CN104178478A - Liquid-phase hybrid capture enriching liquor for genome DNA sequencing library and hybrid method adopting liquid-phase hybrid capture enriching liquor - Google Patents
Liquid-phase hybrid capture enriching liquor for genome DNA sequencing library and hybrid method adopting liquid-phase hybrid capture enriching liquor Download PDFInfo
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Abstract
The invention discloses a liquid-phase hybrid capture enriching liquor for a genome DNA sequencing library and a hybrid method adopting the liquid-phase hybrid capture enriching liquor. The hybrid enriching liquor comprises phosphate buffer, sodium citrate buffer (SSC), SDS (lauryl sodium sulfate), EDTA (ethylene diamine tetraacetic acid) and Denhardt liquor. The hybrid liquor and the corresponding hybrid method are low in cost, simple and convenient to implement; the hybrid time can be effectively shortened, the needed DNA fragment can be precisely extracted, influences of a background value on a signal value can be reduced, and the hybrid target enriching effects can be optimized.
Description
Technical field
The invention belongs to biology field, in particular to a kind of formula and hybridizing method of genomic dna sequencing library solution hybridization trapping enrichment solution.
Background technology
In the past few years, there is the transformation of an essence in DNA sequencing technology, from traditional Sanger order-checking, developed into so-called " order-checking of future generation " technology (Next Generation Sequencing, NGS).Its outstanding feature is large-scale parallel order-checking.NGS technology allows millions of even sequencing reaction of hundreds of millions to carry out simultaneously, thereby reaches the jumbo growth of sequencing throughput.NGS technology platform need to build the fragment library that is applicable to large-scale parallel order-checking.The process that builds sequencing library comprises DNA fragmentation, DNA reparation and end processing (flush end or A overhang) subsequently.Finally, the specificity joint of selecting according to concrete technology platform connects.
Although compare with traditional Sanger sequencing, the cost that NGS technology is relevant significantly reduces, and it is higher that genome sequencing remains cost.In addition, there are many application not need genome sequencing, but need to check order for the specific region of one or more samples.Therefore, if interested region is, also or in a large number the sample of numbers need to be analyzed for a genomic part, we usually tend to only the special subset of a part for sequencing library be checked order, rather than whole genomic library is checked order, thereby reduce unnecessary cost and labor force.For example effectively full exon (encoding parts of all genes) order-checking is at present than major application, but is also widely used for the enrichment order-checking of less genome or genome area.In the process of target enrichment, uninterested DNA fragmentation is removed to greatest extent, and by enrichment the genome sequencing library from initial out, the sequencing result that follow-up NGS order-checking is produced mainly concentrates on interested target area in target area.
In order to apply better NGS, carry out target order-checking, a plurality of flow processs of carrying out target enrichment before order-checking are developed out.Conventionally to carry out the enrichment of a plurality of steps so that final target sequencing library to be provided.At present, conventional target enriching method comprises pcr amplification method (PCR amplification), selectivity cyclisation method (selective circularization), and hybridization mass trapping (hybrid capture).Different target beneficiation technologies have different performances and ease for use.The most important characteristic of every kind of method, also the ultimate challenge that has reflected conversely target enrichment, comprise the time that target enrichment sequencing library consumes that obtains, obtain the total cost of each target base of useful sequencing result, the parameter of enrichment, comprises specificity (ratio of target area Yu Fei target area in sequencing result), fraction of coverage (the order-checking degree of depth), covering uniformity coefficient in all targets target area, the total amount of the repeatable and needed input DNA of method flow.
The enrichment of PCR-based principle as its name suggests, is to utilize the PCR primer for target area to come amplified target to sequence, and then specific PCR product is checked order.This method need to be permitted multicycle sex change conventionally, anneals and extends, and often causes the skewed popularity of non-specific product and amplification.In addition, for the hundreds of or thousands of substance PCR reaction of operation, need to use special for automatic equipment or subregion equipment (for example technology of Fruidigm and RainDance company).Limited multiplex PCR is carried out in being chosen as in a reaction of other.Therefore, the enrichment of PCR-based can not expand target area easily, thereby is used for less target area (several kilobase are to several million scopes).
Selectivity cyclisation method is also molecule inversion probe (MIP).Each cyclisation probe for enrichment has the few nucleic acid chains of a single stranded DNA, and the sequence that is not connected with target complement sequence is contained respectively at its two ends, and is contrary linear precedence.The specific hybrid of probe and target sequence has formed cyclic DNA structure.This structure is further formed the single-stranded cyclic DNA of sealing by gap-fill and ligation.For one section of consensus sequence on ring and the PCR reaction meeting carried out finally increase these target enrichments region and produce the sequencing library of target enrichment.The great advantage of selectivity cyclisation method is its high specific, but its most outstanding shortcoming be for each target area to catch homogeneity poor, far below hybrid capture.And its cost is higher, the size of the target area that can detect limited (several kilobase are to several million scopes).
When hybridization mass trapping is the DNA probe specific hybrid complementary with it of target nucleotide sequence in inputting DNA sample and customization, target dna sequence will catching and separating by physical property.Because hybridization mass trapping can synthesize a large amount of specificity DNA probing needles easily, and carry out in a reaction system simultaneously, and multiple DNA sequencing library sample can mix simultaneously and process by connecting the sequence measuring joints of different coding, thereby the method for hybridization trapping is used for the target area (1 to 60Mb) of medium-to-large or compared with Multi-example.Along with the widespread use of full exon order-checking, hybridization mass trapping more and more should be used, also more and more for its various experiment flows and commercialization reagent.
Hybridization trapping enrichment can be divided into again solid phase chip hybridization and solution hybridization.In solid phase chip hybridization, genomic dna sequencing library is hybridized mutually with the DNA probe being fixed on chip.Can not in conjunction with non-specific DNA library fragment from chip, washed away, and the DNA library of target enrichment be eluted after a while for order-checking.In solution hybridization, the specific combination of biotin labeled oligonucleotide capture probe of the target sequence in DNA sequencing library and customization.Add subsequently Streptavidin MagneSphere to be combined with biotinylated probe, thereby make the specific target sequence of combination with it utilize magnetic-adsorption and separate.DNA library after enrichment is eluted for order-checking.
Though solid phase chip hybrid method has many places that are better than pcr amplification enrichment, but the cost compare of chip is high, and experiment process needs expensive hardware device, for example chip incubator.In addition, due to the necessary while of the chip that starts to hybridize the together DNA library of wash-out enrichment simultaneously, can simultaneously treated number of chips be also very limited at every turn.Finally, in order to obtain a hybridization enrichment, react required enough DNA library amounts, we need the input DNA of about 10-15ug to prepare genomic library, and its requirement to experiment material initial amount is huge.By contrast, solution hybridization, because of its easy handling, only needs again a small amount of input DNA library, particularly when sample has in limited time, and is applied by increasing people.Solution hybridization is because its capture probe is excessive with respect to DNA library, thereby promoted fully carrying out of hybridization.The homogeneity of solution hybridization and specificity are all hybridized higher than solid phase chip in addition.Finally, solution hybridization is caught does not need special instrument, only needs the thermal cycler of laboratory routine, and can process a large amount of samples with the pattern of 96 orifice plates simultaneously.
Because DNA capture probe used is designed to and the complementary hybridization of target regional sequence, thereby make enrichment depend on the physics and chemistry characteristic of Nucleotide, for example molecular mass of Nucleotide, its GC content, secondary structure, fusing and annealing temperature, and the concentration of Nucleotide and salt concn.These all factors all produce material impact to the binding kinetics of hybridization and specificity.Technology based on Hybridization principle usually needs the higher temperature of fusion close to probe used, and need to improve specificity by longer incubation time.The incubation time scope of standard is not from 10 hours to 72 hours etc.A significant drawbacks of long incubation time is to have increased the time that obtains target enrichment sequencing library, thereby has affected follow-up order-checking.In addition, higher incubation temperature and long incubation time cause as the problem of moisture evaporation, may change the important parameter in hybridization mixture, salt concn for example, especially works as the volume of hybridization smaller time.Current many companies have developed the test kit of commercial solution hybridization trapping enrichment genomic dna sequencing library.But the capture probe that its hybridization solution and hybridizing method spininess are produced for our company, not etc., and it is not underground to fill a prescription for from 60 to 150 bases of its capture probe length, expensive.The hybridization temperature of each test kit, incubation time, and input library amount is all not identical.Thereby a kind of low cost of necessary invention, simple to operate, repeatable high, cycle is short, high specific, be applicable to again the hybridization solution of genomic dna sequencing library that several different methods builds and capture probe and hybridizing method correspondingly thereof, to meet the growing requirement to target order-checking of future generation.
Summary of the invention
The object of this invention is to provide a kind of can at lower cost, simply, fast, realization efficiently hybridization and trap the hybridization solution of enrichment genomic dna sequencing library and the method that adopts this hybridization solution to hybridize.In order to achieve the above object, intend adopting following technical scheme:
One aspect of the present invention relates to a kind of genomic dna sequencing library hybridization trapping enrichment solution, it is characterized in that it comprises sodium phosphate buffer, sodium citrate buffer solution (SSC), sodium lauryl sulphate (SDS), ethylenediamine tetraacetic acid (EDTA) (EDTA) and step on Hart (Denhardt) solution, described component independent packaging.
In a preferred embodiment of the present invention, genomic dna sequencing library hybridization trapping enrichment solution contains or does not contain other component.
In another preferred embodiment of the present invention, described sodium phosphate buffer be 0.4-0.6M sodium phosphate buffer, its pH value is between 6.8-7.2.
In another preferred embodiment of the present invention, the concentration of SDS is 0.8-1.2%; The concentration of EDTA is 1-3mM.
In another preferred embodiment of the present invention, described genomic dna sequencing library hybridization trapping enrichment solution comprises 1 part of sodium phosphate buffer, 2 parts of sodium citrate buffer solutions (SSC), 1 part of SDS, 1 part of EDTA and 4 parts of Denhardt solution.
In a preferred embodiment of the present invention, the formula of 1M sodium phosphate buffer (pH7.0): 57.7ml1M Na
2hPO
4with 42.3ml1M NaH
2pO
4mix.
In a preferred embodiment of the present invention, 20X SSC formula: 3M NaCl, 300mM Trisodium Citrate; Regulate pH value to 7.0.
In a preferred embodiment of the present invention, 50X Denhardt solution: contain 1% ficoll (Ficoll in this solution, 400 types), 1% polyvinylpyrrolidone (polyvinylpyrrolidone), and 1% bovine serum albumin (BSA).
The present invention also relates to a kind of hybridizing method on the other hand, it is characterized in that adopting the enrichment of said gene group DNA sequencing library hybridization trapping enrichment solution.
In a preferred embodiment of the present invention, described hybridizing method comprises the steps (Fig. 1):
(1) genomic DNA fragment is turned to 350-550bp (Mechanical Crushing, as supersound process, hydraulic shear, or enzymatic fragmentation all can; Its required fragment length depends on the order-checking length that follow-up plan adopts);
(2) according to follow-up used NGS order-checking platform, according to standard test flow process, prepare gene order-checking library.
(3) pond, total amount 250-2000ng gene order-checking library is dry in 0.2mL PCR pipe, in this pond, library, can mix a plurality of gene order-checkings library sample with different index sequence joint.
(4) following composition added to the sequencing library of having dried and it fully dissolved in standing 10 minutes:
| The strand Nucleotide capture probe 3pMole of biotin modification |
| 2-5 μ g mankind Cot-l DNA |
| Each 1mM of respective ends sealing polymeric blends |
| Nuclease free deionized water, mends cumulative volume to 10 μ L |
(5) add 10 μ L said gene group DNA sequencing library hybridization trapping enrichment solutions, and fully mix with liquid-transfering gun.
(6) above-mentioned reaction system is put into thermal cycler operation follow procedure:
This hybridization solution formula is simple, easily obtains, and has reduced production cost and has easily preserved.
Of the present invention method is simple, only needs the conventional thermal cycler using in laboratory.The hybridization of spending the night is convenient to the design of flow process, and the selection of 65 ℃ of hybridization temperatures is applicable to the strand Nucleotide capture probe of conventional 80-120 base length.
Hybridization solution formula of the present invention and hybridizing method applicable to multiple different methods, prepare (for example Illumina test kit, NEB test kit, KAPA test kit etc.) genomic dna sequencing library catch enrichment.And can while a plurality of libraries of enrichment sample in a hybridization.Total input library sample only need to be no less than the DNA of 250ng.
By using this hybridization solution and hybridizing method, the background value producing in the time of can controlling well hybridization, improves enrichment signal, optimizes crossbreeding effect.Experimental results show that, utilize this hybridization solution to carry out the target enrichment (about 1Mb) of one group of specific gene to the complete genome DNA sequencing library of normal people's bone-marrow-derived lymphocyte system, improving hybridization temperature, when shortening hybridization time, the hybridization signal of target gene significantly increases (as Fig. 2 A), and non-specific background value significantly reduces (as Fig. 2 B) simultaneously.Compare with certain commercialization hybridization buffered soln, this hybridization solution formula and method can significantly improve the enrichment times of target gene and effectively reduce background value (as Fig. 2 C) on the basis of shortening the operating time.
Improved hybridization solution formula and corresponding hybridizing method cost are lower, simple and easy to do, have effectively shortened hybridization time, and can extract accurately needed DNA fragmentation, reduce the impact that background value produces signal value, optimize hybridization target concentration effect.
Accompanying drawing explanation:
Fig. 1, utilize hybridization solution formula of the present invention to carry out the schema of genomic dna sequencing library hybridization trapping enrichment.(1) genomic DNA fragment.(2) build genomic dna sequencing library.(3) mix a plurality of gene order-checkings library sample with different index sequence joint.(4) add blocker and sealing polymkeric substance.(5) configuration hybridization system.(6) in thermal cycler, carry out the hybridization of target sequence and probe.
Fig. 2, the simultaneous test of utilizing this hybridization solution and commercial hybridization solution to carry out target enrichment.A and B are for this hybridization solution formula is in the performance under the time without hybridization temperature and hybridization, thereby the hybridizing method of this hybridization solution is used in optimization.C is for being used the simultaneous test of this hybridization solution and corresponding hybridizing method and certain commercialization hybridization solution and hybridizing method thereof.The enrichment times of target gene and non-target gene detects by real-time quantitative PCR (realtime-qPCR).Detected result is three independent revision test averaging of income value ± standard errors (mean ± SEM).
Embodiment
In conjunction with embodiment, describe embodiments of the present invention in detail, but technical scope of the present invention is not limited to following embodiment, is not changing under the prerequisite of its main points, can make various changes and implement.
Embodiment 1
Prepare genomic dna sequencing library hybridization trapping enrichment solution, it is by sodium phosphate buffer, sodium citrate buffer solution (SSC), SDS, EDTA and Denhardt solution composition.At-20 ℃, can preserve for a long time.The concrete formula of its 2X concentration solution is as follows:
0.5M sodium phosphate buffer, pH7.0 (1)
1％SDS
2mM?EDTA
2X?SSC，pH7.0?(2)
4X Denhardt solution (3)
(1) formula of 1M sodium phosphate buffer (pH7.0): 57.7ml1M Na
2hPO
4with 42.3ml1M NaH
2pO
4mix
(2) 20X SSC formula: 3M NaCl, 300mM Trisodium Citrate; Regulate pH value to 7.0.
(3) 50X Denhardt solution: Denhardt solution is a kind of mixture for the closed reagent of hybridizing.In this solution, contain 1% ficoll (Ficoll, 400 types), 1% polyvinylpyrrolidone (polyvinylpyrrolidone), and 1% bovine serum albumin (BSA).
The method that above-mentioned hybridization solution is hybridized, by following step (Fig. 1):
(1) genomic DNA fragment is turned to 550bp (Covaris ultrasonication).
(2) according to Illumina TruSeq PCR-free, prepare gene order-checking library.
(3) pond, total amount 500ng gene order-checking library vacuum in 0.2mL PCR pipe is dried at a high speed.In this pond, library, can mix a plurality of gene order-checkings library sample with different index sequence joint, each sequencing library is no less than 25ng.
(4) following composition added to the sequencing library of having dried and make it fully dissolve (approximately 10 minutes):
(5) add 10 μ L2X hybridization solutions (have Precipitation as 2X hybridization solution at room temperature thaws, please dissolve for 10 minutes in 55 ℃ of Heating Waters), and fully mix with liquid-transfering gun.Hybridization cumulative volume is 20ul.
(6) above-mentioned reaction system is put into thermal cycler operation follow procedure:
65 ℃ of insulations (spending the night for 16-24 hour)
The above is the preferred embodiments of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a genomic dna sequencing library hybridization trapping enrichment solution, is characterized in that it comprises sodium phosphate buffer, sodium citrate buffer solution (SSC), SDS, EDTA and Denhardt solution, described component independent packaging.
2. enrichment solution is traped in genomic dna sequencing library hybridization according to claim 1, and genomic dna sequencing library hybridization trapping enrichment solution contains or do not contain other component.
3. genomic dna sequencing library according to claim 1 hybridization trapping enrichment solution, described sodium phosphate buffer be 0.4-0.6M sodium phosphate buffer, its pH value is between 6.8-7.2.
4. enrichment solution is traped in genomic dna sequencing library hybridization according to claim 1, and the concentration of SDS is 0.8-1.2%; The concentration of EDTA is 1-3mM.
5. enrichment solution is traped in genomic dna sequencing library hybridization according to claim 1, described genomic dna sequencing library hybridization trapping enrichment solution comprises 1 part of sodium phosphate buffer, 2 parts of sodium citrate buffer solutions (SSC), 1 part of SDS, 1 part of EDTA and 4 parts of Denhardt solution.
6. enrichment solution, the formula of 1M sodium phosphate buffer (pH7.0): 57.7ml1M Na are traped in genomic dna sequencing library hybridization according to claim 1
2hPO
4with 42.3ml1M NaH
2pO
4mix.
7. genomic dna sequencing library hybridization according to claim 1 is traped enrichment solution, 20X SSC formula: 3M NaCl, 300mM Trisodium Citrate; Regulate pH value to 7.0.
8. enrichment solution is traped in genomic dna sequencing library hybridization according to claim 1,50X Denhardt solution: contain 1% ficoll (Ficoll in this solution, 400 types), 1% polyvinylpyrrolidone (polyvinylpyrrolidone), and 1% bovine serum albumin (BSA).
9. a hybridizing method, is characterized in that adopting the enrichment of said gene group DNA sequencing library hybridization trapping enrichment solution.
10. hybridizing method according to claim 9, described hybridizing method comprises the steps:
(1) genomic DNA fragment is turned to 350-550bp (Mechanical Crushing, as supersound process, hydraulic shear, or enzymatic fragmentation all can; Its required fragment length depends on the order-checking length that follow-up plan adopts);
(2) according to follow-up used NGS order-checking platform, according to standard test flow process, prepare gene order-checking library;
(3) pond, total amount 250-2000ng gene order-checking library vacuum in 0.2mL PCR pipe is dry at a high speed, in this pond, library, can mix a plurality of gene order-checkings library sample with different index sequence joint;
(4) following composition added to the sequencing library of having dried and it fully dissolved in standing 10 minutes:
(5) add 10 μ L said gene group DNA sequencing library hybridization trapping enrichment solutions, and fully mix with liquid-transfering gun;
(6) above-mentioned reaction system is put into thermal cycler operation follow procedure:
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| PCT/CN2015/086005 WO2016023430A1 (en) | 2014-08-13 | 2015-08-04 | Liquid-phase hybridization capture enriching solution for genome dna sequencing library and hybridization method |
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| CN107236727A (en) * | 2017-05-31 | 2017-10-10 | 江苏为真生物医药技术股份有限公司 | The single-stranded probe preparation method of polygenes capture sequencing |
| CN107304448A (en) * | 2016-04-22 | 2017-10-31 | 张彦伟 | A kind of hybrid capture method of genome target region sequencing |
| CN107304444A (en) * | 2016-04-22 | 2017-10-31 | 张彦伟 | The hybridization enrichment solution and kit of sequencing are captured for genome target region |
| CN107354207A (en) * | 2017-07-11 | 2017-11-17 | 大连晶泰生物技术有限公司 | Solution hybridization capture agent box, washing reagent box and its application based on double-chain probe |
| CN107988317A (en) * | 2017-12-14 | 2018-05-04 | 中源协和基因科技有限公司 | A kind of buffer solution that hybrid capture is carried out to target set nucleic acid region |
| CN107988350A (en) * | 2017-12-14 | 2018-05-04 | 中源协和基因科技有限公司 | A kind of hybridization buffer for the capture of DNA sequencing Library hybridization |
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| CN112955568A (en) * | 2018-08-15 | 2021-06-11 | Illumina公司 | Compositions and methods for improved library enrichment |
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| CN107304448B (en) * | 2016-04-22 | 2021-03-23 | 张彦伟 | Hybridization capture method for sequencing genome target region |
| CN107304444B (en) * | 2016-04-22 | 2021-03-23 | 张彦伟 | Hybridization enrichment solution and kit for capture sequencing of genome target region |
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| CN111961711A (en) * | 2020-08-31 | 2020-11-20 | 伯科生物科技有限公司 | Universal hybridization enhancers and methods for targeted sequencing |
| CN112980938A (en) * | 2021-03-02 | 2021-06-18 | 浙江大学 | Method for capturing genetic deafness gene sequence in targeted manner and sequencing method |
| CN113622033A (en) * | 2021-08-06 | 2021-11-09 | 成都佰维生物科技有限公司 | Preparation method and application of nucleic acid library for low host background interference |
| CN113493932A (en) * | 2021-09-09 | 2021-10-12 | 北京贝瑞和康生物技术有限公司 | Method and kit for constructing capture library with high detection performance |
| WO2023036271A1 (en) * | 2021-09-09 | 2023-03-16 | 北京贝瑞和康生物技术有限公司 | Method for constructing capture library having high test performance, and kit |
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