A kind of R-Loop high-throughput sequencing libraries construction methodTechnical field
The invention belongs to biotechnologies, and in particular to a kind of R-Loop high-throughput sequencing libraries construction method.
Background technology
R-loop refers to the complementary strand pairing worked as in a RNA molecule and its double chain DNA molecule, while will be anotherDNA chain excludes and the cyclic structure that is formed.R-loop is a kind of special three chain nucleic acid structure, by a RNA/DNA heterozygosisChain and a single stranded DNA are formed.More and more evidences find this unique chromatin Structure in protokaryon and eukaryon at presentGenerally existing in biology, plays critical function in many crucial biological processes, including chromatin modification, transcriptional control,DNA damage reparation and Genome stability etc..
Find the R-loop structures being stabilized in a variety of organisms in recent years, and more and more research shows that R-The accumulation of loop is related with many human diseases, such as neurodegenerative disease, amplification oligonucleotide disease and cancer etc..R-loopRemoving adjusted by many factors, wherein ribalgilase RNase H can specifically remove the RNA in DNA/RNA heterozygosis chains,It is one of the Main Factors for removing R-loop structures.
Existing R-Loop full-length genomes detection method is there are serious technological deficiency, and the quality of data is bad, to resultGenerate very important influence.
Invention content
In view of the problems of the existing technology, the present invention provides a kind of R-Loop high-throughput sequencing libraries construction method, leads toCross DNA/RNA heterozygosis chains co-immunoprecipitation and high throughput sequencing technologies that single stranded DNA builds library, can specificity in genomeDNA/RNA heterozygosis chains are captured and are enriched with and carried out library construction, have accurate, efficiently, sensitive, reproducible, operation letterThe advantages that single.
The R-Loop high-throughput sequencing library construction methods of the present invention, follow the steps below：
(1) extraction genomic DNA carries out fragmentation processing to it；
(2) magnetic bead is added afterwards in step (1) and carries out fragmentation products purifying；
(3) capture magnetic bead, specific capture dna-RNA heterozygosis chains are added afterwards in step (2)；
(4) magnetic bead is added afterwards in step (3) and carries out capture product purification；
(5) 1 reaction system of enzyme is added afterwards in step (4), carries out end reparation；
(6) 2 reaction system of enzyme is added afterwards in step (5), carries out connector connection reaction；
(7) magnetic bead is added afterwards in step (6) and carries out connector connection product purifying；
(8) PCR amplification system is added afterwards in step (7), carries out the amplification in library；
(9) magnetic bead is added afterwards in step (8) to purify the library after amplification, product after purification is upper machine textLibrary.
Wherein the fragmentation uses Physical or enzyme process, and the Genome DNA content of extraction is at least 50ng, by geneGroup DNA is broken into 200-500bp.
The capture magnetic bead includes three parts, is [S9.6] Antibody, Protein A and special sex modification respectivelyMagnetic bead, the wherein group of the special sex modification of magnetic bead surfaces can combine closely with Protein A.
1 system of enzyme includes T4PNK, T4DNA Polymerase, dNTP and T4DNA ligase Buffer, endIt is 37 DEG C to hold repairing condition, 72 DEG C after 30min, 20min.
2 reaction system of enzyme includes T4DNA ligase, T4DNA ligase Buffer, PEG6000 and RNAAdapter, it is 20 DEG C that connector, which connects reaction condition, 15min；65 DEG C, 10min；The connector includes Universal AdapterIt is P5 end connectors, including P5 binding sequences and Read1 reading sequences with RNA Adapter, wherein Universal AdapterRow, RNA Adapter are P7 end connectors, including P7 binding sequences, and Index sequences and Read2 read sequence.
The amplification system includes buffer solution, archaeal dna polymerase, dNTPs and a pair of of upstream and downstream primer, sense primerP5, is library P5 end connector specific primer sequences, and downstream primer P7 is library P7 end connector specific primer sequences.
The library size is 400-500bp.
The upper machine library be suitable for Roche, Illumina, ThermoFisher, Pacific Biosciences,Hua Da gene, Oxford Nanopore Technologies, Hua Yinkang, big desert gene high-flux sequence platform.
Compared with prior art, the features of the present invention and advantageous effect are：
The present invention provides a kind of R-Loop library constructing methods, and during building library, each reagent component is reacted in controlDosage and reaction temperature and time, using special Enzyme 1 and Buffer 1, the formula of Enzyme 2 and Buffer 2,And the base sequence of each primer is designed, including Universal Adapter, RNA Adapter, P5 and P7.
The Library Quality higher of R-Loop library constructing methods structure of the present invention, data more preferably, are adapted to different speciesWith a variety of sample types, there are accurate, efficiently, sensitive, reproducible, simple operation and other advantages, be advantageously implemented automation behaviourMake and extensive library concentrates structure, the banking process provided to can be used for different types of microarray dataset, applicability is wide, easilyIn popularization.
Description of the drawings
Fig. 1 is the schematic diagram for establishing R-Loop high-throughput sequencing libraries of the present invention；
Fig. 2 is the flow chart for establishing R-Loop high-throughput sequencing libraries of the present invention；
Fig. 3 is 2% agarose gel electrophoresis detection library figure；
Wherein：M:100bp DNA ladder (precious bioengineering (Dalian) Co., Ltd)；1:The libraries R-loop.
Specific implementation mode
Now by taking Illumina platforms as an example, the present invention is further described in conjunction with the embodiments.
Nucleotide sequence involved in the present embodiment is as shown in sequence table.
Embodiment 1：
The method for building R-loop high-throughput sequencing libraries based on single stranded DNA of the present embodiment, follows the steps below：
Universal Adapter nucleic acid sequences are as shown in sequence 1：
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG
ATCT-3’；
RNA Adapter nucleic acid sequences such as sequence 2 is shown：
5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG-3'；
P5 nucleic acid sequences such as sequence 3 is shown：5'-AATGATACGGCGACCACCGAGA-3'；
P7 nucleic acid sequences such as sequence 4 is shown：5'-CAAGCAGAAGACGGCATACGA-3'.
(1) extraction patient with breast cancer's canceration tissue gene group DNA carries out fragmentation processing；
Patient's cancerous issue genome is carried out according to blood/tissue/cellular genome extracts kit extraction stepExtract (Tiangeng, article No.：DP304)；Fragmentation processing is carried out to the genome of extraction, interrupts 200-500bp；
(2) magnetic bead is added afterwards in step (1) and carries out fragmentation products purifying；
Use NF-H2Fragmentation products are complemented to 50 μ L by O, 90 μ L AMPure XP beads are added, simultaneously room temperature is quiet for mixingSet 5min.Reaction tube is placed on magnetic frame, stands 3-5min.Supernatant is abandoned, 200 μ L, 80% ethyl alcohol is added to be eluted, repeats 2~3It is secondary, with 70 μ L NF-H2O is eluted；
(3) capture magnetic bead, specific capture dna-RNA heterozygosis chains are added afterwards in step (2)；
In step (2) eluted product, pretreated [the S9.6]/Protein A magnetic beads of 10 μ L, NF-H is added2O mend to100 μ L, room temperature mixing 20min.After magnetic, supernatant is removed, is cleaned three times with 1mL 1X Bind/Wash Buffer, 30 μ L NF-H2O is eluted, 65 DEG C of incubation 15min, after magnetic, shifts supernatant, the DNA-RNA heterozygosis chains as captured；
(4) magnetic bead is added afterwards in step (3) and carries out capture product purification；
Step (3) after reaction, uses NF-H2O complements to 50 μ L by product is captured, and 90 μ L AMPure XP are addedBeads, mixing are simultaneously stored at room temperature 5min.Reaction tube is placed on magnetic frame, stands 3-5min.Supernatant is abandoned, 200 μ L, 80% second is addedAlcohol is eluted, and is repeated 2~3 times, with 30 μ L NF-H2O is eluted；
(5) 1 reaction system of enzyme is added afterwards in step (4), carries out end reparation；
With 1 reaction system of tabulating.Mixing, 37 DEG C of reactions 30min, 72 DEG C of reaction 20min in thermal cycler.After immediatelyCarry out next step operation.
Repair reaction system in 1 end of table
| Component | Volume (μ L) |
| Upper step product | 30 |
| Buffer 1 | 7 |
| Enzyme 1 | 2 |
| NF-H2O | 11 |
(6) 2 reaction system of enzyme is added afterwards in step (5), carries out connector connection reaction；
With 2 reaction systems of tabulating.Mixing, 20 DEG C of reactions 15min, 65 DEG C of reaction 10min in thermal cycler.After immediatelyCarry out next step operation.
2 connector coupled reaction system of table
| Component | Volume (μ L) |
| Upper step product | 50 |
| RNA Adapter | 2 |
| Buffer 2 | 27 |
| Enzyme 2 | 1 |
(7) magnetic bead is added afterwards in step (6) and carries out connector connection product purifying；
After reaction, 30 μ L AMPure XP beads are added in step (6), and mixing is simultaneously stored at room temperature 5min.Reaction tubeIt is placed on magnetic frame, stands 3-5min.Supernatant is abandoned, 200 μ L, 80% ethyl alcohol is added to be eluted, is repeated 2~3 times, with 50 μ L NF-H2O is eluted, and is and then purified with 1X XP beads, with 23 μ L NF-H2O is eluted；
(8) PCR amplification system is added afterwards in step (7), carries out the amplification in library；
Reaction system is prepared by table 3, amplified library is carried out by following program.
3 PCR reaction systems of table
| Component | Volume (μ L) |
| Upper step product | 23 |
| 2×KAPA HiFi HotStart ReadyMix | 25 |
| P5 | 1 |
| P7 | 1 |
PCR amplification program：
(9) magnetic bead is added afterwards in step (8) to purify the library after amplification, product after purification is upper machine textLibrary.
Step (8) complements to 50 μ L by product is captured after reaction, with 10mM Tris-HCl, and 45 μ L AMPure are addedXP beads, mixing are simultaneously stored at room temperature 5min.Reaction tube is placed on magnetic frame, stands 3-5min.Supernatant is abandoned, 200 μ L 80% are addedEthyl alcohol is eluted, and is repeated 2~3 times, is eluted with 25 μ L DB, and product after purification is upper machine library.
QPCR quality inspections are carried out to upper machine library：
According to KAPA Library Quantifcation Kit (Kapabiosystems, Cat No.KK4854) explanationBook is operated, and is detected using 480 real-time fluorescence quantitative PCR instrument of Roche Light Cycler, with reference to the standard in kitProduct carry out the absolute quantitation of library concentration.
QPCR quantitative analysis results are as shown in table 4：
4 qPCR testing results of table
| Sample | QPCR concentration (nM) |
| The libraries R-Loop | 112 |
The library of structure reaches confidential and seeks concentration, can be used for machine sequencing.
Upper machine sequencing is carried out to upper machine library：
Library denaturation, dilution and sequencing are carried out according to NextSeq sequenator operating processes, the results are shown in Table 5.
5 sequencing data Quality Control of table
Electrophoresis detection analysis is carried out to upper machine library：The results are shown in Figure 3 for electrophoretic analysis, and library master tape understands and mainly dividesFor cloth in 450bp or so, band brightness is bright, consistent with expected results.
Embodiment 2
The method that the present embodiment is sequenced by PCR and a generation captures the RNA in product to [S9.6] and is expanded and be sequencedAnd compared with library sequencing result is built, to verify the sequencing result of embodiment 1.
(1) product is captured to [S9.6] in embodiment 1 and carries out I digestion process of DNase；
(2) 2 reads for randomly selecting sequencing result in embodiment 1, are RL1, RL2 successively；
Detailed sequence is as follows：
RL1 sequences are as shown in sequence 5：
5’-GGGCGGCCGAGGGAGAGGGGAGGGGGCGGCCTCACCTGCGCCACTGCGATGCCCGCCGCCGCCGCCGCCGAGTCATTATCTCGGGCGGAAGCGAGAAGGGCGGCGAGGGAGGGGGTGGAAGCCCGCAGAGACAAGGGTGGGGATGGATCGC-3’
RL2 sequences are as shown in sequence 6：
5’-GACGGAGCAGAGACACGGGACGGGGCAGAGACACGGGACGGAGCAGAGACACGGGACGGAGCAGAGACACGGGGGACGGAGGAGAGACAGGGGGGACGGAAGAGAGACGCGGGGCGGGGCAGAGACCGGGGACGGAGCAGAGACACGGGAC-3’；
(3) according to its nucleic acid sequence, upstream and downstream primer is separately designed, names F-RL1 and R-RL1, F-RL2 and R- successivelyRL2；
Detailed sequence is as follows：
F-RL1 is as shown in sequence 7：5’-GCCGCCGAGTCATTATCT-3’
R-RL1 is as shown in sequence 8：5’-ATCCATCCCCACCCTTGT-3’
F-RL2 is as shown in sequence 9：5’-AGACACGGGACGGGGCAGAG-3’
R-RL2 is as shown in sequence 10：5’-GTCTCTGCTCCGTCCCGTGTCT-3’；
(4) RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, goods is respectively adoptedNumber：) and AmpliTaq Gold 360DNA Polymerase (Applied Biosystems, article No. K1622：4398892) intoRow cDNA synthesis and PCR reactions.
Generation sequencing is carried out to amplification and is analyzed.
R-Loop builds the comparison of library sequencing result and RNA amplification generation sequencing result.
6 nucleic acid sequence alignment result of table
| Title | RL1 | RL2 |
| Compare logarithm | 100% | 100% |
As a result show that R-Loop builds the corresponding sequences of random 2 reads of library sequencing data and a RNA amplification generation measuresSequence alignment number equal 100%, thus illustrate the accuracy in the libraries R-Loop that the present invention is built.
The common R-Loop library constructing methods comparison of R-Loop library constructing methods of the present invention and market is as shown in table 7,
7 sequencing data Quality Control of table
| Banking process | Common R-Loop builds library method | R-Loop of the present invention builds library method |
| It uncaps number | 20 | 13 |
| Tube number | 8 | 5 |
| Experimental procedure | 8 | 5 |
| Test the used time (h) | 6.5 | 4.5 |
| Experimental cost | It is high | It is low |
From 7 comparing result of table it is found that R-Loop of the present invention, which builds library method, not only simplifies experimental implementation flow and step, and shortenTime saves human cost, while also can avoid pollution and sample sample mixing between sample, reduces because Library development flow complexity causesFaulty operation risk improves the repeatability and accuracy of experimental result.