CN116162690B

CN116162690B - One-tube targeting high-throughput sequencing method

Info

Publication number: CN116162690B
Application number: CN202211486785.9A
Authority: CN
Inventors: 陈文浩; 姚丹丹; 杨鑫斌; 童亮
Original assignee: Boke Biotechnology Co ltd
Current assignee: Boke Biotechnology Co ltd
Priority date: 2022-11-24
Filing date: 2022-11-24
Publication date: 2023-12-26
Anticipated expiration: 2042-11-24
Also published as: CN116162690A

Abstract

The invention discloses a tubular targeting high-throughput sequencing method, which utilizes novel hybridization buffer solution and cleaning buffer solution, develops a tubular capturing method, ensures that the whole flow including pre-library construction and hybridization capturing does not need to replace a reaction tube, reduces the types and the cleaning times of the cleaning buffer solution, has excellent capturing data performance, and is a high-throughput gene targeting sequencing method suitable for automatically carrying out large-scale sample detection.

Description

One-tube targeting high-throughput sequencing method

Technical Field

The invention relates to a gene targeting sequencing method, in particular to a simpler and convenient gene targeting high-throughput sequencing method based on hybrid capture.

Background

The targeted sequencing method based on hybrid capture (targeted capture) has the advantages of high accuracy, economy, high flux and the like, and is suitable for detecting different types of genetic variation, such as Single Nucleotide Variation (SNV), copy Number Variation (CNV) and the like. Currently, targeted capture technology has been widely used for diagnosis of genetic diseases, tumors, and complex diseases.

The whole process of the targeted capture technology is complex [ Giannoukos G, brockman W, lepro E M, et al solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing ], and the whole process is divided into two links of pre-library construction and hybrid capture. The pre-library construction comprises the steps of repairing, adding dA at the tail end, connecting a joint and enriching by PCR, and obtaining a pre-library after purification; and then entering a hybridization capture link, wherein the hybridization capture includes the steps of pre-blocking (pre-library, cot-1DNA, and linker blocking reagent are mixed and then evaporated to dryness), hybridization of the probe and the pre-library, capture (streptavidin magnetic bead capture), heat cleaning, room temperature cleaning and PCR enrichment.

Although the prior research has simplified part of pre-library construction links, the product still needs to be transferred to a new reaction tube before the PCR enrichment step, and the reaction tube is replaced again to carry out hybridization capture links after the PCR enrichment is finished; in addition, in the cleaning step of the hybridization link, a plurality of reagents are used, the cleaning steps are more, and the operation is complicated. Thus, current targeted capture procedures are not conducive to large-scale sample detection and development of automated procedures.

The information in the background section is only for the purpose of illustrating the general background of the invention and is not to be construed as an admission or any form of suggestion that such information forms the prior art that is well known to those of ordinary skill in the art.

Disclosure of Invention

The present invention aims to simplify and shorten methods for genome-targeted high throughput sequencing. More specifically, the invention aims to complete all experimental steps including pre-library construction and hybridization capture in one reaction tube (one tube) by optimizing reagent components, and no tube rotation operation is needed in the middle; at the same time, the method has the advantages of simplicity, high efficiency and simultaneously ensuring the capturing performance, along with fewer reagent types and operation steps. Specifically, the present invention includes the following.

The invention provides a tubular targeted high-throughput sequencing method, in particular to a genome targeted sequencing method, which comprises the following steps:

(1) Constructing a pre-library, namely fragmenting a sample to be detected, repairing, adding dA at the tail end, connecting a sequencing joint, adding purified magnetic beads to purify a connecting product, and then adding an adsorption solution to purify the product to obtain the pre-library combined with the magnetic beads;

(2) Hybridization capture, which includes hybridizing a pre-library with capture probes in hybridization solution, then utilizing separation magnetic bead capture probes-target fragment complexes, then using a cleaning buffer solution for cleaning, and obtaining a sequencing library after PCR enrichment and purification;

(3) High throughput sequencing, which comprises sequencing the sequencing library to obtain captured data.

In certain embodiments, a tube-type targeted high-throughput sequencing method according to the present invention, wherein the purified magnetic beads are carboxyl-functional modified magnetic beads.

In certain embodiments, a tube-type targeted high-throughput sequencing method according to the present invention, wherein the isolated magnetic beads comprise streptavidin magnetic beads.

In certain embodiments, a tube-type targeted high throughput sequencing method according to the present invention, wherein the sample to be detected in step (1) comprises DNA, in particular genomic DNA, comprising fragmenting the sample to be detected into 100-300bp, preferably 150-250bp, such as 200bp, then repairing and adding dA at the end, ligating sequencing adaptors, adding purified magnetic bead purified ligation products, then performing Index PCR enrichment in the original reaction tube and adding sample tags.

In certain embodiments, a tube-type targeted high throughput sequencing method according to the present invention, wherein the sample to be tested in step (1) comprises RNA, such as total RNA, comprising reverse transcription of the RNA to cDNA, followed by fragmentation into fragments of 100-300bp, preferably 150-250bp, such as 200bp, followed by repair and addition of dA tails at the ends, ligation of sequencing adaptors, and Index PCR enrichment and addition of sample tags.

In certain embodiments, a tube-type targeted high throughput sequencing method according to the present invention, wherein the ligation product purified in step (1) is in the form of a nucleic acid magnetic bead complex, and Index PCR enrichment is performed in a magnetic bead-containing system.

In certain embodiments, a tube-type targeted high-throughput sequencing method according to the present invention, wherein the adsorption solution comprises PEG and NaCl, and the weight-volume concentration of PEG is 10-30%, such as 12%, 15%, 18%, 20%, 24%, 26%, etc., and the concentration of NaCl is 1-5M, such as 2M, 3M, 4M. The ion bridge of DNA-salt ion-carboxyl is formed in PEG/high-salt ion buffer system, so that DNA can be reversibly adsorbed. In an exemplary embodiment, the adsorption solution comprises 20% (w/v) PEG and 2.5M NaCl.

In certain embodiments, a tube-type targeted high-throughput sequencing method according to the present invention, wherein the hybridization solution further comprises a blocking DNA, a linker blocking reagent, and a hybridization buffer. The pre-library is in the form of a pre-library/magnetic bead complex prior to hybridization. Blocking DNA refers to DNA that has the function of blocking the genomic repeat sequence, examples of which include, but are not limited to, cot-1DNA and/or salmon sperm DNA. The linker blocking reagent may be an Index one-to-one targeted oligonucleotide or a universal blocking oligonucleotide compatible with different indices.

In certain embodiments, the capture probe is a 5' end modified 120nt DNA probe. In some aspects, the modification of the probe may be a 3' modification. In some aspects, the probe may have a length of 60-150nt,90-130nt, preferably 120nt. In some aspects the probe may also be an RNA probe, the modification of which may be random base modification.

In certain embodiments, a tube-type targeted high throughput sequencing method according to the present invention, wherein the hybridization buffer comprises 0.1-0.8M MOPS (3-morpholinopropanesulfonic acid), 0.5-1.5M Trehalose (Trehalose), 0.5-1.5M tetramethyl ammonium chloride (TMACL), 10-30% (v/v) Formamide (Formamide). Exemplary hybridization buffers are composed of 0.5M MOPS, 1.0M Trehalose (Trehalose), 1.0M TMACL, 20% (v/v) Formamide (Formamide). In some aspects, MOPS is a buffer with better buffering capacity, examples of which include, but are not limited to, HEPES, MES, and the like. In some aspects, trehalose is used to reduce the annealing temperature of high GC target regions, including but not limited to Betaine, DMSO, and the like. In some aspects, TMACl is used to increase the annealing temperature of the low GC target region, including but not limited to tea cl, TPACl, and the like. In some aspects, the concentration of the different components may be a range interval.

In certain embodiments, a tube-type targeted high-throughput sequencing method according to the present invention, wherein the wash buffer comprises a hot wash buffer and a room temperature wash buffer; wherein the hot wash buffer composition comprises 0.05-1.15M MOPS, 0.05-0.15% Tween-20, 0.05-0.15% SDS, pH6.3, and the room temperature wash buffer composition comprises 0.05-0.15M MOPS, pH5.8. Illustratively, the hot wash buffer composition is 0.1M MOPS, 0.1% Tween-20, 0.1% SDS pH6.3. The room temperature wash buffer composition was 0.1M MOPS pH5.8.

The invention develops a One-Tube (One-Tube) capturing method by utilizing the novel hybridization buffer solution and the cleaning buffer solution, the whole process (pre-library construction and hybridization capturing) does not need to replace a reaction Tube, meanwhile, the cleaning buffer solution is reduced from 4 types to 2 types, the cleaning step is reduced from 6 times to 4 times, and the captured data is excellent in performance, so that the method is a high-throughput gene targeting sequencing method suitable for automatically carrying out large-scale sample detection.

Drawings

FIG. 1 is a schematic flow diagram of an exemplary targeted capture method and a conventional method of the present invention.

FIG. 2 is a comparison of the exemplary method of the present invention with conventional methods in experimental procedures and hybridization and wash buffer compositions.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

As used herein, the term "relative depth of coverage" refers to a relative depth of coverage equal to the depth of coverage of a gene divided by the average depth of coverage of all genes. For example, the average value of the coverage Depth of all genes is 100x, the coverage Depth of a certain site is 100x, the relative coverage Depth is equal to 1 time of the average Depth (1 x Mean Depth), and if the coverage Depth of a certain site is 20x, the relative coverage Depth is equal to 0.2 time of the average Depth (0.2 x Mean Depth).

As used herein, the term ". Gtoreq.0.2 x Mean Depth ratio" refers to the ratio of the number of sites of 0.2 times the average Depth to the total number of sites. For evaluating coverage uniformity in captured data.

As used herein, the term ". Gtoreq.0.5 x Mean Depth ratio" refers to the number of sites of 0.5 times the average Depth, in proportion to all sites. For evaluating coverage uniformity in captured data.

Example 1

1. Genomic DNA fragmentation:

human genomic DNA (NA 12878, coriell) was sonicated to about 200bp using an M220 sonicator (Covaris).

2.1 DNA repair and end addition of dA

As shown in Table 1, end prepmix 4 (supplier: northenzan; cat. ND 607-02) was added to the sonicated product, vortexed, and briefly centrifuged. The prepared reaction solution was put into a PCR instrument and run according to the procedure shown in Table 2 (hot cap 75 ℃).

TABLE 1

Component (A)	Volume (mu L)
		Ultrasonic products of human genomic DNA	50
End Prep Mix4	15
		Total volume of	65

TABLE 2

2.2 Joint connection

After the reaction was completed, the reagents shown in Table 3 (supplier: northenzan; cat. No. ND 607-02) were added to the product of the previous step, and after vortexing and mixing, the mixture was centrifuged briefly and incubated at 20℃for 30 minutes.

TABLE 3 Table 3

Component (A)	Volume (mu L)
		The product of the last step	65
illuminea linker Vazyme-S	2.5
		Rapid ligation buffer2	25
Rapid DNA ligase	5
		Asepsis water without enzyme	2.5
Total volume of	100

2.3 purification of the linker ligation products

After the reaction, 90. Mu. L VAHTS DNA clear Beads (supplier: northenzan; product number: N411-03) was added to the reaction mixture, and after thoroughly vortexing and mixing, incubated at room temperature for 5 minutes, briefly centrifuged and magnetically adsorbed for 5 minutes, the supernatant was removed by pipetting, and after incubation with 180. Mu.L of 80% ethanol for 30 seconds, the supernatant was removed by pipetting. The reaction tube was centrifuged briefly and placed on a magnetic rack, the residual liquid was removed by suction, and dried at room temperature for 5 minutes.

The traditional method comprises the following steps: add 22. Mu.L of Low-TE buffer, vortex mix, stand at room temperature for 2 minutes, centrifuge briefly, place on a magnetic rack, adsorb for 2 minutes, transfer 20. Mu.L of supernatant to a new PCR tube.

The process of the invention comprises the following steps: add 20. Mu.L of Low-TE buffer, vortex mix well, stand for 2 minutes at room temperature for use.

2.4Index PCR amplification

As shown in Table 4, 25ul VAHTS HiFi amplification mix and 5ul Index Primer (supplier: northenzan; cat# N411-03) were added to 20. Mu.L of the ligation product, and PCR amplification was performed according to the procedure of Table 5.

TABLE 4 Table 4

TABLE 5

2.5 Pre-library purification

Pre-library purification and transfer were performed as shown in Table 6.

The traditional method comprises the following steps: to the reaction solution, 45. Mu. L VAHTS DNA clear Beads (supplier: northenan; cat# N411-03) were added for purification. After the magnetic beads are washed and dried, 42 mu L of sterile water without enzyme is added, vortex mixing is carried out, standing is carried out at room temperature for 2 minutes, after short centrifugation, the mixture is placed on a magnetic rack for 2 minutes of absorption, and 40 mu L of supernatant is transferred to a new centrifuge tube.

The process of the invention comprises the following steps: 45. Mu.L of 20% PEG and 2.5M NaCl buffer are added, after thoroughly vortex mixing, incubation is carried out for 5 minutes at room temperature, centrifugation is carried out briefly, then magnetic force is applied for 5 minutes, the supernatant is sucked and removed, 180. Mu.L of 80% ethanol is added for 30 seconds, the supernatant is sucked and removed, and 180. Mu.L of 80% ethanol is added for 30 seconds, and the supernatant is sucked and removed. The reaction tube is centrifuged briefly and placed on a magnetic rack, residual liquid is sucked and removed, and the reaction tube is dried at room temperature for 5 minutes for later use.

TABLE 6

Reagent(s)	Flow of traditional method	The flow of the invention
			Pre-library purification reagents	45μL VAHTS DNA Clean Beads	45μL 20％PEG,2.5M NaCl
Elution (non-enzyme sterile water)	40μL	Not to be eluted separately
			Transfer of	Transfer to a fresh centrifuge tube	No transfer

3. Pre-library and Gene Panel hybridization capture:

3.1 library Pre-blocking

The traditional method comprises the following steps: the reagents as in Table 7 were added to the centrifuge tube containing the library (step 2.5) and the solution was evaporated to dryness in the centrifuge tube using a vacuum concentrator (Eppendorf) for further use.

The invention comprises the following steps: mu.L of Cot-1DNA (primary, TC 0016) and 2. Mu. L Universal Blockers-ILMN-TS (Du) (primary, TC0010D 9) were added to resuspend the beads and the next step was performed.

TABLE 7

3.2 hybridization of probes to libraries

After addition of the relevant reagents, vortex mixing and incubation for 5 minutes at room temperature was performed as shown in table 8. Subsequently, the mixture was denatured at 95℃for 10 minutes on a PCR apparatus, followed by adding 4. Mu.L of 650Kb Gene Panel (3 pmol 120nt probe Pool, burseraceae), vortexing, and incubating at 65℃for 16 hours.

TABLE 8

3.3 streptavidin magnetic bead preparation

Streptavidin beads (supplier: bovidae; cat. No. TC 00322.1) were removed from the refrigerator (4 ℃) and brought to room temperature (about 30 minutes). Vortex and mix for 15 seconds. 50. Mu.L of streptavidin magnetic beads were added to a new 1.5mL low adsorption centrifuge tube. The centrifuge tube was placed on a magnetic rack until the solution was clear.

The supernatant was aspirated and the beads were not disturbed. The streptavidin magnetic beads were washed as follows:

(1) The centrifuge tube was removed from the magnet holder, 100. Mu.L of 1X Beads Wash Buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA, 0.1% (v/v) Tween-20) was added, and vortexed for 10 seconds.

(2) The centrifuge tube was centrifuged instantaneously and placed on a magnetic rack until the solution was clear, the supernatant was aspirated and the beads were not disturbed.

Repeating the steps (1) and (2).

The centrifuge tube is removed from the magnet rack, hybridization buffer (10. Mu.L) corresponding to the hybridization step is added, for example, if "conventional hybridization buffer" is used for hybridization, then "conventional hybridization buffer" is added, and then the mixture is blown and sucked for later use.

3.4 streptavidin magnetic bead Capture

Blowing and sucking the mixed streptavidin magnetic beads, and transferring the mixed streptavidin magnetic beads into a hybridization tube (step 3.2). The mixture was gently sucked 10 times with a pipette and mixed. The incubation was carried out for 45 minutes at 65℃using a PCR apparatus (the temperature of the hot cap was set at 75 ℃). Vortex mixing was performed for 3 seconds every 12 minutes to ensure that the beads were in suspension.

3.5 post capture cleaning

As shown in Table 9, the conventional washing buffer contains 4 reagents, and requires 6 steps of washing; the cleaning buffer solution contains 2 reagents and needs 4 steps of cleaning. The detailed reagent formulations are shown in table 10.

TABLE 9

Table 10

3.5.1 washing with conventional washing buffer

A heat cleaning step at 1.65 ℃):

mu.L of preheated Wash Buffer I was added to the centrifuge tube containing the hybridization mixture. After blowing, sucking and mixing evenly, placing the centrifuge tube on a magnetic rack until the solution is clear, and sucking and discarding the supernatant.

The cleaning is carried out continuously according to the following steps:

(1) 150. Mu.L of preheated Wash Buffer S was added, and after air-aspiration mixing, incubated at 65℃for 5 minutes. The tube was placed on a magnetic rack by instantaneous centrifugation until the solution was clear, and the supernatant was aspirated off.

(2) 150. Mu.L of preheated Wash Buffer S was added again, and after air-aspiration mixing, incubated at 65℃for 5 minutes. The tube was placed on a magnetic rack by instantaneous centrifugation until the solution was clear, and the supernatant was aspirated off.

2. Room temperature cleaning

150. Mu.L Wash Buffer I was added and vortexed for 2 minutes. The centrifuge tube was centrifuged briefly and placed on a magnetic rack until the solution was clear, and the supernatant was aspirated off. 150. Mu. LWAsh Buffer II was added and vortexed for 1 minute. The centrifuge tube was centrifuged briefly and placed on a magnetic rack until the solution was clear, and the supernatant was aspirated off. 150 mu L Wash Buffer III was added and vortexed for 30 seconds. The centrifuge tube was centrifuged briefly and placed on a magnetic rack until the solution was clear, and the supernatant was aspirated off.

3. Magnetic bead resuspension

Immediately 20. Mu.L of sterile water was added. And (4) blowing and sucking for 10 times by using a pipette, re-suspending the magnetic beads, and entering the step (4).

3.5.2 washing with the washing buffer according to the invention

A heat cleaning step at 1.65 ℃):

(1) 150. Mu.L of preheated Wash Buffer H was added, and after air-aspiration mixing, incubated at 65℃for 5 minutes. The tube was placed on a magnetic rack by instantaneous centrifugation until the solution was clear, and the supernatant was aspirated off.

(2) 150. Mu.L of preheated Wash Buffer H was added again, and after air-aspiration mixing, incubated at 65℃for 5 minutes. The tube was placed on a magnetic rack by instantaneous centrifugation until the solution was clear, and the supernatant was aspirated off.

2. Room temperature cleaning

(1) 150. Mu.L of preheated Wash Buffer R was added and vortexed for 2 minutes. The tube was placed on a magnetic rack by instantaneous centrifugation until the solution was clear, and the supernatant was aspirated off.

(2) 150. Mu.L of preheated Wash Buffer R was added again and vortexed for 2 minutes. The tube was placed on a magnetic rack by instantaneous centrifugation until the solution was clear, and the supernatant was aspirated off.

PCR amplification

The PCR reaction system was prepared according to Table 11.

TABLE 11

Vortex mixing to keep the magnetic beads in suspension state, and immediately enter the PCR step. The PCR apparatus was run according to the procedure of Table 12, with a hot lid temperature of 105 ℃.

Table 12

PCR product purification

Each PCR tube was added with 75. Mu. L VAHTS DNA clear Beads (supplier: northenzan; cat# N411-03) and the capture library was purified. Elution was performed using 22. Mu.L Tris-HCl (10 mM, pH 8.5). Transfer 20 μl of eluate containing the capture library to a new 1.5mL low adsorption centrifuge tube (Eppendorf).

6. Capture library quality control

Library concentrations were measured using a Qubit fluorometer 3.0 (thermo fisher). Library fragment lengths were measured using Agilent 2100, with product major bands centered at 320bp, linker-free dimerization.

7. High throughput sequencing

The library after capture was sequenced in PE150 mode using an Illumina NovaSeq6000 sequencer.

8. Data analysis

And (3) removing the linker and the low-quality sequence by using Trimmomatic to obtain clean data, extracting reads of a 650Kb Gene Panel target region by using Samtools, and counting the targeting rate and coverage uniformity of the target Gene.

The middle target rate refers to the proportion of the base data of the target area in the captured data to the total data, under the traditional flow, the middle target rate of the traditional hybridization buffer solution and the traditional cleaning buffer solution is 46.49-47.43%, under the flow of the invention (One-Tube), the middle target rate of the buffer solution and the cleaning buffer solution is 56.78-57.51%, and the invention is obviously superior to the traditional method. Under the process of the invention (One-Tube), the intermediate target rate of the traditional hybridization and washing buffer is 25.90-28.02%, which is obviously lower than the conditions of the hybridization buffer and the washing buffer of the invention, and the hybridization and washing buffer of the invention is proved to be more suitable for the One-Tube process. In addition, under the condition that the hybridization buffer solution of the invention is adopted in the process of the invention (One-Tube), the targeting rate of the washing buffer solution of the invention is equivalent to that of the traditional washing buffer solution (56.01-57.42%), and the working efficiency of the washing buffer solution of the invention is equivalent to that of the traditional washing buffer solution, but the types and the washing steps of the washing buffer solution of the invention are fewer. The coverage uniformity of the target area showed the same trend as the mid-target parameters (0.2 x Mean and 0.5x Mean) except for mid-target.

The results showed that (One-Tube) procedure of the present invention + hybridization buffer of the present invention + washing buffer of the present invention) = (One-Tube) procedure of the present invention + hybridization buffer of the present invention + conventional washing buffer) > (conventional procedure + conventional hybridization buffer + conventional washing buffer) > (One-Tube) procedure of the present invention + conventional hybridization buffer + conventional washing buffer).

Table 13 captured data representation

In summary, by optimizing hybridization and wash buffers and adjusting experimental procedures, the invention establishes a tubular targeted capture method that can be adapted to automated procedures. The method can complete the pre-library construction and hybridization capturing links in one reaction tube without transferring reaction liquid and replacing the reaction tube.

In addition, the experiment system is simpler and more convenient to operate, the cleaning buffer solution is composed of 2 reagents and only needs to be cleaned for 4 times, the traditional method is composed of 4 reagents and needs to be cleaned for 6 times, and the experiment flow is simpler and more convenient; therefore, the One-Tube targeted capturing method is more suitable for automatic processing and is high in throughput of large-scale samples.

While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. A method of one-tube targeted high throughput sequencing, comprising the steps of:

(1) The method comprises the steps of fragmenting a sample to be detected, repairing, adding dA at the tail end, connecting a sequencing joint, adding a purified magnetic bead to purify a connection product, then adding an adsorption solution to purify the product, wherein the purified connection product exists in the form of a nucleic acid magnetic bead complex, and carrying out Index PCR enrichment in a raw reaction tube containing a magnetic bead system to obtain a pre-library combined with the magnetic beads, wherein the purified magnetic beads in the step (1) are carboxyl functional group modified magnetic beads, the adsorption solution comprises PEG and NaCl, the weight volume concentration of the PEG is 10-30%, and the concentration of the NaCl is 1-5M;

(2) Hybridization capture, which comprises hybridizing a pre-library with capture probes in a hybridization solution, then capturing probe-target fragment complexes with separation magnetic beads, then washing with a washing buffer, enriching and purifying by post PCR, to obtain a sequencing library, wherein the hybridization solution further comprises a blocking DNA, a linker blocking reagent and a hybridization buffer, the hybridization buffer comprises 0.3-0.8M MOPS, 0.5-1.5M trehalose, 0.5-1.5M TMACl, 20% (v/v) formamide, and the washing buffer comprises a hot washing buffer and a room temperature washing buffer; wherein the hot wash buffer composition comprises 0.05-1.15M MOPS, 0.05-0.15% Tween-20, 0.05-0.15% SDS, pH6.3, and the room temperature wash buffer composition comprises 0.05-0.15M MOPS, pH5.8;

(3) High throughput sequencing, comprising sequencing the sequencing library to obtain captured data;

wherein enrichment from Index PCR to post PCR performed from the original reaction tube in which the ligation product was purified was completed in one reaction tube.

2. The method of claim 1, wherein the separation beads comprise streptavidin beads.

3. The method of claim 1, wherein the sample to be detected in step (1) comprises DNA, which comprises fragmenting the sample to be detected to 100-300bp, then repairing and adding dA at the end, ligating sequencing adaptors, adding purified magnetic beads to purify the ligation products, and then performing Index PCR enrichment and adding sample tags in the original reaction tube.

4. The method of claim 1, wherein the sample to be tested in step (1) comprises RNA, which comprises reverse transcription of the RNA to obtain cDNA, followed by fragmentation into fragments of 100-300bp, followed by repair and addition of dA tails at the ends, ligation of sequencing adaptors, and Index PCR enrichment and addition of sample tags.