阅读说明：本技术 一种目标区域捕获方法、试剂盒及测序方法 (Target area capturing method, kit and sequencing method ) 是由 黄标 刘晨 刘群 周荣芳 丁斐 黄金 田志坚 于 2020-05-15 设计创作，主要内容包括：本申请公开了一种目标区域捕获方法、试剂盒及测序方法。本申请的目标区域捕获方法,包括根据目标区域核酸序列设计向导核酸序列,将向导核酸序列与CAS9酶组合,采用向导核酸序列与CAS9酶的复合物对待处理核酸样品进行酶切消化,根据片段大小回收获得目标区域。本申请的目标区域捕获方法,流程短、操作简单,节省了交付周期和人力成本；并且,捕获效率高,增加了有效数据利用率,节约测序成本。本申请利用CRISPR/Cas9系统进行目标区域捕获,整个捕获过程无需采用PCR扩增,能够保留目标区域基因的甲基化等原始修饰信息。(The application discloses a target area capturing method, a kit and a sequencing method. The target region capturing method comprises the steps of designing a guide nucleic acid sequence according to a target region nucleic acid sequence, combining the guide nucleic acid sequence with a CAS9 enzyme, carrying out enzyme digestion on a nucleic acid sample to be processed by adopting a compound of the guide nucleic acid sequence and a CAS9 enzyme, and recovering and obtaining a target region according to the size of a fragment. The target area capturing method is short in flow, simple to operate and capable of saving delivery cycle and labor cost; in addition, the capture efficiency is high, the effective data utilization rate is increased, and the sequencing cost is saved. According to the method, the CRISPR/Cas9 system is used for capturing the target region, PCR amplification is not needed in the whole capturing process, and original modification information such as methylation of the target region gene can be reserved.)

1. A target area capturing method, characterized by: designing a guide nucleic acid sequence according to a target region nucleic acid sequence, combining the guide nucleic acid sequence with a CAS9 enzyme, carrying out enzyme digestion on a nucleic acid sample to be processed by adopting a compound of the guide nucleic acid sequence and a CAS9 enzyme, recovering a nucleic acid fragment in a product of the enzyme digestion and digestion, wherein the nucleic acid fragment is in accordance with the designed length of the target region nucleic acid sequence of the enzyme digestion and digestion, namely, capturing to obtain the target region.

2. The target area capturing method of claim 1, wherein: the guide nucleic acid sequence is formed by annealing and assembling a crRNA primer and a tracrRNA primer.

3. The target area capturing method of claim 1, wherein: magnetic bead purification is carried out on the digestion products of the enzyme digestion, then nucleic acid fragment screening is carried out, and a target area is captured;

preferably, the nucleic acid fragments in the digestion products are recovered, and particularly, the Blue Pippin automatic nucleic acid recovery system is adopted to sort the nucleic acid fragments in the target area.

4. A target area capture kit, characterized in that: comprises a crRNA primer designed aiming at a nucleic acid sequence of a target region and a universal tracrRNA primer.

5. The target area capture kit of claim 4, wherein: also comprises at least one of CAS9 enzyme, crRNA primer and tracrRNA primer annealing assembly reagent, guide nucleic acid sequence and CAS9 enzyme combination reagent, CAS9 enzyme digestion reagent and magnetic bead purification reagent.

6. A sequencing method, comprising: comprising performing target region capture using the target region capture method of any one of claims 1-3 or the target region capture kit of claim 4 or 5, the captured target region nucleic acid fragments being used directly in sequencing library construction and sequencing.

7. The sequencing method of claim 6, wherein: the sequencing library was constructed using Pacbio.

8. A method for detecting methylation of a target region, comprising: comprising sequencing the target region by the sequencing method of claim 6 or 7, and analyzing the methylation of the target region based on the sequencing result.

9. A human ATXN10 gene capturing kit is characterized in that: comprises ATXN10 gene specific crRNA primer and universal tracrRNA primer;

the ATXN10 gene-specific crRNA primer comprises an oligonucleotide fragment of a nucleic acid sequence shown in SEQ ID No.1 and an oligonucleotide fragment of a nucleic acid sequence shown in SEQ ID No. 2;

SEQ ID No.1：5’-TGTTCCACCAGCCTTTGCCA-3’

SEQ ID No.2：5’-TAAATTTCACCTGATCAAGG-3’。

10. the human ATXN10 gene capture kit according to claim 9, characterized in that: also comprises at least one of CAS9 enzyme, ATXN10 gene-specific crRNA primer and tracrRNA primer annealing assembly reagent, guide nucleic acid sequence and CAS9 enzyme combination reagent, CAS9 enzyme digestion reagent and magnetic bead purification reagent.

Technical Field

The application relates to the technical field of target area capture, in particular to a target area capture method, a kit and a sequencing method.

Background

Target region sequencing is a research strategy of high-throughput sequencing after enriching Target region DNA by customizing probes of genomic regions of interest, hybridizing with genomic DNA. The research on target areas of a large number of samples is helpful for finding and verifying candidate genes or related sites related to diseases, and the method has great application potential in the aspects of clinical diagnosis and drug development. The principle of hybrid capture is to artificially design a DNA or RNA probe, which can be partially or completely complementary with a target segment; mixing the sample and the probe, wherein the probe captures a target section, and the section without the designed probe is eluted and discarded; the probe and capture segments are then separated by denaturation, typically by adjusting the pH to alkaline; the captured fragments can be subjected to secondary sequencing library construction. The target area capture is suitable for large sample analysis of diseases, including research on various diseases such as Parkinson, dysnoesia, dilated cardiomyopathy, breast cancer and the like.

The target region capture technology for target region sequencing is mainly characterized in that in a solution, a target DNA fragment is directly hybridized with a probe with a biotin label, and then the target DNA fragment is anchored on a microbead with avidin through the reaction of biotin avidin; non-target DNA is washed away, and after elution, the enriched DNA is used for sequencing. The existing target region capture technology is developed and designed aiming at a second-generation sequencing platform for short-read long-sequence sequencing. Including Pacbio official and other companies, when sequencing a target region, the development of a third-generation capture sequencing technology is carried out on the basis of second-generation sequencing, due to the limitation of the method, the capture efficiency is extremely low, and basically, the capture is a short fragment and the advantage of long reading length of third-generation sequencing is not fully utilized.

For example, Pacbio target region sequencing technology comprises the steps of extracting total DNA from human blood as a starting sample, adding a joint by using a KAPA Hyper Prep Kits for Illumina sequencing kit after G-tube is broken, carrying out PCR amplification and enrichment of total genomic DNA, capturing a target region by using an RNA probe marked by IDT biotin, carrying out second PCR amplification and enrichment of the target region, carrying out damage repair and end repair on a captured PCR product, connecting the captured PCR product with a known joint SMRT-BELL, carrying out enzyme reaction digestion, carrying out BluePippin sorting, finally obtaining a dumbbell-shaped library, carrying out Pacbio on-machine sequencing after being detected to be qualified by Agilent2100 and Qubit HS, and carrying out CCS data correction after data downloading. The whole process is detailed as follows:

(1) designing a probe, wherein the design principle of the probe is as follows: an RNA type IDT probe with a target region nucleic acid sequence, low comparison rate with a host genome and a length of 50 nt;

(2) breaking the genome DNA into 1-5 kb;

(3) using a KAPA Hyper Prep Kits for Illumina sequencing kit to carry out end repair, adding A and adding Y-shaped joints, namely pacbio nucleic acid sequence;

(4) performing PCR amplification on the genome DNA according to the Primer on the Y-shaped joint;

(5) sample mixing and pooling;

(6) capturing and capturing a target region nucleic acid sequence by using the specific probe designed in the step (1);

(7) capturing a target area by an M-270 magnetic bead;

(8) amplifying and enriching the captured nucleic acid sequence of the target area by PCR;

(9) damage repair, end repair, addition of an SMRT-BELL linker, digestion with enzyme III, enzyme VII, all using Pacbio published nucleic acid sequences;

(10) preparing and processing: adding primer and sequencing polymerase;

(11) data splitting: and (5) CCS calibration.

The third generation capture sequencing technology developed on the basis of the second generation sequencing has the following defects:

A. the test production flow is long, and the cost of manpower and material resources is high;

B. in the library building process, PCR amplification needs to be carried out twice, then machine sequencing is carried out, sequencing data dup is high, and more data waste is caused;

C. because a PCR process exists in the library building process, methylation information is lost in the PCR process, and methylation modification of a target region cannot be detected;

D. due to the limitation of the length of the probe, the length of the target region capable of being captured is generally 1-5Kb, and the advantage of the third generation sequencing long read length cannot be exerted.

Disclosure of Invention

The purpose of the present application is to provide a novel target region capture method, kit and sequencing method.

The application specifically adopts the following technical scheme:

the first aspect of the application discloses a target region capturing method, which comprises the steps of designing a guide nucleic acid sequence according to a target region nucleic acid sequence, combining the guide nucleic acid sequence with a CAS9 enzyme, carrying out enzyme digestion on a nucleic acid sample to be processed by adopting a compound of the guide nucleic acid sequence and a CAS9 enzyme, recovering a nucleic acid fragment which is in accordance with the designed enzyme digestion target region nucleic acid sequence length in an enzyme digestion product, and capturing to obtain a target region.

The target region capture method provided by the application comprises the steps of carrying out enzyme digestion on a target region by using a CRISPR/Cas9 system to obtain a target region nucleic acid fragment with an expected size, and then obtaining the target region by adopting a nucleic acid fragment separation method. The target area capturing method is short in flow, and not only is the delivery cycle saved, but also the labor cost is saved; moreover, the capture efficiency is improved, the utilization rate of effective data is increased, and the sequencing cost is saved; in addition, PCR amplification is not needed in the whole capturing process of the capturing method, the original modification information of the genome DNA can be reserved, and information such as methylation of a target region can be directly detected.

It should also be noted that the target region capture method of the present application is not only suitable for second-generation sequencing, but also suitable for third-generation sequencing, breaks through the technical limitation of the development of the third-generation sequencing target region capture technology on the basis of the second-generation sequencing in the prior art, and solves the technical problem that the third-generation sequencing for the target region is limited by the second-generation sequencing capture technology. The target region capture methods of the present application, the target region products captured thereby, can be used in any sequencing or detection technique; for example, different sequencing adapters can be added to accommodate different sequencing platforms.

Preferably, in the target region capture method of the present application, the guide nucleic acid sequence is formed by annealing and assembling a crRNA primer and a tracrRNA primer.

It is understood that in the CRISPR/Cas9 system, the guide nucleic acid sequence is formed by annealing and assembling a designed crRNA primer and a universal tracrRNA primer; wherein, the designed crRNA primer is an oligonucleotide which is designed aiming at the target region and accurately targets the target region; the tracrRNA primer is an oligonucleotide nucleic acid sequence which anneals with the designed crRNA primer to form a hairpin structure, and the crRNA primer and the tracrRNA primer are annealed to assemble a guide nucleic acid sequence.

Preferably, the target region capture method further comprises performing magnetic bead purification on the digested product, and then performing nucleic acid fragment screening to capture the target region.

It should be noted that the purpose of magnetic bead purification is to facilitate subsequent nucleic acid fragment screening, and it is understood that other methods of nucleic acid purification besides magnetic bead purification are not excluded.

Preferably, in the target region capture method of the present application, the nucleic acid fragments in the digested product are recovered, and specifically, the Blue Pippin automated nucleic acid recovery system is used to sort the nucleic acid fragments in the target region.

It should be noted that Blue Pippin is only a nucleic acid recovery system specifically used in one implementation manner of the present application, and does not exclude that other methods or systems for separating nucleic acid fragments of different sizes may also be used.

In a second aspect of the application, a target region capture kit is disclosed, comprising a crRNA primer designed for a target region nucleic acid sequence and a universal tracrRNA primer.

The key point of the target region capture method is to design a crRNA primer and a universal tracrRNA primer aiming at a target region; therefore, in one implementation of the present application, the designed crRNA primer and the universal tracrRNA primer are used as a kit for convenient use. It will be appreciated that other reagents used in the subject area capture methods of the present application may be purchased on their own or combined in whole or in part into the kits of the present application for ease of use.

Therefore, preferably, the kit of the present application further comprises at least one of CAS9 enzyme, crRNA primer and tracrRNA primer annealing assembly reagent, guide nucleic acid sequence and CAS9 enzyme combination reagent, CAS9 enzyme digestion reagent and magnetic bead purification reagent.

In a third aspect of the present application, a sequencing method is disclosed, comprising performing target region capture using the target region capture method of the present application or the target region capture kit of the present application, wherein the captured target region nucleic acid fragments are directly used for sequencing library construction and sequencing.

The sequencing method of the present application does not develop a capture target region based on the second-generation sequencing, and thus, does not have the limitation of the second-generation sequencing method. The sequencing method of the present application, due to the target region capture method or the kit of the present application, can obtain very long target region nucleic acid fragments in principle, which is particularly suitable for long-read testing methods, such as third-generation sequencing, and can fully exert the advantages of the third-generation sequencing long-read. The sequencing method can improve the capture efficiency, increase the effective data utilization rate and save the sequencing cost; moreover, original modification information of the genomic DNA can be reserved, and information detection and analysis such as methylation of a target region can be realized.

Preferably, in the sequencing method of the present application, the Pacbio library is used for sequencing library construction.

It should be noted that the Pacbio library is adopted because a Pacbio platform in a third generation sequencing platform is specifically adopted in one implementation manner of the present application. It will be appreciated that if different sequencing platforms are used, the sequencing library construction protocol may be adapted and adapted accordingly, with particular reference to the instructions for use of each sequencing platform. For example, the target area capture methods and kits of the present application may also be used on an ONT nanopore sequencing platform; thus, sequencing library construction can also be performed with reference to the ONT nanopore sequencing platform.

The fourth aspect of the present application discloses a method for detecting methylation of a target region, which comprises sequencing the target region by using the sequencing method of the present application, and analyzing the methylation condition of the target region according to the sequencing result.

It should be noted that the sequencing method of the present application has the greatest characteristic that the original modification information of the target region, including information such as methylation, can be retained without using PCR during target region capture. Thus, the sequencing methods of the present application can also be used for methylation detection of target regions.

The fifth aspect of the application discloses a human ATXN10 gene capturing kit, which comprises an ATXN10 gene specific crRNA primer and a universal tracrRNA primer; the ATXN10 gene-specific crRNA primer comprises an oligonucleotide fragment of a nucleic acid sequence shown in SEQ ID No.1 and an oligonucleotide fragment of a nucleic acid sequence shown in SEQ ID No. 2;

SEQ ID No.1：5’-TGTTCCACCAGCCTTTGCCA-3’

SEQ ID No.2：5’-TAAATTTCACCTGATCAAGG-3’。

it should be noted that the human ATXN10 gene capture is only an experimental scheme specifically designed in one implementation of the present application in order to verify the target region capture method of the present application. It is understood that the present invention can be used not only for capturing the human ATXN10 gene, but also for capturing other target regions. Moreover, the ATXN10 gene-specific crRNA primers of the nucleic acid sequences shown in SEQ ID No.1 and SEQ ID No.2 are also only primers specifically adopted in one implementation mode of the application; on the basis of the inventive concept of the present application, different primers can be designed for ATXN10 gene, and are not specifically limited herein.

It should also be noted that, on the basis of designing the specific crRNA primer, the tracrRNA primer can be obtained according to the conventional design scheme of CRISPR/Cas9 system; for example, in one implementation of the present application, on the basis of designing the crRNA primer specific to ATXN10 gene, the functional nucleic acid sequence of the tracrRNA primer is a universal nucleic acid sequence provided by IDT corporation, which is referenced to the universal sequence provideddby IDT: 1072532.

preferably, the human ATXN10 gene capture kit of the present application further comprises at least one of CAS9 enzyme, annealing assembly reagent of ATXN10 gene specific crRNA primer and tracrRNA primer, combination reagent of guide nucleic acid sequence and CAS9 enzyme, digestion reagent of CAS9 enzyme and purification reagent of magnetic beads.

The beneficial effect of this application lies in:

the target area capturing method is short in flow, simple to operate and capable of saving delivery cycle and labor cost; in addition, the capture efficiency is high, the utilization rate of effective data is increased, and the sequencing cost is saved. The method breaks through the technical limitation that the existing target region capture is generally based on the basic development of the second-generation sequencing technology, creatively utilizes the CRISPR/Cas9 system to carry out the target region capture, does not need PCR amplification in the whole capture process, and can retain original modification information such as methylation of the target region gene.

Drawings

FIG. 1 is a block diagram of a target region capture method and library sequencing process according to an embodiment of the present application.

Detailed Description

In the current practice, when the target region is captured and separated, the target region is captured by a target region capture scheme developed and designed for a second-generation sequencing platform for short-read-length sequencing. The schemes have the advantages of low capture efficiency, long flow, high cost and low sequencing data utilization rate; moreover, basically, the captured fragments are short fragments, and the advantages of a long-read long sequencing method (such as third-generation sequencing) cannot be exerted; more importantly, the whole capture process requires PCR amplification, which results in loss of original modification information such as methylation of the target region.

The CRISPR/Cas system is an immune system of prokaryotes to combat the invasion of foreign genetic material, such as phage viruses and foreign plasmids. At the same time, it provides acquired immunity to the bacteria: this is similar to secondary immunization of mammals, where bacteria are invaded by viruses or foreign plasmids, and a corresponding "memory" is created, which can resist their re-invasion. The CRISPR/Cas system can recognize exogenous DNA, cut off the exogenous DNA and silence the expression of exogenous genes. Due to this precise targeting function, the CRISPR/Cas system was developed as a highly efficient gene editing tool. In nature, CRISPR/Cas systems have multiple categories, with the CRISPR/Cas9 system being the most studied and mature category. By virtue of the advantages of low cost, convenient operation, high efficiency and the like, the CRISPR/Cas9 is rapidly popular with global laboratories, and becomes a powerful helper for biological scientific research.

Based on the research and the recognition, the CRISPR/Cas9 system is creatively introduced into the target region for capture, the CRISPR/Cas9 system is utilized for accurate targeted cutting, a crRNA primer and a universal tracrRNA primer are designed aiming at the nucleic acid sequence of the target region, the target region is accurately cut, and then the target region is recovered according to the size of the cut fragment, so that the target region capture is realized.

In one implementation of the present application, the whole target region capture and sequencing, as shown in fig. 1, comprises the following steps:

(1) the nucleic acid sample preparation 01 step is mainly to perform quality inspection on the extracted nucleic acid sample, and the total amount of the nucleic acid sample is required to be more than 5 mu g, the fragment length is required to be more than 40Kb, and no tailing phenomenon occurs.

(2) CRISPR Cas9 preparation 02 step, assembling designed crRNA primer and universal tracrRNA primer to form guide DNAoligo, i.e. guide nucleic acid sequence, which is combined with Cas9 enzyme to form enzyme complex; wherein, the crRNA primer is designed with a cutting site at certain intervals, so that the length of a target region to be captured is between 1 and 35 Kb; the length can well play the advantage of long reading length of third generation sequencing.

(3) A nucleic acid sample digestion and cutting treatment 03 step, namely adding a complex of the guide DNA oligo and the CAS9 enzyme into the nucleic acid sample for digestion treatment; in one implementation manner of the present application, the method further includes performing magnetic bead purification on the digested product.

(4) A step 04 of fragment sorting, wherein in an implementation manner of the method, a Blue Picpin fragment is specifically adopted to sort a target region, and a proper cutting range is selected according to the length of the target region; for example, the length of the capture gene in this embodiment is 5Kb, and the capture gene can be cut at 1Kb upstream and downstream, that is, the cutting range is 4-6Kb, so as to design a crRNA primer, and then nucleic acid fragments are selected according to the range to obtain a target region by capture;

(5) the conventional Pacbio library construction mainly comprises a step of repairing damage/tail ends 05, a step of adding a joint 06 and a step of double enzyme digestion 07, and can refer to the conventional Pacbio library construction.

(6) And 08, performing library quality inspection, namely performing quality inspection on the constructed sequencing library, judging whether the sequencing library meets the sequencing requirement, and performing the quality inspection according to a conventional sequencing library quality inspection method.

(7) The third generation of sequencing 09 step, that is, sequencing by using a third generation of sequencing platform, in an implementation manner of the present application, a Pacbio platform is specifically used for sequencing, in principle, the target region capture method of the present application may also be applied to other sequencing platforms, for example, an ONT nanopore sequencing platform, and the corresponding library building step needs to be adjusted according to the requirements of the sequencing platform, which is not specifically limited herein.

The capture method has the advantages of high capture efficiency, short flow, low cost and high sequencing data utilization rate; and the size of the target region fragment can be designed according to requirements, PCR amplification is not required in the whole capturing process, and original modification information such as methylation of the target region can be well reserved. The target region capturing method is used for third-generation sequencing, and the advantage of long reading length of third-generation sequencing can be well played. Of course, the target region capture method of the present application is not limited to third generation sequencing, but can be used for other sequencing or detection.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

This example uses human standard NA12878 to capture an ATTCT tandem repeat nucleic acid sequence, ATXN10, on chromosome 22 using the CRISPR/Cas9 system. Firstly, ATXN10 is designed to capture the nucleic acid sequences of the required crRNA primer and tracrRNA primer, the crRNA primer and the tracrRNA primer are annealed and then assembled with CAS9 enzyme, and then the nucleic acid molecules are used for enzyme digestion of the target region on the nucleic acid molecules. Finally, Blue Picpin is used for carrying out fragment sorting, and the fragments with the target length are screened out to be capture products. In this example further, Pacbio library sequencing and sequencing results analysis were performed on the captured products. The details are as follows:

1. preparation of sample preparation

This example was tested using human standard NA12878 genomic DNA. If nucleic acid is extracted from a whole blood sample, the quality of the extracted nucleic acid needs to be tested, the length of a fragment needs to be more than 40Kb, no tailing phenomenon occurs, the total amount is more than 5 mug, and a sample is colorless, clear and transparent and can be used for subsequent CRISPR treatment and library construction sequencing.

CRISPR-Cas9 primer preparation

(1) Primer design

Two ATXN10 gene-specific crRNA primers are designed aiming at the ATXN10 gene and are respectively named as ATXN10.DC.1 and ATXN10.DC.2, the ATXN10.DC.1 is a nucleic acid sequence shown by SEQ ID No.1, and the ATXN10.DC.2 is a nucleic acid sequence shown by SEQ ID No. 2. Both crRNA primers were synthesized by IDT corporation, and the tracrRNA primer commonly used in this example, i.e., the kit universal sequence provided by IDT corporation, i.e., IDT: 1072532. two ATXN10 gene-specific crRNA primers are shown in Table 1.

TABLE 1ATXN10 Gene-specific crRNA primers

The crRNA primer nucleic acid sequence of ATXN10 was designed on the following website:

https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design。

(2) primer dilution

The primers were diluted to 50. mu.M with purified water for storage, and stored at-20 ℃ after dilution.

(3) Annealing of primers

Firstly, preparing a primer according to the reaction volume shown in the table 2, then reacting at 95 ℃ for 5min, and then placing a reaction system at room temperature for natural cooling to finish annealing of the crRNA primer and the tracrRNA primer. The annealed primer was named guide DNA oligo, i.e.guide nucleic acid sequence.

TABLE 2 primer annealing System

Name of reagent	Storage concentration	Volume of use
			ATXN10.DC.1 or ATXN10.DC.2	50μM	1μL
tracrRNA	50μM	1μL
			Nuclease-Free Duplex Buffer	-	8μL

Cas9 digest of the digest enzyme

This example was digested with the Vazyme Cas9 Nuclease (Lot: EN301-01/02) kit as detailed below:

(1) combination of Cas9 enzyme and guide DNA oligo: the reaction system was prepared according to table 3, mixed and centrifuged, incubated at 37 ℃ for 10min, and then placed on ice for use.

TABLE 3 guide DNA oligo and CAS9 enzyme combination System

Name of reagent	Storage concentration	Volume of use
			NEBuffer3.1	10×	2.0μL
guide DNA oligo	5μM	8.0μL
			Cas9 Nuclease	20μM	2.0μL
Water(nuclease-free)	-	8.0μL

(2) Target area digestion: the complex of Cas9 enzyme and guide DNAoligo was added to 80. mu.L of nucleic acid, mixed well and centrifuged, and then incubated at 37 ℃ for 60min, and then placed on ice for use.

(3) Cas9 inactivation treatment: add 5. mu.L EDTA with a concentration of 0.5M, mix well, centrifuge and place on ice for use.

(4) Magnetic bead purification of digestion products: in this example, magnetic bead purification was carried out using Novozan VAHTSTM DNA Clean Beads (lot: N411) kit. Specifically, 100. mu.L of Ampure PB Beads magnetic Beads were added to the sample, after binding for 10min, washed twice with 75% absolute ethanol, and finally the nucleic acids were redissolved from the Beads with 30. mu.L of water.

4. Fragment screening

Screening nucleic acid molecules with the target length between 4 and 6Kb by using Blue Picpin; since the capture gene of this example is 5Kb in length and is designed to cleave at 1Kb upstream and downstream of the target region, i.e., in the range of 4 to 6Kb, the target region can be captured by screening nucleic acid molecules of a desired length between 4 and 6 Kb.

Pacbio library construction

(1) Injury repair response

In this example, the Pacbio library construction Kit SMRTbell Template Prep Kit 1.0(lot:100-259-100) was used for sequencing library construction. Wherein, M6630L NEBNext FFPE DNA Repair NEB (NEB, 1000002572) kit is adopted for damage Repair.

In this example, a reaction system was prepared as shown in Table 4, and then reacted at 37 ℃ for 60 minutes to repair the damage.

TABLE 4 Damage repair reaction System

Reagent	Dosage (mu L)
		DNA Damage Repair Buffer	5
NAD+	0.5
		ATP high	5
dNTP	0.5
		DNA Damage Repair Mix	1
DNA (fragment screening product)	38

(2) End repair reactions

The Damage Repair reaction product 50. mu.L was mixed with 2. mu.L of DNA Damage Repair Mix, and then reacted at 25 ℃ for 10 minutes to perform end Repair.

(3) Add the piecing

A joint-added reaction system was prepared as shown in Table 5, and then reacted at 25 ℃ for 12 to 16 hours, specifically 12 hours in this example, to connect joints.

TABLE 5 Tab reaction System

Reagent	Dosage (mu L)
		End-repaired DNA	20
Annealed Blunt Adapter(20uM)	10
		Template Prep Buffer	4
ATP low	2
		Ligase	1
H2O	3

(4) Double enzyme digestion

The linker-added products were subjected to enzymatic digestion using ExoIII and ExoVII, and the reaction system of the enzymatic digestion is shown in table 6. After the reaction system was prepared, it was digested with enzyme at 37 ℃ for 60 minutes.

TABLE 6 enzymatic digestion reaction System

Reagent	Dosage (mu L)
		Adaptor-added DNA	40
ExoIII	1
		ExoVII	1

6. Sequencing on machine

This example uses the Pacbio platform of the three generation sequencing platforms for sequencing.

After the data are downloaded, CCS data correction is carried out, and statistics and analysis of the amount of downloaded data, the number of downloaded reads, the number of effective reads, the capture efficiency, the double rates and the methylation modification degree of m6A are carried out.

Comparative test

In this example, the same ATXN10 gene capture, library construction and sequencing were performed on the same human standard NA12878 using the existing Pacbio target region capture, library construction and sequencing technology. A Pacbio target region capturing and library building sequencing technology comprises the steps of carrying out G-tube cutting on human standard NA12878 genome DNA, adding a joint by using a KAPA HyperPrep Kits for illuminating sequencing kit, carrying out PCR amplification and enrichment on total genome DNA, capturing a target region by using an RNA probe marked by IDT biotin, carrying out second PCR amplification and enrichment on the target region, carrying out damage repair and end repair on a second PCR amplification product, connecting the second PCR amplification product with a known joint SMRT-BELL, carrying out enzyme reaction digestion, carrying out BluePippin sorting, finally obtaining a dumbbell-shaped library, carrying out Pacbio on-machine sequencing after being detected to be qualified by Agilent2100 and Qubit HS, and carrying out CCS data correction after data is downloaded. The details are as follows:

1. probe design

Designing an RNA probe with a target region with a biotin label aiming at the ATXN10 gene, wherein the design principle of the probe is as follows: an RNA type IDT probe having a length of 50 nt. Probe design and synthesis was done by IDT corporation.

2. Disruption of genomic DNA

This example uses the G-tube, 13000rpm/min disruption parameter to disrupt genomic DNA to 1-5 Kb.

3. End repair, add A and add Y-shaped joint

This example uses the KAPA Hyper Prep Kits for Illumina sequencing kit for end repair and A addition, the Y-shaped linker is a pacbio nucleic acid sequence.

(1) End repair and addition of A

A reaction system for repairing the tail end and adding A is prepared according to the reaction system in the table 7, and then the temperature is kept in a PCR reactor for 30min at 20 ℃ and 30min at 65 ℃, and the reaction system is kept at 4 ℃ for standby.

TABLE 7 end repair and A addition reaction System

Composition (I)	Dosage (mu L)
		Sheared DNA	50
End Repair&A-Tailing Buffer	7
		End Repair&A-Tailing Enzyme Mix	3
Total volume	60

(2) Y-shaped joint connection

A Y-shaped linker reaction system was prepared according to the reaction system of Table 8, and then a linker addition reaction was carried out at 20 ℃ for 15 min.

TABLE 8 Tab reaction System

Composition (I)	Volume (μ L)
		End Repair&A-Tailing reaction product	60
PCR-grade water	5
		Ligation Buffer	30
DNA Ligase	10
		Annealed Barcoded Adapter	5

4. First PCR amplification

Carrying out PCR amplification on the genome DNA according to the Primer on the Y-shaped joint, wherein the PCR amplification reaction system refers to the genome amplification of a pacbio nucleic acid sequence by the Y-shaped joint, and the PCR amplification reaction conditions are as follows: denaturation at 95 ℃ for 2min, then 6 cycles: 95 ℃ 20seconds, 62 ℃ 20seconds, 68 ℃ 10min, 68 ℃ 5min after the cycle is over, 4 ℃ Hold.

After the first PCR amplification, the biotin-labeled probe of ATXN10 gene provided by IDT company was used to capture the target region nucleic acid sequence of ATXN10 gene as follows:

A. add 5. mu.L of COT Human DNA (1mg/mL) to a 1.5mL centrifuge tube;

B. adding 2.0 μ g of the library with the adaptor added in front, namely the first PCR amplification product, into the centrifuge tube in the previous step;

C. adding 10. mu.L of a primer, i.e., a primer nucleic acid sequence on the Pacbio Y-shaped adaptor, to the centrifuge tube of the previous step, mixing and centrifuging, wherein the primer is annealed to the Y-shaped adaptor before hybridization, and is used for reducing non-specific capture in a probe capture method;

D. concentrating the sample into dry powder by using a concentrator;

E. preparing a hybridization reaction reagent: 8.5 mu L of xGen 2X hyb buffer, 2.7 mu L of xGen hyb buffer Enhancerr and 1.8 mu L of water;

F. adding the hybridization reaction reagent into the concentrated dry powder, uniformly mixing and centrifuging, and incubating for 10 minutes at 95 ℃;

G. add 4. mu.L of probe, mix well, centrifuge, incubate at 65 ℃ for 4 hours.

5. Elution preparation of streptavidin magnetic beads

The beads were incubated at room temperature for more than 30 minutes and washed twice with 200. mu.L of Wash buffer I. The beads were then thawed back using 100. mu.L of Wash buffer I.

6. Target area capture

Combining the probe and the incubation product of the adapter library by using magnetic beads with the same volume, wherein the combination condition is 45min at room temperature; then, washing once with 1 × Stringent Wash Buffer, incubating at 65 ℃ for 10 min; washing once by using 1 XWash Buffer I, and incubating for 10 minutes at 65 ℃; washing once with 1 × Wash BufferII, and incubating at 65 deg.C for 10 min; wash once with 1 × Wash Buffer III and incubate at 65 ℃ for 10 min.

And (3) performing second PCR amplification: after being cleaned by 1 multiplied by the Wash Buffer III, the product is directly subjected to PCR amplification, and a capture and enrichment target area is obtained.

The reaction system of the second PCR amplification is shown in Table 9, and the reaction conditions of the PCR amplification are as follows: denaturation at 95 ℃ for 2min, then 15 cycles: 95 ℃ 20seconds, 62 ℃ 20seconds, 68 ℃ 10min, 68 ℃ 5min after the cycle is over, 4 ℃ Hold.

TABLE 9 Tab reaction System

Composition (I)	Volume (μ L)
		Captured Library	50
10×LA PCR Buffer	20
		2.5mM each dNTPs	16
100μM PacBio Universal Primer	2
		Takara LA Taq DNA polymerase	1.2
Water	110.8

Pacbio library construction

(1) Injury repair response

In this example, the Pacbio library construction Kit SMRTbell Template Prep Kit 1.0(lot:100-259-100) was used for sequencing library construction. Wherein, M6630L NEBNext FFPE DNA Repair NEB (NEB, 1000002572) kit is adopted for damage Repair.

In this example, a reaction system was prepared as shown in Table 4, and then reacted at 37 ℃ for 60 minutes to repair the damage.

(2) End repair reactions

The Damage repair reaction product 50. mu.L was mixed with 2. mu.L of DNA Damage RepiarMix, and then reacted at 25 ℃ for 10 minutes to perform end repair.

(3) Add the piecing

A joint-added reaction system was prepared as shown in Table 5, and then reacted at 25 ℃ for 12 to 16 hours, specifically 12 hours in this example, to connect joints.

(4) Double enzyme digestion

The linker-added products were subjected to enzymatic digestion using ExoIII and ExoVII, and the reaction system of the enzymatic digestion is shown in table 6. After the reaction system was prepared, it was digested with enzyme at 37 ℃ for 60 minutes.

8. Sequencing on machine

This example uses the Pacbio platform of the three generation sequencing platforms for sequencing.

After the data are downloaded, CCS data correction is carried out, and statistics and analysis of the amount of downloaded data, the number of downloaded reads, the number of effective reads, the capture efficiency, the double rates and the methylation modification degree of m6A are carried out.

This example comparatively analyzes the sequencing results of the target region capture method of this example and the sequencing results of comparative experiments using the current Pacbio target region capture pooling sequencing, as shown in Table 10.

TABLE 10 comparison of sequencing results

As can be seen from the results in Table 10, in both the target region capturing method of the present example (hereinafter referred to as "experimental group") and the comparative test method (hereinafter referred to as "control group"), the data yield was greater than 5Gb, and both capturing methods did not adversely affect the sequencing result in terms of data yield, indicating that the target region capturing method of the present example did not adversely affect the sequencing result.

The proportion of the repetitive sequences aligned to the genome is duplex rate on genome (%), and compared with 8.01% of the control group, the data available increases as the experimental group is optimized and then decreases to 0.42%. The method of the experimental group can obviously reduce the repetitive sequence ratio, thereby improving the utilization rate of data.

In the case of methylation modification, the control group lost modification information due to the PCR process during the capture process, and therefore, m6A methylation modification information on the gene could not be detected. In the present example, the CRISPR method is a capture method that does not require PCR, so that the methylation modification information on the gene is retained.

From the above test and comparison results, it can be seen that the target region capture method of the present embodiment reduces the PCR process, reduces the generation of repeated sequences (duplicate), and simultaneously retains the modification information such as methylation of the target region, thereby effectively improving the data utilization rate and greatly saving the sequencing cost.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> Wuhanhua university medical laboratory Co., Ltd

<120> target area capturing method, kit and sequencing method

<130> 19I29248

<160> 2

<170> PatentIn version 3.3

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<211> 20

<212> DNA

<213> Artificial sequence

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<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

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