阅读说明：本技术 一种构建高检测性能捕获文库的方法和试剂盒 (Method and kit for constructing capture library with high detection performance ) 是由 陈迪 李东宁 张建光 于 2021-09-09 设计创作，主要内容包括：本发明涉及一种构建适用于二代测序平台的捕获文库的方法,包括以下步骤：(1)获得片段化的DNA；(2)将片段化的DNA与Y型接头连接,获得预文库；(3)预文库与探针进行杂交,获得杂交产物；和(4)杂交产物洗脱,获得捕获文库。本发明还涉及用于实施上述方法的试剂盒。(The invention relates to a method for constructing a capture library suitable for a second-generation sequencing platform, which comprises the following steps: (1) obtaining fragmented DNA; (2) connecting the fragmented DNA with a Y-shaped joint to obtain a pre-library; (3) hybridizing the pre-library with the probe to obtain a hybrid product; and (4) eluting the hybridization product to obtain a capture library. The invention also relates to a kit for carrying out the above method.)

1. A method of constructing a capture library suitable for use in a second generation sequencing platform, comprising the steps of:

(1) obtaining fragmented DNA;

(2) connecting the fragmented DNA with a Y-shaped joint to obtain a pre-library;

(3) hybridizing the pre-library with a hybridization probe to obtain a hybridization product;

(4) eluting the hybrid product to obtain a capture library;

wherein the step (4) comprises the step (4 a): the hybridization product is subjected to alkaline denaturation.

2. The method of claim 1, wherein the fragmented DNA is selected from naturally occurring short fragment DNA or short fragment DNA obtained by artificial disruption of genomic DNA.

3. The method of claim 2, wherein the naturally occurring short-fragment DNA is selected from the group consisting of peripheral blood-free DNA, tumor-free DNA, or naturally degraded genomic DNA; the artificial disruption of genomic DNA is achieved by sonication, mechanical disruption or by enzymatic digestion.

4. The method of claim 1, wherein the fragmented DNA is derived from a sample from the group consisting of: blood, serum, plasma, synovial fluid, semen, urine, sweat, saliva, stool, cerebrospinal fluid, ascites, pleural fluid, bile, or pancreatic fluid.

5. The method according to any one of claims 1 to 4, wherein the fragmented DNA is 150-400bp in length.

6. The method according to claim 1, further comprising a step of subjecting the obtained fragmented DNA to end repair and/or end-to-end a addition after step (1).

7. The method of claim 1, wherein the Y-junction is a long Y-junction comprising an amplification primer, an index tag sequence, a read 1/read 2 sequencing primer, and an index read sequencing primer.

8. The method according to claim 1, wherein step (4) further comprises, after step (4 a):

step (4 b): incubation;

step (4 c): removing the magnetic beads; and

step (4 d): and (4) neutralizing.

9. The method according to claim 1, wherein the alkaline reagent used for alkaline denaturation in step (4 a) is selected from one or more of the following group: NaOH, KOH, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, lithium carbonate, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, ammonium hydrogen carbonate, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide or potassium tert-butoxide.

10. The method of claim 9, wherein the alkaline reagent concentration is 0.05M to 1M.

11. The method according to claim 1, wherein in the step (4 a), an alkali reagent is added to the hybridization product to perform alkali denaturation; wherein, the pH value of the system after the alkali reagent is added is 11-14.

12. The method of claim 8, wherein in step (4 d), the neutralization is performed with a neutralizing agent selected from one or more of Tris-HCl, acetic acid, citrate buffer, phosphate buffer, or acetate buffer.

13. A kit for constructing a capture library, comprising:

(a) reagents for attaching linkers, including Y-linkers;

(b) reagents for hybridization; and

(c) reagents for elution of hybridization products.

14. The kit of claim 13, wherein the Y-linker is a long Y-linker; the long Y-shaped joint comprises an amplification primer sequence, an index label sequence, a read 1/read 2 sequencing primer sequence and an index read sequencing primer sequence.

15. The kit of claim 13, wherein the kit further comprises reagents for performing end repair and/or end-plus-a.

16. The kit of claim 13, wherein the reagents for hybridization comprise a hybridization buffer, Cot-1DNA, and a hybridization probe; wherein the reagents for hybridization do not include blocking sequences.

17. The kit of claim 16, wherein the blocking sequence comprises a sequence designed to be reverse complementary to a linker and/or tag sequence.

18. The kit of claim 13, wherein the reagents for elution of hybridization products comprise a denaturant and a neutralizer; wherein the denaturant is an alkali denaturant.

19. The kit of claim 18, wherein the alkaline denaturing agent is selected from one or more of NaOH, KOH, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, lithium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide, or potassium tert-butoxide; the neutralizing agent is selected from one or more of Tris-HCl, acetic acid, citrate buffer, phosphate buffer or acetate buffer.

20. A capture library constructed according to the method of any one of claims 1 to 12, or constructed using the kit of any one of claims 13 to 19.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a method and a kit for constructing a hybrid capture library.

Background

Exon capture is a technology of capturing and enriching DNA sequences of exon regions by using probes, and is widely applied to the fields of scientific research and clinical detection. Compared with whole genome sequencing, the method has the advantages of lower cost, shorter period, better coverage, more economy and high efficiency. The construction of a traditional exon capture library typically comprises the following steps: fragmenting genome DNA, carrying out end repair and end adding A, then connecting a linker and a tag sequence, and obtaining a pre-library through a first round of PCR amplification; the pre-library was hybridized with hybridization probes, purified and amplified by a second round of PCR to obtain the final capture library (see FIG. 1: schematic of conventional exon capture scheme).

The enrichment of DNA by PCR is a common technique in the field of Next-generation sequencing (NGS). PCR is applied to exon capture, amplification of captured products is realized, the amount of a library required by computer is obtained, amplification errors and preference are brought, and an original genome sequence cannot be perfectly presented. For example, preferential amplification of DNA of certain characteristics causes failure of uniform amplification of DNA, which in turn causes non-uniform coverage of the target region by captured data, and finally causes detection errors or omission. The application of the PCR-Free technology can perfectly overcome the defects, and the steps of amplification and the like are saved, so that the experimental process is simplified, and the library building cost is reduced. The traditional PCR-Free technique is more applied to direct machine sequencing after the fragmented DNA is connected with a linker. The PCR-Free technique has high requirements on the input of the DNA template, and the capture efficiency of the probe capture process needs to be considered, so that a great deal of waste of the DNA template is caused. At present, the probe capture experiment method is generally covered in the flow, the initial DNA input amount needs to be as large as 500ng-3 mug, and PCR amplification needs to be involved before and after the probe capture step. Before the present invention, the inventor realizes no PCR reaction before capture under the condition of low DNA yield by technical optimization (see FIG. 2: schematic diagram of exon capture flow after optimization in this laboratory), but still does not solve the problem that PCR amplification is still needed after hybrid capture.

Therefore, there is still a need to establish a simple and economical PCR-Free hybridization capture process that can avoid amplification errors and preference, realize improvement of detection performance and effective data utilization rate, and realize input of trace amounts of DNA.

Disclosure of Invention

In view of the higher pursuit of performance testing of captured data, the need for further cost savings and simplification of the library construction process, the inventors of the prior invention (described in patent application publication CN110409001A, incorporated herein by reference in its entirety), i.e., on the basis of no need for PCR prior to capture, proposed a method for constructing a captured library without PCR amplification both before and after capture (see fig. 3: flow diagram of the method for constructing a full-library process PCR-Free library of the present invention).

The present invention is based on the following facts found by the inventors:

(1) by using the original invention experiment process without PCR before capture, the PCR cycle number after capture is reduced, and the detection performance of Indel is obviously improved. Further, it is thought whether the optimal detection performance can be achieved if PCR can be completely eliminated.

(2) The theoretical calculation of 50-100ng DNA caused, and captured before and after capture without PCR, hybrid capture library total enough machine.

(3) And connecting a long Y-shaped joint pre-library, and obtaining a single-stranded DNA (deoxyribonucleic acid) which has all sequences required by the computer by hybridization and capture.

(4) After alkali denaturation and neutralization reaction, the low-concentration double-stranded DNA library can be stably stored at the temperature of 20 ℃ below zero for more than ten days, and the stability of the single-stranded library can meet the computer-operating requirement.

(5) The alkali denaturation can open the DNA double strand of the probe and the hybridization product, the DNA double strand of Cot-1 and the hybridization product, and can also open the connection of streptavidin and biotin. After alkaline denaturation, a large number of biotin-labeled probes, Cot-1 and unsuccessfully ligated linker sequences remain in the library, and it is uncertain what effects will be brought about in the subsequent loading process. The inventors found through experiments that these residual probes did not affect the quality of sequencing data.

Accordingly, in a first aspect, the present invention provides a method of constructing a capture library suitable for use in a second generation sequencing platform, comprising the steps of:

(1) obtaining fragmented DNA;

(2) connecting the fragmented DNA with a Y-shaped joint to obtain a pre-library;

(3) hybridizing the pre-library with a hybridization probe to obtain a hybridization product;

(4) eluting the hybrid product to obtain a capture library;

wherein the step (4) comprises the step (4 a) of alkali denaturation of the hybridization product.

In some embodiments, the method is used to construct a second generation sequencing capture library.

In some embodiments, wherein the fragmented DNA is selected from naturally occurring short fragments of DNA or short fragments of DNA obtained by artificial disruption of genomic DNA.

In other embodiments, wherein the naturally occurring short stretch of DNA is selected from the group consisting of peripheral blood-free DNA, tumor-free DNA, or naturally degraded genomic DNA; the artificial disruption of genomic DNA is achieved by sonication, mechanical disruption or by enzymatic digestion.

In some embodiments, wherein the fragmented DNA is derived from a sample of the group consisting of: blood, serum, plasma, synovial fluid, semen, urine, sweat, saliva, stool, cerebrospinal fluid, ascites, pleural fluid, bile, or pancreatic fluid.

Preferably, the fragmented DNA is 150-400bp in length, more preferably 180-230bp in length.

In some embodiments, wherein the method further comprises the step of subjecting the obtained fragmented DNA to end repair and/or end-to-end a after step (1). Preferably, the terminal repair and terminal addition of A are carried out in one reaction system. More preferably, DNA fragmentation, end repair and end-addition of a are performed in one reaction system.

In some embodiments, wherein the Y-junction of step (2) is a long Y-junction comprising an amplification primer, an index tag sequence, a read 1/read 2 sequencing primer, and an index read sequencing primer.

In some embodiments, said step (3) is performed in a liquid phase system.

In some embodiments, wherein step (4) further comprises after step (4 a):

step (4 b): incubation;

step (4 c): removing the magnetic beads; and

step (4 d): and (4) neutralizing.

In a preferred embodiment, wherein the alkaline agent used for alkaline denaturation in step (4 a) is selected from one or more of the following group: NaOH, KOH, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, lithium carbonate, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, ammonium hydrogen carbonate, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide or potassium tert-butoxide. Further preferably, the alkaline agent is NaOH or KOH, preferably an aqueous solution thereof.

In a preferred embodiment, the alkali agent concentration is from 0.05M to 1M, preferably from 0.1 to 0.5M, more preferably 0.2M.

Preferably, in step (4 a), an alkali reagent is added to the hybridization product to perform alkali denaturation, wherein the pH value of the system after the alkali reagent is added is 11-14, more preferably 12-14, and most preferably 12-13.

Further preferably, the neutralizing step is neutralized with a neutralizing agent; preferably, the neutralizing agent is selected from one or more of Tris-HCl, acetic acid, citrate buffer, phosphate buffer or acetate buffer. Preferably, the neutralizing agent is selected from Tris-HCl.

In another aspect, the invention also relates to a kit for constructing a capture library, comprising:

(1) reagents for attaching linkers, including Y-linkers;

(2) reagents for hybridization; and

(3) reagents for elution of hybridization products.

In some embodiments, wherein the Y-linker is a long Y-linker; the long Y-type joint comprises an amplification primer sequence, an index label sequence, a read 1/read 2 sequencing primer sequence and an index read sequencing primer sequence.

In some embodiments, agents for performing end repair and/or end-plus-a are also included.

In some embodiments, wherein the reagents for hybridization comprise a hybridization buffer, Cot-1DNA, and a hybridization probe; preferably, the reagents for hybridization do not include blocking sequences.

In some embodiments, wherein the blocking sequence comprises a sequence designed to be reverse complementary to the linker and/or tag sequence.

In some embodiments, wherein the reagents for elution of hybridization products comprise a denaturing agent and a neutralizing agent. Preferably, the denaturing agent is an alkaline denaturing agent.

In a preferred embodiment, the alkali denaturing agent is selected from one or more of the group consisting of: NaOH, KOH, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, lithium carbonate, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, ammonium hydrogen carbonate, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide or potassium tert-butoxide; NaOH or KOH is preferred, and aqueous solutions thereof are more preferred. The neutralizing agent is selected from one or more of the following: Tris-HCl, acetic acid, citrate buffer, phosphate buffer or acetate buffer; Tris-HCl is preferred.

In another aspect, the invention also includes a capture library constructed according to the method described above, or constructed according to the kit described above. In a preferred embodiment, the capture library is used in a second generation sequencing platform.

The invention has the following excellent technical effects:

(1) the requirement on the content of the initial DNA is low, even can be as low as 25ng, so that the utilization rate of rare samples is greatly improved, and the application range of the invention is expanded.

(2) The library building process is simple, and the method of the invention thoroughly realizes the full process PCR-Free before and after capture, thereby simplifying and shortening the experimental process.

(3) The invention does not relate to PCR amplification, thus saving the library construction cost related to amplification, and the low redundancy advantage of the PCR-Free library increases the effective data utilization rate, thereby avoiding the waste of data volume and realizing the reduction of sequencing cost.

(4) The PCR-Free library can perfectly avoid amplification errors and preference, and show the true appearance of the DNA sequence, so that the mutation detection performance is effectively improved.

The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

Drawings

FIG. 1: schematic diagram of conventional exon capture scheme.

FIG. 2: scheme of exon capture after optimization by the inventors (described in patent application CN 110409001A).

FIG. 3: the invention discloses a flow diagram of a method for constructing a full-library construction process PCR-Free library.

FIG. 4: schematic view of Y-shaped structure.

FIG. 5: microscopic schematic of the hybrid capture system.

FIG. 6: library concentration measurements.

Detailed Description

Method for constructing capture library

In a first aspect, the invention provides a method of constructing a capture library.

At the end of traditional capture library construction, PCR amplification is often performed on the hybrid product, on one hand, the fragment captured by the probe is copied into a liquid phase system in an amplification mode (avidin on streptavidin magnetic beads and biotin on the probe are strongly covalently bound, and the probe and the target fragment form a double-stranded structure through base complementary pairing, see fig. 5: microscopic schematic diagram of the hybrid capture system), and on the other hand, the yield of the final library is improved, so that the on-machine requirement is met. The introduction of post-hybridization PCR not only increases the experimental cost and the operational complexity, but also brings about various disadvantages of PCR amplification (amplification error and preference, unfavorable mutation detection, higher redundancy and increased sequencing cost).

In order to overcome the above disadvantages and realize the real PCR-Free library construction process, the present inventors tried to develop a method (after hybridization reaction) that can directly separate the target fragment (the library captured by the probe) from the streptavidin magnetic bead into the liquid phase system.

First, it is considered that a method using free biotin competes with biotin on a probe to obtain a double strand formed by a part of the detached probe and a target fragment, but it was found through tests that avidin binds to biotin on the probe too tightly, and the concentration of the library obtained through the experiment is extremely low.

And secondly, adopting Deep Vent DNA polymerase with strand displacement activity to try to displace a target fragment from a probe, and obtaining a double-stranded library with higher stability.

Finally, it is considered to cleave the double strand formed by the probe and the target fragment by denaturation (hydrogen bond cleavage of double-stranded base pair). Currently, common denaturation methods are high temperatures and extreme pH. Through gradient test, the high temperature means is used to make the double-chain denaturation process difficult to operate and difficult to control, and the concentration and stability of the library obtained by the experiment are not good.

In the invention, the alkali denaturation experiment process is finally selected, the alkali reagent is added to create the strong alkaline condition for unwinding the double chains, the unwinding of the double chains is promoted by incubation and vortex, and the alkalinity of the system is reduced by neutralization at the end of the experiment process, thereby being beneficial to the preservation of the single-chain library. The library finally obtained meets the requirements of multiple computer operation and long-term preservation.

Thus, the present invention provides a method for constructing a capture library with high detection performance, comprising the following steps:

(1) obtaining fragmented DNA;

(2) connecting the fragmented DNA with a Y-shaped joint to obtain a pre-library;

(3) hybridizing the pre-library with the probe to obtain a hybrid product;

(4) eluting the hybrid product to obtain a capture library;

wherein, the step (4) comprises the step (4 a): the hybridization product is subjected to alkaline denaturation.

In some embodiments, the methods of the invention can be used to construct second generation sequencing capture libraries.

In some embodiments, the fragmented DNA refers to a short fragment of DNA that occurs naturally or is obtained by artificial disruption of genomic DNA.

In some embodiments, the fragmented DNA may be derived from a sample of blood, serum, plasma, synovial fluid, seminal fluid, urine, sweat, saliva, stool, cerebrospinal fluid, ascites fluid, pleural fluid, bile, or pancreatic fluid.

In a preferred embodiment, the natural short piece of DNA is peripheral blood-free DNA, tumor-free DNA, or naturally degraded genomic DNA.

In other embodiments, the genomic DNA may be from a variety of sources, such as from buccal swabs, amniotic fluid, dried blood slices, tissues, peripheral blood, and the like. The person skilled in the art knows methods for disrupting genomic DNA, for example by sonication, mechanical disruption or by enzymatic cleavage etc. Since sonication and mechanical disruption relatively lose more DNA, it is preferred to fragment the DNA enzymatically in cases where the starting DNA content is low (e.g.as low as 50 ng).

In some embodiments, the fragmented DNA is 150-400bp, preferably 180-230bp in length.

In some embodiments, the method of the invention further comprises, prior to the attachment to the Y-linker (i.e., step (2)), step (1'): the fragmented DNA is subjected to end repair and/or end-plus-a. In this embodiment, the DNA may be end-repaired with any enzyme suitable for end-repair known to those skilled in the art, such as T4 DNA polymerase, Klenow enzyme, or mixtures thereof. In this embodiment, the DNA may be terminally A-added with any enzyme suitable for terminal A-addition known to those skilled in the art. Examples of such enzymes include, but are not limited to, Taq enzyme, klenow ex-enzyme, or a mixture thereof. In this embodiment, the terminal repair and the terminal addition of A may be carried out in two reaction systems, that is, after the terminal repair, the terminal addition of A is carried out after purification. Alternatively and preferably, the end-point repair and the end-point addition of A are carried out in one reaction system, i.e., the end-point repair and the end-point addition of A are simultaneously completed, after which the nucleic acid is purified. Alternatively, more preferably, the DNA fragmentation, end repair and end addition A are performed in one reaction system, followed by linker ligation. The method not only simplifies the operation steps and saves the cost, but also reduces the pollution among samples.

In some embodiments, the incubation time and temperature for end-blunting and end-plus-a can be determined by one of skill in the art according to routine techniques, as may be required.

In some embodiments, step (2) may be performed with any suitable enzyme for ligating a linker known to those skilled in the art. Examples of such enzymes include, but are not limited to: t4 DNA ligase, T7 DNA ligase or mixtures thereof. The conditions under which the ligation reaction is carried out are well known to those skilled in the art.

In the context of the present invention, a "Y-linker" refers to a linker formed by two strands that are not completely complementary, one end of the linker forming a double strand due to the base complementarity of the two strands, and the other end not forming a double strand due to the incomplete complementarity between the bases of the two strands. The invention is applicable to common Y-type adaptors (True Seq adaptors), as shown in FIG. 4: schematic view of Y-shaped structure.

The common Y-type joint mainly comprises an amplification primer sequence (P5/P7), an index tag sequence, a read 1/read 2 sequencing primer sequence and an index read sequencing primer sequence, wherein the sequences of the read 1/read 2 sequencing primer sequence and the index read sequencing primer are not completely complementary to form a part of double strands.

For example, the Y-linker useful in the invention comprises the following sequences for both strands:

in which the portions of the two strands that are complementary to the bases are underlined.

Methods for phosphorylation modification of oligonucleotides are well known to those skilled in the art. For example, the 5 'end of the oligonucleotide may be phosphorylated by polynucleotide kinase, or a phosphate group may be added directly to the 5' end when the primer is synthesized.

In some embodiments, step (3) of the methods of the invention is performed in a liquid phase hybridization system.

Through the prior technical optimization of the laboratory, under the condition that a pre-library is prepared without PCR pre-amplification and a Y-shaped joint is adopted, the better capture efficiency can be realized without adding any closed sequence in a hybridization system, so that the pre-library amplification library building process before hybridization is omitted, the analysis performance is optimized, and the experiment cost is reduced.

Thus, in some embodiments, the system for hybridization includes a hybridization buffer, Cot-1DNA, and a hybridization probe, but does not include a blocking sequence. The conditions for hybridization, such as hybridization temperature, hybridization time, etc., can be adjusted by those skilled in the art according to actual needs. The general principles of designing and preparing hybridization probes are also well known to those skilled in the art.

In some embodiments, step (4) of the method of the invention further comprises a step of eluting the hybridization products to obtain a final library, in particular, said step (4) comprises:

step (4 a): performing alkali denaturation on the hybridization product;

step (4 b): incubation;

step (4 c): removing the magnetic beads; and

step (4 d): and (4) neutralizing.

In a preferred embodiment, the alkaline denaturing agent and its concentration that can be used during the alkaline denaturation process are well known and selected by those skilled in the art. Preferably, for example, the agent used for alkali denaturation is an inorganic base or an organic base. Further, the inorganic base is selected from one or more of NaOH, KOH, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, lithium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate; the organic base is selected from one or more of sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide or potassium tert-butoxide.

In a further preferred embodiment, the alkali denaturing agent is selected from inorganic bases, preferably from NaOH or KOH; more preferably, it is an aqueous solution of the above-mentioned reagent. Further, the alkali denaturing agent concentration is in the range of 0.05M to 1M, preferably 0.1 to 0.5M, more preferably 0.2M.

In a preferred embodiment, in the alkali denaturation step, an alkali agent is added to adjust the pH of the system so as to denature the hybridization product. The pH adjustment range of the system is well known to those skilled in the art. For example, preferably, the pH of the system can be adjusted to a value of 11 to 14, more preferably 12 to 14, and most preferably 12 to 13.

In the context of the present invention, the term "pH" includes the conventional usage of the term, such as the logarithm of the reciprocal hydrogen ion concentration. In this context, "pH" also includes the observation of pH, i.e. the pH measured by contacting the medium with a conventional acid-base meter of known type, suitably calibrated by known methods. Typically, the medium used in the measurement method of the present invention will contain some water. As used herein, the "pH" of the solution generally refers to, for example, the pH at room temperature (25 ℃).

In some embodiments, the addition of the alkaline denaturing agent further comprises the following process: incubating the reaction system; after the incubation is finished, adsorption is carried out to remove magnetic beads, and finally, a neutralizing agent is added to reduce the alkalinity of the system. Preferably, for example, the incubation is performed at room temperature. The skilled person will be able to select any suitable reagent for use in order to neutralize the above-mentioned alkaline denaturing agents and determine the concentration range thereof, for example Tris-HCl, acetic acid, citrate buffer, phosphate buffer or acetate buffer, etc.

In a preferred embodiment, the neutralizing agent is selected from Tris-HCl; the Tris-HCl concentration is preferably in the range of 100nM to 10mM, more preferably 100nM to 500nM, most preferably 400 nM.

Reagent kit

In a second aspect, the present invention also provides a kit for constructing a capture library, comprising:

(1) reagents for attaching linkers, including Y-linkers;

(2) reagents for hybridization; and

(3) reagents for elution of hybridization products.

In some embodiments, wherein the Y-linker is a long Y-linker; the long Y-type joint comprises an amplification primer sequence, an index label sequence, a read 1/read 2 sequencing primer sequence and an index read sequencing primer sequence.

In some embodiments, agents for performing end repair and/or end-plus-a are also included.

In some embodiments, wherein the reagents for hybridization comprise a hybridization buffer, Cot-1DNA, and a hybridization probe; preferably, the reagents for hybridization do not include blocking sequences. Wherein the blocking sequence comprises a sequence designed to be reverse complementary to the linker and/or tag sequence.

In some embodiments, wherein the reagents for elution of hybridization products comprise a denaturing agent and a neutralizing agent. Preferably, the denaturing agent is an alkaline denaturing agent.

In a preferred embodiment, the alkali denaturing agent is selected from one or more of the group consisting of: NaOH, KOH, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, lithium carbonate, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, ammonium hydrogen carbonate, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide or potassium tert-butoxide. The neutralizing agent is selected from one or more of the following: Tris-HCl, acetic acid, citrate buffer, phosphate buffer or acetate buffer.

In a further preferred embodiment, the alkaline denaturing agent is selected from NaOH or KOH, more preferably an aqueous solution thereof, and the neutralizing agent is selected from Tris-HCl. Specifically, the concentration of the NaOH or KOH aqueous solution is 0.1-0.5M, more preferably 0.2M; the Tris-HCl concentration is preferably 100nM to 500nM, more preferably 400 nM.

In some embodiments, the capture libraries made according to the methods of the invention, or the capture libraries made by the kits of the invention, are suitable for use in a variety of second generation sequencing platforms, including, but not limited to, e.g., Roche/454 FLX, Illumina/Hiseq, Miseq, NextSeq, and Life Technologies/SOLID systems, PGM, Proton, and the like.

Examples

Example 1: constructing a library by using different template input quantities, performing machine sequencing, performing data analysis, and comparing sequencing data quality control and performance analysis results of different input quantities.

This example describes the construction of a capture library with an initial DNA input of 25, 50, 75, 100, 150, 200ng, respectively, using the genomic DNA of the standard cell line GM24385 as a template according to the method of the invention. The library was sequenced on the NextSeq CN500 sequencing platform (150 bp paired-end sequencing). And analyzing sequencing results by utilizing bioinformatics, and analyzing library quality of different template input quantities.

Step 1: obtaining fragmented DNA, end repair and end-plus A

The reaction system shown in Table 1 was prepared using the 5 XWGS Fragmentation Mix kit (enzymes, cat. Y9410L) and a complementary 10 Xfragmentation Buffer kit (enzymes, B0330) according to the manufacturer's instructions to accomplish Fragmentation, end-point repair and end-point addition of A in one step.

The reaction system was subjected to the following reaction according to the procedure of Table 2 below.

Step 2: connecting joint

The library adaptors constructed in the present invention were ordinary Y-adaptors (True Seq adaptors), and the ligation systems shown in Table 3 were prepared using the reaction product of step 1 using WGS Ligase kit (enzymics, cat # L6030-600000), and incubated at 20 ℃ for 15 minutes and then maintained at 4 ℃.

After the ligation reaction was completed, the ligation product was purified using a Beckman Agencour AMPure XP kit (Beckman, cat # A63882).

And step 3: capture hybridization

Using the XGen Lockdown Reagents kit (IDT, cat # 1072281), and Streptavidin Dynbeads M270 magnetic beads (Thermo Fisher Scientific, cat # 35302), 14.5. mu.l of hybridization reagent (9.5. mu.l of XGen 2 Xhybridization buffer, 3. mu.l of XGen hybridization buffer enhancer, and 2. mu.l of Cot-1 DNA) was added to the purified product of step 2, mixed well, and incubated at room temperature for 10 minutes. After incubation, 12.75. mu.l of the supernatant was taken to a new low-sorption 0.2mL centrifuge tube, and then 4.25. mu.l of hybridization probe was added. After incubation, the cells were mixed well and then transiently detached, and capture hybridization was performed according to the procedure described in Table 4 below.

After hybridization, the hybridization products (i.e., magnetic beads bound to the target sequence) were washed and purified using the xGen Lockdown Reagents kit (IDT, cat # 1072281) according to the manufacturer's instructions.

And 4, step 4: elution of the fragment of interest

To the purified hybridization product of step 3 (with beads) were added 3.5. mu.L NaOH solution (0.2M) and 15. mu.L enzyme-free water, mixed well and then incubated at room temperature for 10 minutes, during which mixing was vortexed alternately. After the incubation is finished, placing the mixture on a magnetic frame for adsorption for 5 minutes, taking 18 mu L of supernatant into a low-adsorption 0.2mL centrifuge tube added with 4 mu L of Tris-HCl (400 nM), and carrying out vortex mixing to obtain a final capture library.

The captured library was qPCR quantified and then sequenced using the NextSeq CN500 sequencing platform (150 bp paired end sequencing) according to the standard sequencer protocol, giving 2.5G data for each sample. The sequencing results, including the basic control and SNV & INDEL performance analysis of the standard cell line, are shown in Table 5.

As can be seen from the above table, the capture library constructed according to the method of the present invention has no significant difference in the basic quality control and performance analysis except for the qPCR concentration in the range of 25ng-200ng DNA template input under the same data amount. This shows that samples with starting DNA contents as low as 25ng can be used according to the method of the invention and that the prepared capture library fully satisfies the requirements of on-machine sequencing and subsequent data analysis. "homogeneity" in the table, i.e., IQR (interquartile range), refers herein to the difference in sequence coverage between 25% and 75% of the density depth distribution. This value is a measure of the variability of the results, reflecting the heterogeneity of the coverage of the entire data set. High IQR indicates high variability in genome coverage, while low IQR reflects more uniform sequence coverage.

Comparative example 1: after completion of step 3 in the protocol of example 1, a PCR amplification step (12 cycles, see Table 7 for details) was used instead of step (4) in example 1, and different template inputs were used to construct the library, which was subjected to sequencing, data analysis, comparison of the quality of the sequencing data and the performance analysis results for the different inputs (50 ng, 75ng and 100ng, respectively) and comparison of the above results with the analysis results for the same template inputs in example 1.

In this control example, a final library was prepared by PCR amplification of purified hybrid products (with beads) using genomic DNA of the standard cell line GM24385 as a template after step 3 in the procedure of example 1, and capture libraries were constructed with an initial DNA input of 50ng, 75ng, and 100ng, respectively. The library was sequenced on the nextsseq CN500 sequencing platform (150 bp paired end sequencing). And analyzing sequencing results by utilizing bioinformatics, and analyzing library quality of different template input quantities.

Specifically, using the 2 xKAPA HiFi Hot Start Ready Mix kit (KAPA, cat # KK 2602), amplification systems as shown in Table 6 were prepared according to the manufacturer's instructions, with the following sequences of the preamplification primers:

and PCR was performed according to the procedure of Table 7 below.

After the PCR procedure was completed, the final capture library was obtained by purification using Beckman Agencour AMPure XP kit (Beckman, cat # A63882).

The capture library prepared in control example 1 was qPCR quantified and then the library was sequenced using the NextSeq CN500 sequencing platform (150 bp paired-end sequencing) according to the standard sequencer protocol, giving 2.5G data per sample. The sequencing results are shown in table 8.

As can be seen from the above table, the data fluctuation of the library constructed by the present invention (PCR-Free in Table 8) and the data fluctuation of the introduced PCR amplification library at different template amounts are not obvious under the same data amount. The PCR-Free library was significantly lower in repetition rate than the procedure incorporating PCR (1.73% on average for template at 50/75/100ng and 4.35% on average for PCR incorporation), so that the data on the computer could be used effectively. The library constructed by the invention has improved detection performance of variation, and especially has obviously improved detection performance of INDEL (the mean values of sensitivity and accuracy under the template amounts of 50, 75 and 100ng are 93.37 percent and 97.91 percent respectively, and the mean values of sensitivity and accuracy under the template amounts of 50, 75 and 100ng are 81.57 percent and 90.63 percent respectively when the library constructed by introducing the PCR process is introduced).

Comparative example 2: after completion of step 3 in the flow of example 1, the target fragment was eluted using a saturated solution of free biotin (also called D-biotin), and instead of step (4) in example 1, a library was constructed using a template input of 100ng, and then qPCR concentration quantification was performed, and the above results were compared with the library concentration results corresponding to the same template input in example 1.

In the control example, standard cell line GM24385 genomic DNA is used as a template, and after step 3 in the flow of example 1 is finished, a saturated solution of free avidin is added to the purified hybridization product (with beads) and the mixture is heated and incubated, so that the final library is eluted.

Specifically, dissolving free biotin (ThermoFisher, Cat. No. B1595) in non-enzyme water to prepare a saturated solution (about 0.2 mg/mL), adding 22.5 mu L of the saturated solution of free biotin into the purified hybridization product (with beads) obtained in the step 3, incubating at 37 ℃ for 30 minutes after fully and uniformly mixing, placing on a magnetic frame for adsorption for 5 minutes after incubation is finished, taking 22 mu L of supernatant into a 0.2mL low-adsorption centrifuge tube, and performing vortex mixing to obtain the final capture library.

The capture library prepared in control example 2 was subjected to qPCR quantification and the library concentrations are shown in table 9.

As can be seen from the above table, at the same input amount of template DNA, the concentration of the library obtained by using the free biotin substitution method is too low to meet the requirement of the on-machine method.

Comparative example 3: after completion of step 3 in the flow of example 1, strand displacement reaction of the target fragment was performed using Deep Vent DNA polymerase, instead of step (4) in example 1, a library was constructed using a template input of 100ng, and then qPCR concentration quantification was performed, and the above results were compared with the library concentration results in example 1 corresponding to the same template input.

In this control example, a final library was prepared by strand displacement reaction of purified hybridization products (with beads) after completion of step 3 in the procedure of example 1 using genomic DNA of the standard cell line GM24385 as a template.

Specifically, strand displacement systems as shown in Table 10 were prepared using Deep Vent DNA Polymerase (NEB, cat No. M0258S) according to the manufacturer's instructions, and the sequences of the strand displacement primers were as follows:

and the reaction was carried out according to the procedure of the following table 11.

After the PCR procedure was completed, the final capture library was obtained by purification using Beckman Agencour AMPure XP kit (Beckman, cat # A63882).

The capture library prepared in control example 3 was subjected to qPCR quantification and the library concentrations are shown in table 12.

As can be seen from the above table, at the same input amount of template DNA, the concentration of the library obtained by using the strand displacement method is about 31.66% of that of the alkali-denaturing method, the concentration of the library has large difference, and the requirement of multiple computer-installing is not met.

Comparative example 4: after completion of step 3 in the flow of example 1, the target fragment was eluted at different incubation times (2 minutes, 5 minutes) by using a high temperature heat denaturation method, and instead of step (4) in example 1, a library was constructed using a template input amount of 100ng, and then qPCR concentration quantification was performed, and the results were compared with the library concentration results corresponding to the same template input amount in example 1.

In this control example, standard cell line GM24385 genomic DNA was used as a template, and after step 3 in the procedure of example 1 was completed, 22.5. mu.l of enzyme-free water was added to the purified hybridization product (with beads), and after carrying out DNA double strand helicity denaturation at high temperature, magnetic beads were rapidly removed, thereby finally achieving the purpose of final library elution.

Specifically, 22.5 μ L of enzyme-free water is added into the purified hybridization product (with beads) in step 3, the mixture is fully mixed, incubated at 98 ℃ for 2 minutes and 5 minutes respectively, after the incubation is finished, the mixture is placed on a magnetic frame to be adsorbed for 1 minute, 22 μ L of supernatant is taken and put into a 0.2mL low-adsorption centrifuge tube, and the final capture library is obtained by vortex mixing.

The capture library prepared in control example 4 was subjected to qPCR quantification and the library concentrations are shown in table 13.

As can be seen from the above table, at the same template DNA input amount, the library obtained by using the thermal denaturation method has too low concentration and does not meet the on-machine requirement.

Example 2: performing on-machine sequencing on the library constructed by adopting the initial DNA input amount of 100ng in the example 1 and the library constructed by introducing the same template DNA input amount of PCR amplification in the comparison example 1, respectively intercepting the data amounts of 2, 2.5, 3, 3.5, 4, 4.5 and 5G, performing data analysis, and comparing the sequencing data quality control and performance analysis results of two different library construction processes at different sequencing depths. The results of the analyses are shown in tables 14(a) and 14(b), respectively.

As can be seen from the above two tables, as the amount of sequencing data increases, the sequencing depth of the sample will change greatly, and the change in the sequencing depth can optimize the detection performance of SNP and INDEL to some extent. Comparing the data in the two tables can further show that the invention has great advantages for INDEL detection performance, and the flow of the invention can obtain more stable detection performance under the data volume of 2.5G.

Example 3: this example uses the genomic DNA of the standard cell line GM24385 as a template to construct a capture library with an initial DNA input of 100 ng. The library construction method of this example is the same as that of example 1, except that the room temperature incubation time for eluting the target fragment of step 4 is 5 minutes, 10 minutes and 15 minutes, respectively. The library was sequenced on the NextSeq CN500 sequencing platform (150 bp paired-end sequencing). And analyzing sequencing results by utilizing bioinformatics, and analyzing library quality at different incubation times. The sequencing results are shown in table 15.

As can be seen from the above table, the concentration of the library increases with the increase of the incubation time, and the library obtained under 3 incubation conditions all meets the requirement of 3 times of operation. Under the same data volume, the basic quality control of the library obtained under 3 incubation conditions and the detection performance of SNP/INDEL are not greatly different, the library comparison rate and the key gene 20 multiplied coverage are higher under the incubation time condition of 10min, and the experimental operation time is more reasonable, so the invention recommends the flow of incubation time at room temperature of 10 minutes.

Example 4: the library construction method of this example is the same as that of example 1 (constructing a capture library with an initial DNA input of 100 ng), except that the DNA template is changed from the original standard cell line to a clinically positive sample. The capture libraries were prepared with buccal swab DNA, amniotic fluid DNA, dried blood slice DNA, tissue DNA, peripheral blood DNA, respectively. The captured library was qPCR quantified and then sequenced using the NextSeq CN500 sequencing platform (150 bp paired end sequencing) according to the standard sequencer protocol, giving 2.5G data for each sample. The sequencing results are shown in table 16.

The basic quality control of the library of 5 clinical positive samples adopted in the table reaches the standard and is correctly detected. Therefore, the method for constructing the sequencing library can be applied to various sample types, especially samples with less DNA content.

Example 5: study on the stability of the qPCR concentration of the library

Libraries were constructed according to the method described in example 1 (constructing a capture library with an initial DNA input of 100 ng) using six standard cell line genomic DNA as templates (GM 24385, GM24694, GM12878, GM24631, GM24143, and GM 24695) and the library qpCR concentration determinations were performed at intervals of 0, 20, and 40 days (the library was stored at-20 ℃). The effect of storage time on the qPCR concentration of the library was explored. The results of concentration measurement are shown in table 17 and fig. 6.

As can be seen from Table 17 and FIG. 6, the library constructed by the process of the present invention has no obvious change in the concentration and good stability when stored at-20 ℃ for 40 days.

Example 6: library recombinant detection stability exploration

Six standard cell line genomic DNA libraries constructed in example 5 were used to sequence the libraries using the NextSeq CN500 sequencing platform (150 bp paired end sequencing) according to the standard protocol of the sequencer, with 2.5G data per sample. And reloaded (same data size) after 25 days of storage at-20 ℃ to investigate the effect of storage time on library stability. The sequencing results are shown in table 18.

As can be seen from the above table, the quality of on-machine sequencing of the library constructed by the process of the present invention has no obvious change when the library is stored at-20 ℃ for 25 days. In example 5, it can be seen that the library constructed by the process of the present invention is stable and can be stored for a long time.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention, and those skilled in the art will appreciate that various modifications and changes can be made to the present invention. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.