTCTTCTGCTTG-3'。-AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTCGTATGCCGTCTTCTGCTTG-3'.
华大测序平台测序文库定量用标准品结构序列：The structural sequence of the standard for quantification of sequencing libraries on the BGI sequencing platform:
5'-CTCTCAGTACGTCAGCAGTTCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTTT5'-CTCTCAGTACGTCAGCAGTTCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTTT
-序列插入片段（SEQ ID NO.1）-Sequence insert (SEQ ID NO.1)
-AAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAACTGATAAGGTCGCCATGC-3'。-AAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAACTGATAAGGTCGCCATGC-3'.
本发明提供的高通量测序文库定量用标准品在多个区段范围内的碱基占比均是均衡的，使用该测序文库定量用标准品进行实时荧光定量PCR时，其扩增效率高且稳定，有利于避免因测序文库定量用标准品序列复杂而导致的扩增效率不佳或不稳定。The high-throughput sequencing library quantification standard provided by the present invention has a balanced base ratio within multiple segments. When the sequencing library quantification standard is used for real-time fluorescence quantitative PCR, the amplification efficiency is high and stable, which is conducive to avoiding poor amplification efficiency or instability caused by the complex sequence of the sequencing library quantification standard.
优选地，PCR扩增后的目标序列拷贝浓度≥0.5nM。Preferably, the copy concentration of the target sequence after PCR amplification is ≥ 0.5 nM.
优选地，PCR扩增后，使用数字PCR进行目标特定序列文库的定量，并将目标序列的拷贝浓度进行梯度稀释。Preferably, after PCR amplification, digital PCR is used to quantify the target specific sequence library, and the copy concentration of the target sequence is graded diluted.
在本发明提供的一种较为具体的实施方式中，稀释得到一组文库定量用标准品：使用数字PCR进行目标特定序列文库的定量，并将拷贝浓度换算为摩尔浓度。将文库定量用标准品稀释至一系列浓度梯度（如：0.5nM、0.05nM、0.005nM、0.0005nM、0.00005nM），配制过程中加入一定浓度的poly(dN)（N=A/C/T/G碱基）。Poly(dN)优选Poly(dA)，Poly(dA)的浓度为0.1-100ng/μL，优选1-10ng/μL，优选1ng/μL；dA碱基数为5-100，优选10-50，优选18-30。In a more specific embodiment provided by the present invention, a set of standards for library quantification is obtained by dilution: digital PCR is used to quantify the target specific sequence library, and the copy concentration is converted into a molar concentration. The standard for library quantification is diluted to a series of concentration gradients (such as: 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM), and a certain concentration of poly(dN) (N=A/C/T/G base) is added during the preparation process. Poly(dN) is preferably Poly(dA), and the concentration of Poly(dA) is 0.1-100ng/μL, preferably 1-10ng/μL, preferably 1ng/μL; the number of dA bases is 5-100, preferably 10-50, preferably 18-30.
第二方面，本发明提供了所述高通量测序文库定量用标准品的制备方法，由包括序列插入片段的模板DNA经PCR扩增得到，所述序列插入片段的核苷酸序列如SEQ ID NO.1所示。In a second aspect, the present invention provides a method for preparing the standard for high-throughput sequencing library quantification, which is obtained by PCR amplification of a template DNA including a sequence insert fragment, and the nucleotide sequence of the sequence insert fragment is shown in SEQ ID NO.1.
优选地，用于PCR扩增的引物对的上游引物核苷酸序列如SEQ ID NO.2所示，下游引物核苷酸序列如SEQ ID NO.3所示。Preferably, the nucleotide sequence of the upstream primer of the primer pair used for PCR amplification is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3.
本发明提供的制备方法简单快捷、稳定可靠。获得的高通量测序文库定量用标准品序列单一、无需文库片段筛选，批间差异小；且稳定性高，便于使用和保存。The preparation method provided by the present invention is simple, fast, stable and reliable. The obtained high-throughput sequencing library quantification standard has a single sequence, does not require library fragment screening, has small batch differences, and has high stability, and is easy to use and store.
第三方面，本发明提供了所述高通量测序文库定量用标准品或者所述制备方法制备得到的高通量测序文库定量用标准品在文库定量中的应用。In a third aspect, the present invention provides the use of the standard for high-throughput sequencing library quantification or the standard for high-throughput sequencing library quantification prepared by the preparation method in library quantification.
优选地，本发明所述应用将所述高通量测序文库定量用标准品与待检文库样本一同进行实时荧光定量PCR检测，根据所述高通量测序文库定量用标准品的摩尔浓度和Ct值绘制标准曲线，再根据标准曲线和待检文库样本的Ct值计算待检文库样本的摩尔浓度。Preferably, the application of the present invention performs real-time fluorescence quantitative PCR detection on the high-throughput sequencing library quantification standard together with the library sample to be tested, draws a standard curve based on the molar concentration and Ct value of the high-throughput sequencing library quantification standard, and then calculates the molar concentration of the library sample to be tested based on the standard curve and the Ct value of the library sample to be tested.
优选地，在本发明所述应用中，所述标准曲线的扩增效率为95-105%，线性相关系数r＞0.999。Preferably, in the application of the present invention, the amplification efficiency of the standard curve is 95-105%, and the linear correlation coefficient r>0.999.
有益效果：Beneficial effects:
本发明提供了一种高通量测序文库定量用标准品及其制备方法和应用。所述高通量测序文库定量用标准品由包括序列插入片段的模板DNA经PCR扩增得到，该序列插入片段及其扩增得到的测序文库定量用标准品均无法比对到人、常见动植物和微生物，可有效避免多种原因可能导致的测序文库定量用标准品相关序列的污染，提高待检文库样本测序数据结果分析的准确率。同时，本发明提供的高通量测序文库定量用标准品在多个区段范围内的碱基占比均是均衡的，使用该测序文库定量用标准品进行实时荧光定量PCR时，其扩增效率高且稳定，有利于避免因测序文库定量用标准品序列复杂而导致的扩增效率不佳或不稳定。The present invention provides a standard product for high-throughput sequencing library quantification and a preparation method and application thereof. The standard product for high-throughput sequencing library quantification is obtained by PCR amplification of a template DNA including a sequence insert fragment. The sequence insert fragment and the standard product for sequencing library quantification obtained by amplification cannot be matched to humans, common animals, plants and microorganisms, which can effectively avoid the contamination of the sequence related to the standard product for sequencing library quantification that may be caused by various reasons, and improve the accuracy of the analysis of the sequencing data results of the sample library to be tested. At the same time, the base ratios of the standard product for high-throughput sequencing library quantification provided by the present invention are balanced within the range of multiple sections. When the standard product for sequencing library quantification is used for real-time fluorescence quantitative PCR, the amplification efficiency is high and stable, which is conducive to avoiding the poor amplification efficiency or instability caused by the complex sequence of the standard product for sequencing library quantification.
具体实施方式Detailed ways
本发明提供了一种高通量测序文库定量用标准品及其制备方法和应用。所述高通量测序文库定量用标准品由包括序列插入片段的模板DNA经PCR扩增得到。The present invention provides a standard product for high-throughput sequencing library quantification and a preparation method and application thereof. The standard product for high-throughput sequencing library quantification is obtained by PCR amplification of a template DNA including a sequence insert fragment.
在本发明提供的一种具体实施方式中，所述高通量测序文库定量用标准品的制备及应用方法包括以下步骤：In a specific embodiment provided by the present invention, the preparation and application method of the standard for high-throughput sequencing library quantification comprises the following steps:
（1）合成1种特定序列文库质粒：质粒上的特定序列文库包括双端接头及特定序列插入片段（SEQ ID NO.1）。其中特定序列插入片段的长度为300bp（测序文库定量用标准品长度会由于不同测序平台接头长度不同而不同），且特定序列插入片段的第1~60bp、第61~120bp、第121~180bp、第181~240bp、第241~300bp各分区段，以及第1~300bp整个区段的四种碱基（A/C/T/G）占比均为各25%。同时该特定序列插入片段均无法比对到人、常见动植物和微生物，可有效防止数据污染。(1) Synthesis of a specific sequence library plasmid: The specific sequence library on the plasmid includes a double-ended adapter and a specific sequence insert (SEQ ID NO.1). The length of the specific sequence insert is 300 bp (the length of the standard for sequencing library quantification will vary due to the different adapter lengths of different sequencing platforms), and the four bases (A/C/T/G) in the 1st to 60bp, 61st to 120bp, 121st to 180bp, 181st to 240bp, 241st to 300bp of the specific sequence insert, as well as the entire 1st to 300bp segment, account for 25% each. At the same time, the specific sequence insert cannot be matched to humans, common animals, plants, and microorganisms, which can effectively prevent data contamination.
（2）获得1种目标特定序列文库：使用PCR方法获得质粒上的1种目标特定序列文库。该目标特定序列文库包含双端接头及特定序列插入片段（SEQ ID NO.1）。该目标特定序列文库的序列是单一且固定的，且特定序列的第1~60bp、第61~120bp、第121~180bp、第181~240bp、第241~300bp各分区段，以及第1~300bp整个区段的四种碱基（A/C/T/G）占比均为各25%。该目标特定序列文库需与待检文库样本的长度接近（长度最多相差±100bp），避免差异过大，以使目标特定序列文库与待检文库样本在文库定量时进行无差异扩增反应。(2) Obtaining a target specific sequence library: Use the PCR method to obtain a target specific sequence library on the plasmid. The target specific sequence library contains a double-end adapter and a specific sequence insert (SEQ ID NO.1). The sequence of the target specific sequence library is single and fixed, and the four bases (A/C/T/G) in the 1st to 60bp, 61st to 120bp, 121st to 180bp, 181st to 240bp, 241st to 300bp segments of the specific sequence, as well as the entire 1st to 300bp segment, account for 25% each. The target specific sequence library should be close to the length of the sample to be tested (the maximum length difference is ±100bp), to avoid too large a difference, so that the target specific sequence library and the sample to be tested can undergo indifferent amplification reactions during library quantification.
（3）稀释得到一组文库定量用标准品：使用数字PCR进行目标特定序列文库的定量，并将拷贝浓度换算为摩尔浓度。将文库定量用标准品稀释至一系列浓度梯度（如：0.5nM、0.05nM、0.005nM、0.0005nM、0.00005nM），配制过程中加入一定浓度的poly(dN)（N=A/C/T/G碱基）。Poly(dN)优选Poly(dA)，Poly(dA)的浓度为0.1~100ng/μL，优选1~10ng/μL，优选1ng/μL；dA碱基数为5~100，优选10~50，优选18~30。(3) Dilute to obtain a set of standards for library quantification: Use digital PCR to quantify the target specific sequence library, and convert the copy concentration into molar concentration. Dilute the library quantification standards to a series of concentration gradients (such as: 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM), and add a certain concentration of poly(dN) (N=A/C/T/G base) during the preparation process. Poly(dN) is preferably Poly(dA), and the concentration of Poly(dA) is 0.1~100ng/μL, preferably 1~10ng/μL, preferably 1ng/μL; the number of dA bases is 5~100, preferably 10~50, preferably 18~30.
本发明提供的高通量测序文库定量用标准品的制备方法可快速、稳定、准确制备出特定序列及长度的高通量测序文库定量用标准品，可有效降低待测序文库定量不准确带来的风险。The method for preparing a standard product for high-throughput sequencing library quantification provided by the present invention can quickly, stably and accurately prepare a standard product for high-throughput sequencing library quantification of a specific sequence and length, and can effectively reduce the risk caused by inaccurate quantification of the library to be sequenced.
本发明将获得的高通量测序文库定量用标准品应用于文库定量：将所述高通量测序文库定量用标准品与待检文库样本一同进行实时荧光定量PCR检测。qPCR数据下机后先使用一组测序文库定量用标准品结果制作标准曲线，其X轴、Y轴分别是测序文库定量用标准品的摩尔浓度的对数值、测序文库定量用标准品的Ct值，通过直线拟合得到标准曲线公式（Y=kX+b，其中k、b分别为标准曲线的斜率与截距）。再将待检文库样本的Ct值代入线性标准曲线中，即可换算得到待检文库样本的摩尔浓度（单位与文库标准品的单位一致）。定量后的待检文库样本即可根据高通量测序的要求进行上机。The present invention applies the obtained high-throughput sequencing library quantification standard to library quantification: the high-throughput sequencing library quantification standard and the sample of the library to be tested are subjected to real-time fluorescence quantitative PCR detection. After the qPCR data is downloaded, a set of sequencing library quantification standard results are used to prepare a standard curve, wherein the X-axis and Y-axis are the logarithmic value of the molar concentration of the sequencing library quantification standard and the Ct value of the sequencing library quantification standard, respectively, and the standard curve formula (Y=kX+b, where k and b are the slope and intercept of the standard curve, respectively) is obtained by linear fitting. Then, the Ct value of the sample of the library to be tested is substituted into the linear standard curve to convert the molar concentration of the sample of the library to be tested (the unit is consistent with the unit of the library standard). The sample of the library to be tested after quantification can be put on the machine according to the requirements of high-throughput sequencing.
测序文库定量用标准品序列长度单一且固定，同时在多个区段范围内的均衡碱基有助于提高实时荧光定量PCR的扩增效率和稳定性，保证待检文库样本定量结果的准确性和稳定性，从而快速稳定地获得相较准确的高质量测序数据，有效避免因测序文库定量用标准品序列复杂而导致的扩增效率不佳或不稳定。The length of the standard sequence for sequencing library quantification is single and fixed, and the balanced bases in multiple segments help improve the amplification efficiency and stability of real-time fluorescence quantitative PCR, ensuring the accuracy and stability of the quantitative results of the library samples to be tested, thereby quickly and stably obtaining relatively accurate high-quality sequencing data, and effectively avoiding poor or unstable amplification efficiency caused by the complex sequence of the standard sequence for sequencing library quantification.
由于不同测序平台对应的测序文库的接头可能不同，因此以下以illumina测序平台为例说明实施方式。Since the adapters of the sequencing libraries corresponding to different sequencing platforms may be different, the implementation method is described below using the Illumina sequencing platform as an example.
该制备方法主要步骤如下：The main steps of the preparation method are as follows:
S1、采用基因合成法，合成1个含有特定序列插入片段的大肠杆菌克隆质粒（或长链DNA分子）。S1. Use gene synthesis to synthesize an E. coli cloning plasmid (or long-chain DNA molecule) containing a specific sequence insertion fragment.
以质粒为例说明实施方式：质粒载体为克隆性质粒，包括但不限于pUC57质粒。质粒上的插入片段包括测序文库的双端接头及特定序列插入片段（SEQ ID NO.1）。该特定序列插入片段均无法比对到人、常见动植物和微生物，可有效防止数据污染。通过Sanger测序法确认质粒插入片段序列合成正确。Taking plasmid as an example to illustrate the implementation method: the plasmid vector is a cloning plasmid, including but not limited to pUC57 plasmid. The inserted fragments on the plasmid include double-end adapters of the sequencing library and a specific sequence inserted fragment (SEQ ID NO.1). The specific sequence inserted fragments cannot be matched to humans, common animals, plants and microorganisms, which can effectively prevent data contamination. The plasmid inserted fragment sequence is confirmed to be correctly synthesized by Sanger sequencing.
S2、采用分子生物学常规方法进行质粒提取纯化（例如使用商品化试剂盒）、检测质粒的质量浓度（例如使用分光光度计、荧光计等）。S2. Use conventional molecular biology methods to extract and purify the plasmid (for example, using commercial kits) and detect the mass concentration of the plasmid (for example, using a spectrophotometer, fluorometer, etc.).
S3、PCRS3, PCR
（1）将制备测序文库定量用标准品的上述特定序列文库质粒样本以及下表中试剂取出，室温解冻，完全融化后轻柔振荡混匀，短暂离心后置于冰盒上备用。(1) Take out the above-mentioned specific sequence library plasmid samples for preparing sequencing library quantification standards and the reagents in the table below, thaw at room temperature, gently shake to mix after complete melting, centrifuge briefly, and place on ice for later use.
（2）在0.2 mL的离心管中，按照下表配制PCR反应体系。其中质粒模板用量为：1pg-50ng，优选1pg-1ng，优选10pg。使用高保真DNA聚合酶，以确保DNA序列在扩增过程中的保真性。(2) In a 0.2 mL centrifuge tube, prepare the PCR reaction system according to the following table. The amount of plasmid template is: 1pg-50ng, preferably 1pg-1ng, preferably 10pg. Use a high-fidelity DNA polymerase to ensure the fidelity of the DNA sequence during amplification.
表 1 PCR反应体系Table 1 PCR reaction system
具体引物序列如下所示：The specific primer sequences are as follows:
正向引物（P5）：AATGATACGGCGACCACCGAGForward primer (P5): AATGATACGGCGACCACCGAG
反向引物（P7）：CAAGCAGAAGACGGCATACGAGAReverse primer (P7): CAAGCAGAAGACGGCATACGAGA
（3）将配制好的反应体系振混离心后，按照如下反应条件进行PCR反应，热盖105℃。(3) After the prepared reaction system is shaken and centrifuged, the PCR reaction is performed according to the following reaction conditions, with the heated lid at 105°C.
表 2 PCR反应条件Table 2 PCR reaction conditions
注：对应的特定序列文库质粒模板用量越少，循环数应对应增加。Note: The smaller the amount of plasmid template of the corresponding specific sequence library, the greater the number of cycles should be.
S4、PCR反应后的磁珠纯化S4. Magnetic bead purification after PCR reaction
PCR反应结束后，将样本转移至操作台，进行下一步纯化操作。根据产品说明书，采用VAHTS®DNA Clean Beads纯化磁珠纯化PCR产物。After the PCR reaction is completed, the sample is transferred to the operating table for the next purification operation. According to the product instructions, VAHTS® DNA Clean Beads purification magnetic beads are used to purify the PCR product.
S5、制备测序文库定量用标准品S5. Preparation of standards for sequencing library quantification
（1）取1 µL 特定序列文库进行质量浓度的检测（例如使用分光光度计、荧光计等）并根据下述公式先粗略计算出对应的摩尔浓度。(1) Take 1 µL of the specific sequence library to measure the mass concentration (for example, using a spectrophotometer, fluorometer, etc.) and roughly calculate the corresponding molar concentration according to the following formula.
摩尔浓度（nM）=质量浓度（ng/μL）÷分子量（g/mol）*10^-6Molar concentration (nM) = mass concentration (ng/μL) ÷ molecular weight (g/mol) * 10^-6
（2）根据粗略计算出的特定序列文库的摩尔浓度，稀释至数字PCR检测所需的浓度范围。(2) Based on the roughly calculated molar concentration of the specific sequence library, dilute it to the concentration range required for digital PCR detection.
（3）将下表中试剂取出，室温解冻，完全融化后轻柔振荡混匀，短暂离心后置于冰盒上备用。(3) Take out the reagents in the table below, thaw at room temperature, and gently shake to mix after they are completely thawed. Centrifuge briefly and place on ice for later use.
（4）在0.2 mL的离心管中，按照下表分别配制数字PCR反应体系。(4) In a 0.2 mL centrifuge tube, prepare the digital PCR reaction system according to the table below.
表 3 数字PCR反应体系Table 3 Digital PCR reaction system
在数字PCR反应体系中：In the digital PCR reaction system:
PCR正向引物（P5）：AATGATACGGCGACCACCGAG；PCR forward primer (P5): AATGATACGGCGACCACCGAG;
PCR反向引物（P7）：CAAGCAGAAGACGGCATACGAGA。PCR reverse primer (P7): CAAGCAGAAGACGGCATACGAGA.
（5）将配制好的数字PCR反应体系振混离心后，按照如下反应条件进行数字PCR反应。(5) After shaking and centrifuging the prepared digital PCR reaction system, perform the digital PCR reaction according to the following reaction conditions.
表 4 PCR反应条件Table 4 PCR reaction conditions
（6）将数字PCR得到的拷贝浓度换算为摩尔浓度。摩尔浓度（mol/L）=拷贝浓度（拷贝/L）÷阿伏伽德罗常数。(6) Convert the copy concentration obtained by digital PCR to molar concentration. Molar concentration (mol/L) = copy concentration (copies/L) ÷ Avogadro constant.
（7）将上述特定序列文库依次按照10倍梯度浓度进行稀释，同时加入一定浓度的poly(dN)，制成一组测序文库定量用标准品（如：0.5nM、0.05nM、0.005nM、0.0005nM、0.00005nM）。Poly(dN)优选Poly(dA)，Poly(dA)的浓度为0.1-100ng/μL，优选1-10ng/μL，优选1ng/μL；dA碱基数为5-100，优选10-50，优选18-30。(7) The above-mentioned specific sequence library is diluted in 10-fold gradient concentrations, and a certain concentration of poly(dN) is added to prepare a set of standards for sequencing library quantification (e.g., 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM). Poly(dN) is preferably Poly(dA), and the concentration of Poly(dA) is 0.1-100ng/μL, preferably 1-10ng/μL, preferably 1ng/μL; the number of dA bases is 5-100, preferably 10-50, preferably 18-30.
S6、将测序文库定量用标准品与待检文库样本一同进行实时荧光定量PCR检测。qPCR数据下机后先使用一组测序文库定量用标准品结果制作标准曲线，其X轴、Y轴分别是测序文库定量用标准品的摩尔浓度的对数值、测序文库定量用标准品的Ct值，通过直线拟合得到标准曲线公式（Y=kX+b，其中k、b分别为标准曲线的斜率与截距）。再将待检文库样本的Ct值代入线性标准曲线中，即可换算得到待检文库样本的摩尔浓度（单位与文库标准品的单位一致）。S6. Perform real-time fluorescence quantitative PCR detection on the sequencing library quantification standard and the sample of the library to be tested. After the qPCR data is downloaded, a set of sequencing library quantification standard results is used to make a standard curve, whose X-axis and Y-axis are the logarithmic value of the molar concentration of the sequencing library quantification standard and the Ct value of the sequencing library quantification standard, respectively. The standard curve formula is obtained by linear fitting (Y=kX+b, where k and b are the slope and intercept of the standard curve, respectively). Then, the Ct value of the sample of the library to be tested is substituted into the linear standard curve to convert the molar concentration of the sample of the library to be tested (the unit is consistent with the unit of the library standard).
S7、定量后的待检文库样本即可根据高通量测序的要求进行上机。S7. After quantification, the library samples to be tested can be loaded onto the machine according to the requirements of high-throughput sequencing.
S8、针对下机后数据，可通过序列比对，将测序文库定量用标准品的已知序列从测序结果中剔除，避免影响待检文库样本测序结果分析。S8. For the data after being taken off the machine, the known sequences of the standards used for sequencing library quantification can be removed from the sequencing results through sequence alignment to avoid affecting the sequencing result analysis of the library samples to be tested.
为了便于理解本发明，下面将对本发明实施例中的技术方案进行清晰完整的描述，但是不能把它们理解为对本发明保护范围的限定。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂和仪器未注明生产厂家者，均为可以通过市售购买获得的常规产品。In order to facilitate the understanding of the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below, but they should not be understood as limiting the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents and instruments are not specified, they are all conventional products that can be purchased commercially.
实施例1Example 1
本实施例以illumina测序平台为例，按照具体实施方式所述内容制备测试文库定量用标准品，选择3天分批次制备，每天制备一个批次。测试文库定量用标准品设计为一组5个标准品，浓度依次为：0.5nM、0.05nM、0.005nM、0.0005nM、0.00005nM。Poly(dN)选用Poly(dA)，Poly(dA)的浓度为1ng/μL；dA碱基数为18。In this example, the illumina sequencing platform is used as an example. The standard for quantitative test library is prepared according to the content described in the specific implementation method. The preparation is divided into batches over 3 days, and one batch is prepared every day. The standard for quantitative test library is designed as a group of 5 standards, and the concentrations are: 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM. Poly(dN) is selected as Poly(dA), and the concentration of Poly(dA) is 1ng/μL; the number of dA bases is 18.
将制备好的3个批次测序文库定量用标准品，每批都对同一组10例待检文库样本做3次重复定量（定量试剂为KAPA Library Quantification Kit），每次定量后根据10例待检文库样本的实测浓度进行上机测序。同时设置对照组：即将10例待检文库样本使用Qubit荧光计进行定量，定量后根据10例待检文库样本的实测Qubit浓度进行上机测序（测序仪型号：illumina Nextseq CN500），每一例样本预期数据量为4G。The prepared three batches of sequencing library quantification standards were used, and each batch was quantified three times for the same group of 10 samples of the library to be tested (the quantitative reagent was KAPA Library Quantification Kit). After each quantification, the samples were sequenced according to the measured concentration of the 10 samples of the library to be tested. At the same time, a control group was set up: the 10 samples of the library to be tested were quantified using a Qubit fluorometer, and after quantification, the samples were sequenced according to the measured Qubit concentration of the 10 samples of the library to be tested (sequencing machine model: illumina Nextseq CN500), and the expected data volume for each sample was 4G.
结果显示，使用实时荧光定量PCR方法（qPCR）得到的测序文库定量用标准品进行定量的10例待检文库浓度间的相对标准偏差均不超过10%；而使用对照组（Qubit定量）方法得到的测序文库定量用标准品进行定量的10例待检样本文库浓度间的相对标准偏差最大达22%。由此可以表明：按本发明方法制备出的测序文库定量用标准品制备的批间差异很小。具体结果见下表。The results showed that the relative standard deviations of the concentrations of the 10 libraries to be tested using the real-time fluorescence quantitative PCR method (qPCR) for quantification of the sequencing library quantification standard were no more than 10%; while the relative standard deviations of the concentrations of the 10 sample libraries to be tested using the control group (Qubit quantification) method for quantification of the sequencing library quantification standard were up to 22%. This shows that the batch differences of the sequencing library quantification standard prepared by the method of the present invention are very small. The specific results are shown in the table below.
表 5 不同方法文库定量结果Table 5 Library quantification results of different methods
将上述定量后的10例待检文库样本根据高通量测序的要求进行上机。通过高通量测序仪测序，以illumina Nextseq CN500测序仪为例。结果显示：使用实时荧光定量PCR方法（qPCR）得到的测序文库定量用标准品进行定量的10例待检文库的三批次实际产出数据量与预期数据量的相对偏差均不超过±10%。而使用对照组（Qubit定量，仅上机一次）方法得到的测序文库定量用标准品进行定量的10例待检样本文库的实际产出数据量与预期数据量的相对偏差最大达-30%。由此可以表明：按本发明方法制备出的测序文库定量用标准品制备的批间差异很小，且用其定量的文库测序数据量实际产出与预期更为相符。具体结果见下表。The 10 samples of the library to be tested after the above quantification were put on the machine according to the requirements of high-throughput sequencing. Sequencing was performed by a high-throughput sequencer, taking the illumina Nextseq CN500 sequencer as an example. The results showed that the relative deviation between the actual output data volume and the expected data volume of the three batches of 10 libraries to be tested, which were quantified by the sequencing library quantification standard obtained by the real-time fluorescence quantitative PCR method (qPCR), did not exceed ±10%. The relative deviation between the actual output data volume and the expected data volume of the 10 sample libraries to be tested, which were quantified by the sequencing library quantification standard obtained by the control group (Qubit quantification, only on the machine once), was up to -30%. This shows that the batch differences of the sequencing library quantification standard prepared by the method of the present invention are very small, and the actual output of the library sequencing data volume quantified by it is more consistent with expectations. The specific results are shown in the table below.
表 6 不同方法定量文库的测序数据量对比Table 6 Comparison of sequencing data volume of quantitative libraries by different methods
实施例2Example 2
本实施例按照具体实施方式所述内容制备文库定量用标准品，文库定量用标准品设计为一组5个标准品，浓度依次为：0.5nM、0.05nM、0.005nM、0.0005nM、0.00005nM。Poly(dN)为Poly(dA)，Poly(dA)的浓度为1ng/μL；dA碱基数为18。同时设置对照组，对照组不加入具体实施方式所述的poly(dN)。两组文库定量用标准品同时做实时稳定性测试（保存于-30~-10℃，第0/4/8/12/16/20/24个月）和加速破坏稳定性测试（反复冻融10次、室温（20-30℃）放置2天）。共测试3个批次文库定量用标准品，均分别对同一例待检文库样本进行定量。In this embodiment, the library quantification standard is prepared according to the content described in the specific implementation method. The library quantification standard is designed as a group of 5 standards, and the concentrations are: 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM. Poly(dN) is Poly(dA), and the concentration of Poly(dA) is 1ng/μL; the number of dA bases is 18. A control group is also set up, and the control group does not add poly(dN) described in the specific implementation method. The two groups of library quantification standards are simultaneously tested for real-time stability (stored at -30~-10℃, 0/4/8/12/16/20/24 months) and accelerated destruction stability test (repeated freezing and thawing 10 times, placed at room temperature (20-30℃) for 2 days). A total of 3 batches of library quantification standards were tested, and the same sample of the library to be tested was quantified.
定量结果显示，按照具体实施方式所述进行制备的测试文库定量用标准品，计算得到的标准曲线扩增效率均在95-105%之间，线性相关系数r均大于0.999，且对同一例待检文库样本进行定量的结果，无论是实时稳定性测试，还是加速破坏稳定性，各组间无明显差异；对照组（不加入具体实施方式所述的poly(dN)），计算得到的标准曲线扩增效率部分在90-95%之间，线性相关系数r部分在0.98-0.99之间，且对同一例待检文库样本进行定量的结果，无论是实时稳定性测试，还是加速破坏稳定性，随时间的延长以及加速破坏条件的增加，定量结果出现较大波动。由此可以证明，按本发明方法制备出的测序文库定量用标准品制备的实时稳定性、加速破坏稳定性（反复冻融、室温放置）均良好。具体结果见下表7-10。The quantitative results show that the standard products for quantitative testing of the test library prepared according to the specific implementation method have a calculated standard curve amplification efficiency of 95-105%, a linear correlation coefficient r of greater than 0.999, and the results of quantitative testing of the same sample of the library to be tested, whether it is a real-time stability test or accelerated stability, there is no significant difference between the groups; the control group (poly (dN) described in the specific implementation method is not added), the calculated standard curve amplification efficiency is partly between 90-95%, the linear correlation coefficient r is partly between 0.98-0.99, and the results of quantitative testing of the same sample of the library to be tested, whether it is a real-time stability test or accelerated stability, with the extension of time and the increase of accelerated destruction conditions, the quantitative results fluctuate greatly. It can be proved that the real-time stability and accelerated stability (repeated freezing and thawing, room temperature placement) of the standard products for quantitative sequencing library prepared by the method of the present invention are good. Specific results are shown in Tables 7-10 below.
表 7 文库定量用标准品实时稳定性分析结果（PCR扩增效率和线性相关系数）Table 7 Real-time stability analysis results of library quantification standards (PCR amplification efficiency and linear correlation coefficient)
表 8 文库定量用标准品实时稳定性分析结果（浓度定量结果）Table 8 Real-time stability analysis results of library quantification standards (concentration quantification results)
表 9 文库定量用标准品加速破坏稳定性分析结果（PCR扩增效率和线性相关系数）Table 9 Results of accelerated destruction stability analysis of library quantification standards (PCR amplification efficiency and linear correlation coefficient)
表 10 文库定量用标准品加速破坏稳定性分析结果（浓度定量结果）Table 10 Results of accelerated destabilization analysis of library quantification standards (concentration quantification results)
以上所述实施例仅表达了本发明的几种实施方式，便于具体和详细地理解本发明的技术方案，但并不能因此而理解为对本发明保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solution of the present invention in detail, but cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410277936.2ACN117867086B (en) | 2024-03-12 | 2024-03-12 | Standard substance for quantitative high-throughput sequencing library and preparation method and application thereof |
| Application Number | Priority Date | Filing Date | Title |
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| CN202410277936.2ACN117867086B (en) | 2024-03-12 | 2024-03-12 | Standard substance for quantitative high-throughput sequencing library and preparation method and application thereof |
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| CN117867086A CN117867086A (en) | 2024-04-12 |
| CN117867086Btrue CN117867086B (en) | 2024-06-25 |
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| CN202410277936.2AActiveCN117867086B (en) | 2024-03-12 | 2024-03-12 | Standard substance for quantitative high-throughput sequencing library and preparation method and application thereof |
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| CN (1) | CN117867086B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108203847A (en)* | 2016-12-16 | 2018-06-26 | 深圳华大智造科技有限公司 | For library, reagent and the application of the assessment of two generation sequencing qualities |
| CN110628890A (en)* | 2019-11-07 | 2019-12-31 | 中国人民解放军军事科学院军事医学研究院 | Sequencing quality control standards and their applications and products |
| CN114703266A (en)* | 2022-04-06 | 2022-07-05 | 杭州杰毅医学检验实验室有限公司 | Internal reference, kit containing internal reference and application of kit |
| CN116179664A (en)* | 2023-02-10 | 2023-05-30 | 青岛锐翌精准医学检验有限公司 | High-throughput detection method, system and kit for determining microorganisms based on internal reference |
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| BRPI0701529A2 (en)* | 2007-04-05 | 2008-11-18 | Uniao Brasileira De Educacao E Assistencia Ubea | nucleotide sequence reference standard, process for detection and / or quantitation of nucleotide sequences, and kit |
| CN111235244A (en)* | 2019-11-28 | 2020-06-05 | 广州微远基因科技有限公司 | Sequencing internal standard molecule and preparation method and application thereof |
| CN111500691A (en)* | 2020-04-24 | 2020-08-07 | 中国食品药品检定研究院 | Quality control standard substance and quality control method for microbial high-throughput DNA sequencing data |
| CN116732151A (en)* | 2023-04-23 | 2023-09-12 | 阿吉安(福州)基因医学检验实验室有限公司 | Target detection semi-quantitative method of multiple PCR system |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108203847A (en)* | 2016-12-16 | 2018-06-26 | 深圳华大智造科技有限公司 | For library, reagent and the application of the assessment of two generation sequencing qualities |
| CN110628890A (en)* | 2019-11-07 | 2019-12-31 | 中国人民解放军军事科学院军事医学研究院 | Sequencing quality control standards and their applications and products |
| CN114703266A (en)* | 2022-04-06 | 2022-07-05 | 杭州杰毅医学检验实验室有限公司 | Internal reference, kit containing internal reference and application of kit |
| CN116179664A (en)* | 2023-02-10 | 2023-05-30 | 青岛锐翌精准医学检验有限公司 | High-throughput detection method, system and kit for determining microorganisms based on internal reference |
| Publication number | Publication date |
|---|---|
| CN117867086A (en) | 2024-04-12 |
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