阅读说明：本技术 基于基因捕获技术的测序方法 (Sequencing method based on gene capture technology ) 是由 刘倩 江翱 马海玲 陈晶晶 孙睿 王嫚 曹振 宋东亮 于 2021-10-27 设计创作，主要内容包括：本发明提供了一种基于基因捕获技术的测序方法,原理是使用重组酶复合物在37℃条件下协助3’端封闭的单链DNA探针高效特异性地杂交到双链目标DNA分子中,再利用单链DNA探针上带有的亲和标记对靶DNA进行特异性富集。这种方法既具有探针杂交捕获技术的长探针配对较强和探针设计要求低等优势,又具有CRISPR/dCas9系统操作简单、耗时短、DNA无需热变性、捕获效率高和成本低等优点,且探针不参与后续的扩增,提高鉴定准确性。因此相较于其他捕获技术,RATE-seq具有操作简单、捕获效率高、耗时极短(30 min)、稳定性好和成本低等明显优势,非常适用于基因捕获和自动化应用。除此之外,RATE-seq还能有效分离去除病理样本中的人类宿主DNA,提高病原微生物的检出率。(The invention provides a sequencing method based on a gene capture technology, which adopts the principle that a recombinase complex is used for assisting a single-stranded DNA probe with a closed 3' end to efficiently and specifically hybridize to a double-stranded target DNA molecule under the condition of 37 ℃, and then affinity labels carried on the single-stranded DNA probe are utilized for specifically enriching the target DNA. The method has the advantages of strong long probe pairing, low probe design requirement and the like of the probe hybridization capture technology, has the advantages of simple operation, short time consumption, no thermal denaturation of DNA, high capture efficiency, low cost and the like of the CRISPR/dCas9 system, and improves the identification accuracy because the probe does not participate in subsequent amplification. Therefore, compared with other capture technologies, the RATE-seq has the obvious advantages of simple operation, high capture efficiency, extremely short time consumption (30 min), good stability, low cost and the like, and is very suitable for gene capture and automation application. In addition, the RATE-seq can also effectively separate and remove human host DNA in pathological samples, and improve the detection RATE of pathogenic microorganisms.)

1. A sequencing method based on gene capture technology is characterized by comprising the following steps:

(1) obtaining a total DNA library of a sample;

(2) designing and synthesizing a probe aiming at a target gene to be captured, wherein the probe is strictly complementary and paired with the DNA of the target gene and is provided with a modification convenient for being combined with a capture medium, and the 3' end of the probe is modified by dideoxyribonucleotide, biotin or amino;

(3) pre-binding the probe with a recombinase complex;

(4) binding the probe-recombinase complex to the total DNA library of the sample;

(5) separating the target gene DNA library combined with the probe from the total DNA library by utilizing a capture medium capable of being specifically combined with the probe to obtain an enriched target gene;

(6) amplifying the obtained target gene, and sequencing.

2. The gene capture technology-based sequencing method of claim 1, wherein: the length of the probe is 30-50 nt.

3. The sequencing method based on gene capture technology of claim 1, characterized in that: the 5' end of the probe is modified by biotin or poly-deoxyadenylate.

4. The sequencing method based on gene capture technology of claim 1, characterized in that: the recombinase complex comprises UvsX recombinase and UvsY recombinase accessory proteins and is used at a concentration of 20-200 nM.

5. The sequencing method based on gene capture technology of claim 4, characterized in that: the recombinase complex also includes gp32 single-stranded DNA binding protein, used at a concentration of 10-50 nM.

6. The sequencing method based on gene capture technology of claim 1, characterized in that: the using concentration of the probe is 10-100 nM, and the input amount of the total DNA is 0.1-100 ng/uL.

7. The sequencing method based on gene capture technology of claim 1, characterized in that: pre-combining buffer solution is used in the step (2) and is pre-combined for 5-30min at room temperature, and the proportion of the pre-combining buffer solution is as follows: 10-100 mM of trihydroxymethyl aminomethane, 10-300 mM of potassium acetate, 3-50 mM of magnesium acetate, 0.3-3 mM of dithiothreitol and 1-20 mM of adenosine triphosphate.

8. The sequencing method based on gene capture technology of claim 1, characterized in that: the binding temperature in step (3) is 30-50 ℃.

9. The sequencing method based on gene capture technology of claim 3, characterized in that: when the modification on the probe is biotin modification, streptavidin magnetic beads are used as the capture medium; when the modification on the probe is a poly-deoxyadenylate modification, the capture medium is poly-deoxythymidylate magnetic beads.

10. The sequencing method based on gene capture technology of claim 9, characterized by: the step (4) is specifically as follows: incubating the capture medium and the reaction system for 10-60min at room temperature, separating the capture medium and washing, and then eluting the DNA adsorbed on the capture medium in a high-temperature 98 ℃ elution mode, a 1-20 mM biotin competitive elution mode or a denaturing agent deionized formamide elution mode with a volume concentration of 5% -50%.

11. The sequencing method based on gene capture technology of claim 10, characterized by: the capture medium is washed with a wash buffer comprising 10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20 by volume.

Technical Field

The invention relates to a sequencing method based on a gene capture technology, and belongs to the technical field of biology.

Background

The second generation and third generation sequencing technologies have the advantages of large flux, complete information, simple and convenient operation and the like, and become core technologies in the field of gene diagnosis and treatment. Gene trapping has been a major problem in high-throughput DNA and RNA sequencing technologies. Because human genome information is huge, about 30 hundred million base pairs are included, the proportion of the region of the real coding gene is less than 4%, and the proportion of the exon region and the transcription regulatory element region is less than 1%. Most pathogenic mutations are concentrated in the exon region and the transcription regulatory element region, so that the genetic information of the regions plays a key role in the diagnosis and treatment of diseases. Since these regions are too small, sequence information verification and mutation type analysis using whole genome sequencing techniques have extremely low efficiency and accuracy. Generally, the judgment of one site information requires hundreds to tens of thousands of sequencing coverage depths, which means that effective analysis of gene information by using whole-gene sequencing often requires sequencing data of over hundreds of G, which greatly increases the cost and popularization difficulty of second-generation sequencing and third-generation sequencing in gene diagnosis, and also causes a great amount of unnecessary waste. Therefore, the development of a technology for capturing the gene sequence of the target site is the key for the popularization and application of the second generation and third generation sequencing technologies in the field of gene therapy.

The existing gene trapping technology is mainly divided into two types. One is probe hybridization capture technology, which utilizes long probes (40-120 nt) with modifications (such as biotin) to hybridize with genomic DNA and capture with corresponding affinity media (such as streptavidin magnetic beads). The method is very complex, double-stranded DNA is required to be pre-denatured, then the denatured single-stranded DNA is hybridized with a probe, and the hybridized solution is dried and concentrated in vacuum to capture a target site. The method has the advantages of longer pairing area between the probe and the target DNA, strong binding capacity and low design requirement of the probe. But also has obvious defects, such as complex operation, long time consumption (about two days), high cost, strong dependence on instruments, low capture efficiency, high non-specificity and large DNA loss, and greatly limits the application of capture technology.

In recent years, with the development of a gene editing technology CRISPR/Cas system, various Cas proteins are developed and applied to different fields. dCas9 with completely inactivated cleavage activity becomes an important tool for in vitro gene capture. The method combines the high specificity of a CRISPR/Cas9 gene editing system and also utilizes helicase activity carried by dCas9, so that a target site can be captured by specially modified dCas9 and sgRNA at room temperature. Compared with the traditional probe hybridization capture technology, the capture method has the advantages of low cost, short time consumption (2 h), high capture efficiency, simple operation, no need of thermal denaturation of DNA and the like, but the capture efficiency and specificity are seriously hindered by the nonspecific binding of dCas9 with the DNA and the off-target effect of sgRNA. In addition, the sgRNA is difficult to design (depending on the PAM sequence), and the pairing length with the target site is short (20 nt), which seriously hinders the application of sgRNA in large-scale gene capture. And the poor stability of sgRNA and dCas9 also makes the capture mode difficult to popularize and popularize in a large scale.

Disclosure of Invention

The invention aims to provide a sequencing method based on a gene capture technology.

The technical scheme adopted by the invention is as follows: a sequencing method based on gene capture technology is characterized by comprising the following steps:

(1) obtaining a total DNA library of a sample;

(2) designing and synthesizing a probe aiming at a target gene to be captured, wherein the probe is strictly complementary and paired with the DNA of the target gene and is provided with a modification convenient for being combined with a capture medium, and the 3 'end of the probe is modified by dideoxyribonucleotide, biotin or amino to close the 3' end of the probe;

(3) pre-binding the probe with a recombinase complex;

(4) binding the probe-recombinase complex to the total DNA library of the sample;

(5) separating the target gene DNA library combined with the probe from the total DNA library by utilizing a capture medium capable of being specifically combined with the probe to obtain an enriched target gene;

(6) amplifying the obtained target gene, and sequencing.

It should be noted that, as will be fully appreciated by those skilled in the art, there is no requirement for the order of operation between step (1) and steps (2) and (3), and steps (1), (2) and (3) are labeled before the steps for convenience of description, and in fact, the manner of performing steps (2) and (3) first and then performing step (1) is equivalent to the manner of performing steps (1), (2) and (3). Therefore, those skilled in the art should recognize that the manner of performing steps (2), (3) first, then step (1), and performing steps (1), (2), and (3) are within the scope of the present invention.

Preferably, the length of the probe is 30 to 50 nt.

Preferably, the 5' end of the probe is modified by biotin or poly-deoxyadenosine.

Preferably, the recombinase complex comprises UvsX recombinase and UvsY recombinase helper proteins, used at a concentration of 20-200 nM.

Preferably, the recombinase complex further includes gp32 single-stranded DNA binding protein, used at a concentration of 10-50 nM.

Preferably, the probe is used at a concentration of 10-100 nM, and the total DNA input is 0.1-100 ng/uL.

Preferably, a pre-binding buffer is used in the step (2), and pre-binding is performed for 5-30min at room temperature, wherein the pre-binding buffer comprises the following components in percentage by weight: 10-100 mM of trihydroxymethyl aminomethane, 10-300 mM of potassium acetate, 3-50 mM of magnesium acetate, 0.3-3 mM of dithiothreitol and 1-20 mM of adenosine triphosphate.

Preferably, the binding temperature in step (3) is 30-50 ℃.

Preferably, when the modification on the probe is a biotin modification, the capture medium uses streptavidin magnetic beads; when the modification on the probe is a poly-deoxyadenylate modification, the capture medium is poly-deoxythymidylate magnetic beads.

Preferably, the step (4) is specifically: incubating the capture medium and the reaction system for 10-60min at room temperature, separating the capture medium and washing, and then eluting the DNA adsorbed on the capture medium in a high-temperature 98 ℃ elution mode, a 1-20 mM biotin competitive elution mode or a denaturing agent deionized formamide elution mode with a volume concentration of 5% -50%.

Preferably, the capture medium is washed with a wash buffer comprising 10-100 mM Tris-hydroxymethyl-aminomethane, 10-2000 mM NaCl, Tween 20 at a concentration of 0.1-3% by volume.

The invention has the beneficial effects that:

the invention provides a novel gene capturing technology based on Recombinase and is named as RATE-seq (recombination-Assistant Target entity and Sequencing). The principle of RATE-seq is that a recombinase complex is used to assist a single-stranded DNA probe to hybridize to a double-stranded target DNA molecule efficiently and specifically at 37 ℃, and then affinity labels carried on the single-stranded DNA probe are used to perform specific enrichment on the target DNA. The method has the advantages of strong long probe pairing, low probe design requirement and the like of the probe hybridization capture technology, and has the advantages of simple operation, short time consumption, no thermal denaturation of DNA, high capture efficiency, low cost and the like of the CRISPR/dCas9 system. Therefore, compared with other capture technologies, the RATE-seq has the obvious advantages of simple operation, high capture efficiency, extremely short time consumption (30 min), good stability, low cost and the like, and is very suitable for gene capture and automation application. In addition, the RATE-seq can also effectively separate and remove human host DNA in pathological samples, and improve the detection RATE of pathogenic microorganisms.

The probe used in the invention is a probe with a 3' end modified and sealed, the probe has simple structure and low synthesis cost, and the probe does not participate in the amplification of subsequent target genes, thereby ensuring the original information of a captured sequence and improving the accuracy of the identification of the captured target molecule sequence. Meanwhile, the closed probe can also prevent the probe from building a library by itself, so that the probe is prevented from generating non-specific binding with the joint, and the accuracy of a sequencing result is further improved.

Drawings

FIG. 1 is a schematic diagram of the technical process and principle of RATE gene capture.

FIG. 2 is a schematic diagram of the application of two 5.8S modified probes in RATE gene capture technology.

FIG. 3 qPCR verifies the efficiency and specificity of two 5.8S modified probes in RATE gene capture technology.

FIG. 4 second generation high throughput sequencing verifies the efficiency and specificity of two 5.8S modified probes in RATE gene capture technology.

FIG. 5 qPCR is used for verifying the application effect comparison of two 3' end modified probes on RATE capture technology.

FIG. 6 qPCR verifies the efficiency and specificity of 5.8S modified probe length in RATE gene capture technology.

FIG. 7 qPCR verifies the efficiency and specificity of recombination reaction temperature on RATE gene capture technology.

FIG. 8 qPCR verifies the efficiency and specificity of recombination reaction time on RATE gene capture technology.

FIG. 9 qPCR verifies the efficiency and specificity of the elution mode in the RATE gene capture technique.

FIG. 10 is a comparison of the characteristics of three gene trapping techniques.

FIG. 11 second generation sequencing verifies the capture efficiency and specificity of the three gene capture technologies for the 5.8S gene.

FIG. 12 three gene capture techniques capture the 5.8S gene sequencing signal map.

FIG. 13 shows the efficiency and specificity of the capture of BRCA1 gene by RATE gene technology.

FIG. 14 captures two third-generation sequencing signal plots of the BRCA1 gene using RATE technology.

FIG. 15 alignment results and probe positions of human high abundance Alu sequences.

FIG. 16 removal effect of capture of human host DNA using RATE capture technology targeting Alu sequences.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. The primer sequences used in this example are shown in table 1, and the sgRNA library primer sequences are shown in the attached table.

TABLE 1 linker sequences and modifications

Example 1: and comparing the application effects of the biotin modified probe and the poly A modified probe in the RATE capture technology.

In this example, we compared the capture effect of biotin-modified probe and poly-A-modified probe on RATE capture technology (see FIG. 1 and FIG. 2 in principle), and we picked the target DNA site as double-stranded DNA derived from 5.8S rRNA. The specific implementation mode is as follows:

1) construction of RNA library: 1 ug 293F RNA was subjected to RNA Library construction by Hieff NGS ^ -Ultima Dual-mode RNA Library Prep Kit for Illumina (Cat # 12252) of Saint Netherlands, and then captured by the RATE-seq flow.

2) Biotin modified probe capture

TABLE 2

Components	Dosage of
		Biotin-5.8S probe （SEQ ID NO: 6）	10-100 nM
UvsX recombinase	20-200 nM
		UvsY recombinase accessory proteins	20-200 nM
10 × recombinant buffer	3 µL
		Total volume	20 µL

The 10 × recombinant buffer solution comprises 100-1000 mM tris, 100-3000 mM potassium acetate, 30-500 mM magnesium acetate, 3-30 mM dithiothreitol, 10-200 mM adenosine triphosphate, etc

Standing at room temperature for 5 min.

TABLE 3

Components	Dosage of
		The above reaction volume	20 µL
gp32 single-chain binding protein	10-50 nM
		Library constructed in step (1)	0.1-100 ng/uL
Total volume	30 µL

Reacting at 35-45 deg.c for 10 min.

After the reaction, 10 ul of Dynabeads ™ M-270 streptavidin magnetic beads washed according to the instructions were added, mixed well and incubated at room temperature for 10 min with rotation. Separating magnetic beads and supernatant on magnetic separation rack, washing magnetic beads with washing buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20) for 3 times, adding 20 ul ddH2O, and collecting supernatant at 98 deg.C for 5 min. The recovered DNA was subjected to quantitation and NGS sequencing analysis, the quantitation primers are shown in Table 1, and the results are shown in FIGS. 3 and 4.

3) Poly-deoxyadenylate modified probe capture

TABLE 4

Components	Dosage of
		polyA-5.8S probe 39	10-100 nM
UvsX recombinase	20-200 nM
		UvsY recombinase accessory proteins	20-200 nM
10 × recombinant buffer	3 µL
		Total volume	20 µL

The 10 × recombinant buffer solution comprises 100-1000 mM tris, 100-3000 mM potassium acetate, 30-500 mM magnesium acetate, 3-30 mM dithiothreitol, 10-200 mM adenosine triphosphate, etc

Standing at room temperature for 5 min.

TABLE 5

Components	Dosage of
		The above reaction volume	20 µL
gp32 single-chain binding protein	10-50 nM
		Library constructed in step (1)	0.1-100 ng/uL
Total volume	30 µL

Reacting at 30-45 deg.c for 10 min.

After the reaction, 10 ul of oligo (dT)23 magnetic beads washed according to the instructions were added, mixed well and incubated at room temperature for 10 min with rotation. Separating magnetic beads and supernatant on magnetic separation rack, washing magnetic beads with washing buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20) for 3 times, adding 20 ul ddH2O, and collecting supernatant at 98 deg.C for 5 min. The recovered DNA was subjected to quantification and NGS sequencing analysis, and the quantitative primers are shown in Table 1, and the results are shown in FIGS. 3 and 4.

As shown in FIG. 3 and FIG. 4, the RATE capture technology can effectively capture 5.8S target gene fragments, the capture efficiency can reach more than 85%, and the capture efficiency is very low for untargeted sites such as 28S and ACTB genes, which indicates that the RATE technology has great application value in gene capture. Among them, biotin-modified probes have higher efficiency in RATE capture.

Example 2: and comparing the application effects of different 3' end modified probes on the RATE capture technology.

In this example, we compared the capture effect of two 3' modified probes, SEQ ID NO: 3 and SEQ ID NO: 2, of dideoxyribonucleotide modified ddN (SEQ ID NO: 3) and NH2C6 (SEQ ID NO: 2), on RATE capture technology, and selected the target DNA site as 5.8S rRNA-derived double-stranded DNA. The specific implementation manner is the same as the capture process of the biotin-modified probe in example 1. As a result, as shown in FIG. 5, both the ddN modification and the NH2C6 modification at the 3' end of the probe have good RATE capture efficiency, and the ddN modified probe has better capture efficiency.

Example 3: compared with the application effect of the biotin modified probes with different lengths in the RATE capture technology.

In this example, we compared the capture effect of biotin-modified probes (SEQ ID NOS: 3-10) of different lengths (30-50 nt) on RATE capture technology, and selected the target DNA site as double-stranded DNA derived from 5.8S rRNA. The specific implementation mode is as follows:

1) construction of RNA library: 1 ug 293F RNA was subjected to RNA Library construction by Hieff NGS ^ -Ultima Dual-mode RNA Library Prep Kit for Illumina (Cat # 12252) of Saint Netherlands, and then captured by the RATE-seq flow.

2) Biotin modified probe capture

TABLE 6

Components	Dosage of
		Biotin-5.8S probe 30-50	10-100 nM
UvsX recombinase	20-200 nM
		UvsY recombinase accessory proteins	20-200 nM
10 × recombinant buffer	3 µL
		Total volume	20 µL

The 10 × recombinant buffer solution comprises 100-1000 mM tris, 100-3000 mM potassium acetate, 30-500 mM magnesium acetate, 3-30 mM dithiothreitol, 10-200 mM adenosine triphosphate, etc

Standing at room temperature for 5 min.

TABLE 7

Components	Dosage of
		The above reaction volume	20 µL
gp32 single-chain binding protein	10-50 nM
		In the step (1)Constructed libraries	0.1-100 ng/uL
Total volume	30 µL

Reacting at 30-45 deg.c for 10 min.

After the reaction, 10 ul of Dynabeads ™ M-270 streptavidin magnetic beads washed according to the instructions were added, mixed well and incubated at room temperature for 10 min with rotation. Separating magnetic beads and supernatant on magnetic separation rack, washing magnetic beads with washing buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20) for 3 times, adding 20 ul ddH2O, and collecting supernatant at 98 deg.C for 5 min. The recovered DNA was subjected to quantitation and NGS sequencing analysis, and the quantitation primers are shown in Table 1, and the results are shown in FIG. 6.

As a result, as shown in FIG. 6, the probes at 36 to 45 nt length had higher capture efficiency and capture specificity.

Example 4: the application effect of the recombination reaction temperature on the RATE capture technology is compared.

In this example, we compared the capture effect of different recombination reaction temperatures (30-45 ℃) on RATE capture technology, and the specific implementation is the same as example 3. As shown in FIG. 7, the recombination reaction showed the highest trapping efficiency at 35-40 ℃.

Example 5: the application effect of the recombination reaction time on the RATE capture technology is compared.

In this example, we compared the capture effect of different recombination reaction times (5-30 min) on RATE capture technology, and the specific implementation is the same as example 3. As a result, as shown in FIG. 8, the recombination reaction time was 10 to 20, and the efficiency and specificity of the capture were higher.

Example 6: the application effect of the magnetic bead elution condition on the RATE capture technology is compared.

In this example, we compared the capture effect of different bead elution conditions on RATE capture technology. The specific implementation mode is as follows:

1) construction of RNA library: 1 ug 293F RNA was subjected to RNA Library construction by Hieff NGS ^ -Ultima Dual-mode RNA Library Prep Kit for Illumina (Cat # 12252) of Saint Netherlands, and then captured by the RATE-seq flow.

2) Biotin modified probe capture

TABLE 8

Components	Dosage of
		Biotin-5.8S probe 45	10-100 nM
UvsX recombinase	20-200 nM
		UvsY recombinase accessory proteins	20-200 nM
10 × recombinant buffer	3 µL
		Total volume	20 µL

The 10 × recombinant buffer solution comprises 100-1000 mM Tris, 100-3000 mM potassium acetate, 30-500 mM magnesium acetate, 3-30 mM dithiothreitol, 10-200 mM adenosine triphosphate, etc.

Standing at room temperature for 5 min.

TABLE 9

Components	Dosage of
		The above reaction volume	20 µL
gp32 single-chain binding protein	10-50 nM
		Library constructed in step (1)	0.1-100 ng/uL
Total volume	30 µL

The reaction was carried out at 37 ℃ for 10 min.

After the reaction, 10 ul of Dynabeads ™ M-270 streptavidin magnetic beads washed according to the instructions were added, mixed well and incubated at room temperature for 10 min with rotation. The magnetic beads were separated from the supernatant on a magnetic separation rack and washed 3 times with wash buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20).

High-temperature elution: 20 ul ddH2O was added thereto at 98 ℃ for 5min, and the supernatant was collected.

Biotin competitive elution: 20 ul of ddH2O containing 1-20 mM biotin was added, and the mixture was incubated at room temperature for 30min with rotation, and the supernatant was collected.

And (3) eluting the denaturant: adding 20 ul ddH2O containing 5% -50% biotin, rotary-incubating at room temperature for 30min, and collecting supernatant.

The recovered DNA was quantitatively analyzed, and the quantitative primers are shown in Table 1.

As a result, as shown in FIG. 9, the high temperature elution exhibited higher capture efficiency and capture specificity.

Example 7: comparison of the three capture methods.

In this example, we compared the capture effect of three gene capture modes on 5.8S DNA. The specific implementation mode is as follows:

1) construction of RNA library: 1 ug 293F RNA was subjected to RNA Library construction by Hieff NGS ^ -Ultima Dual-mode RNA Library Prep Kit for Illumina (Cat # 12252) of Saint Netherlands, and then captured by the RATE-seq flow.

2）RATE-seq

Watch 10

Components	Dosage of
		Biotin-5.8S probe 45	10-100 nM
UvsX recombinase	20-200 nM
		UvsY recombinase accessory proteins	20-200 nM
10 × recombinant buffer	3 µL
		Total volume	20 µL

The 10 × recombinant buffer solution comprises 100-1000 mM Tris, 100-3000 mM potassium acetate, 30-500 mM magnesium acetate, 3-30 mM dithiothreitol, 10-200 mM adenosine triphosphate, etc.

Standing at room temperature for 5 min.

TABLE 11

Components	Dosage of
		The above reaction volume	20 µL
gp32 single-chain binding protein	10-50 nM
		Library constructed in step (1)	0.1-100 ng/uL
Total volume	30 µL

The reaction was carried out at 37 ℃ for 10 min.

After the reaction, 10 ul of Dynabeads ™ M-270 streptavidin magnetic beads washed according to the instructions were added, mixed well and incubated at room temperature for 10 min with rotation. The magnetic beads and supernatant were separated on a magnetic separation rack and the beads were washed 3 times with wash buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20). 20 ul ddH2O was added thereto at 98 ℃ for 5min, and the supernatant was collected.

2）CATE-seq(CRISPR-Assistant Target Enrichment and Sequencing)

TABLE 12

Components	Dosage of
		Biotin-5.8S sgRNA	10-1000 nM
dCas9	20-200 nM
		10×NEB Buffer 3.1	3 µL
Total volume	20 µL

Standing at room temperature for 10 min.

Watch 13

Components	Dosage of
		The above reaction volume	20 µL
Library constructed in step (1)	0.1-100 ng/uL
		Total volume	30 µL

The reaction was carried out at 37 ℃ for 60 min.

After the reaction, 10 ul of Dynabeads ™ M-270 streptavidin magnetic beads washed according to the instructions were added, mixed well and incubated at room temperature for 10 min with rotation. The magnetic beads were separated from the supernatant on a magnetic separation rack and washed 3 times with wash buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20). 20 ul ddH2O, 10 ul 2 mg/mL proteinase K was added and the supernatant was removed at 50 ℃ for 15 min.

3) Traditional probe hybridization capture: hybridization Capture was performed using the Integrated DNA Technologies xGen Hybridization and Wash Kit (Cat # 1080577) according to the protocol, and the probes used biotin 5.8S hygrobe-1 and 2.

The recovered DNA was subjected to quantitation and NGS sequencing analysis, and the quantitation primers are shown in Table 1.

The three gene capturing modes are particularly compared as shown in figure 10, the traditional hybridization capturing method is complicated in operation, long in time consumption, low in capturing efficiency and specificity, DNA needs to be denatured, and high in cost, and the gene capturing technology CATE-seq based on CRISPR/dCas9 enhances the capturing specificity and efficiency, reduces the capturing cost, simplifies the operation process and shortens the time, but the design of the capturing probe depends on the PAM sequence of the sgRNA, and the pairing fragment of the sgRNA and the target DNA is shorter, so that the capturing efficiency and the wide applicability are influenced. The gene capture technology RATE-seq of the gene recombinase well overcomes the following defects, has the characteristics of simple operation, extremely long time, high capture efficiency and specificity, high pairing strength, no need of DNA denaturation, small dependence on DNA sequences, low cost and the like, and is suitable for wide gene capture requirements. In FIG. 11 and FIG. 12, we compared the capture effect of the three capture modes on 5.8S DNA, demonstrating the advantages of RATE-seq in capture efficiency and capture specificity.

Example 8: the RATE capture technique is applied to third-generation sequencing.

In this example, we verified the use of RATE gene capture technology for three-generation sequencing, the target gene we verified was BRCA1, and the probe sequence used was SEQ ID NO: 14-17. The specific implementation mode is as follows:

293F genomic DNA was extracted, DNA fragmentation and end repair were performed.

TABLE 14

Components	Dosage of
		293F genomic DNA	1-10 µg
Smearase® Mix（Yeasen）	10 µL
		Total volume	60 µL

The reaction is carried out for 1 min at the temperature of 30 ℃ and for 5min at the temperature of 72 ℃.

2) RATE Capture

Watch 15

Components	Dosage of
		Biotin-BRCA1 probe 1-4	10-100 nM
UvsX recombinase	20-200 nM
		UvsY recombinase accessory proteins	20-200 nM
10 × recombinant buffer	3 µL
		Total volume	20 µL

Standing at room temperature for 5 min.

TABLE 16

Components	Dosage of
		The above reaction volume	20 µL
gp32 single-chain binding protein	10-50 nM
		Total volume	30 µL

And (3) suspending the magnetic beads in the step (2) by using prepared 30 uL of recombinant reaction solution, and reacting for 10 min at 37 ℃. The supernatant was collected by centrifugation. Add 10 ul Dynabeads ™ M-270 streptavidin magnetic beads washed as described, mix well and then mix in the back chamberIncubate for 10 min with warm rotation. The magnetic beads were separated from the supernatant on a magnetic separation rack and washed 3 times with wash buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20). 20 ul ddH was added₂O, at 98 ℃ for 5min, and taking the supernatant.

3) And (5) constructing a library. The recovered DNA was used to construct Pacbio and Nanopore DNA sequencing libraries as per the instructions, and sequenced and analyzed on the corresponding platforms.

As a result, as shown in FIGS. 13 and 14, the RATE gene trapping technique was able to effectively trap the DNA sequence of BRCA1 gene and was applied to the third generation sequencing of PacBio-seq and Nanopore-seq. This suggests that the RATE-seq developed by us is a simple gene capture technique that can be paired with second and third generation sequencing.

Example 9: the use of RATE capture technology targeting Alu sequences for host genome isolation.

The Alu sequence is a highly repetitive sequence on the human genome with 50-100 ten thousand copies. These repeats are scattered over the genome at an average distance of only about 3 kb. Therefore, Alu sequences are important targets for gene capture and isolation in human hosts. The alignment results and probe positions of the human Alu sequence with higher abundance are shown in FIG. 15. In this example, we used RATE gene capture technology to capture and remove human host genome DNA from a sample of a mock standard containing pathogens such as bacterial, fungal, viral DNA, etc. The specific implementation mode is as follows:

1) preparing a DNA standard substance mixture. Genomic DNA of 293F cells was extracted using MolPure Cell/Tissue DNA Kit (Cat # 18700) from the next saint organism. Genomic DNA of Escherichia coli and Bacillus stearothermophilus was extracted using MolPure Bacterial DNA Kit (Cat # 18806) from the next san Jose organism. Genomic DNA of Pichia pastoris and Gibberella moniliforme was extracted using MolPure Fungal DNA Kit of the next saint organism (Cat # 18812). Mycoplasma genomic DNA standards were purchased from Minerva Biolabs, HBV DNA standards from ZeptoMetrix (Cat # P0060), and T7 phage DNA standards from Applichem (Cat # A5197). The concentration of the standard was determined using Qubit. The above DNAs were mixed in the following ratios:

TABLE 17

Components	Dosage of
		Human 293F gDNA	10 µg
Escherichia coli DNA	100 ng
		Bacillus stearothermophilus DNA	10 ng
Pichia pastoris DNA	100 ng
		DNA of erythromyces moniliforme	10 ng
T7 phage DNA	1 ng
		Mycoplasma genomic DNA	0.1 ng
HBV DNA standard	0.01 ng
		Adding DEPC water to	100 µL

And (3) determining the concentration of the DNA standard substance by the Qubit, and diluting the DNA standard substance into a DNA standard substance of 100 ng/muL by DEPC water.

2) RATE Capture

Watch 18

Components	Dosage of
		Biotin-Alu probe 1 and 2	10-100 nM
UvsX recombinase	20-200 nM
		UvsY recombinase accessory proteins	20-200 nM
10 × recombinant buffer	3 µL
		Total volume	20 µL

Standing at room temperature for 5 min.

Watch 19

Components	Dosage of
		The above reaction volume	20 µL
gp32 single-chain binding protein	10-50 nM
		DNA standard	1-100 ng
Total volume	30 µL

The reaction was carried out at 37 ℃ for 10 min. The supernatant was collected by centrifugation. Add 10 ul Dynabeads ™ M-270 streptavidin magnetic beads washed as described, mix well and incubate at room temperature for 10 min with rotation. The magnetic beads and the supernatant were separated on a magnetic separation rack. The supernatant is reserved for use. The magnetic beads were washed 3 times with wash buffer (10-100 mM Tris, 10-2000 mM NaCl, 0.1-3% Tween 20). 20 ul ddH2O was added thereto at 98 ℃ for 5min, and the supernatant was collected.

3) DNA sequencing: DNA recovered from the captured supernatant and the captured magnetic beads was subjected to DNA Library construction using Hieff NGS OnePet DNA Library Prep Kit for Illumina (Cat # 12203) from the St History organisms and sequenced on the Illumina NovaSeq 6000 platform. The sequencing results were compared with human, E.coli, Bacillus stearothermophilus, yeast, Gibberella leucotrichum, T7 phage, Mycoplasma and HBV DNA, respectively, and the ratios of the pathogenic microorganism DNAs in the sequencing results were calculated, as shown in FIG. 16.

The sequencing result is shown in fig. 16, and the human host DNA sequence in the pathogenic standard can be effectively captured and separated by using the human host DNA capture removal mode of the targeted Alu sequence, so that the sensitivity and the accuracy of pathogenic microorganism detection are improved.

In conclusion, we developed a new Recombinase-based gene capture technology and named RATE-seq (recombination-Assistant Target entity and Sequencing). The principle of RATE-seq is that a recombinase complex is used to assist a single-stranded DNA probe to hybridize to a double-stranded target DNA molecule efficiently and specifically at 37 ℃, and then affinity labels carried on the single-stranded DNA probe are used to perform specific enrichment on the target DNA. The method has the advantages of strong long probe pairing, low probe design requirement and the like of the probe hybridization capture technology, and has the advantages of simple operation, short time consumption, no thermal denaturation of DNA, high capture efficiency, low cost and the like of the CRISPR/dCas9 system. Therefore, compared with other capture technologies, the RATE-seq has the obvious advantages of simple operation, high capture efficiency, extremely short time consumption (30 min), good stability, low cost and the like, and is very suitable for gene capture and automation application. In addition, the RATE-seq can also effectively separate and remove human host DNA in pathological samples, and improve the detection RATE of pathogenic microorganisms.

Sequence listing

<110> Histo Histoste of next (Shanghai) Ltd

<120> sequencing method based on gene capture technology

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tgcgtcgatg aagaacgcag ctagctgcga g 31

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tcgtgcgtcg atgaagaacg cagctagctg cgag 34

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<212> DNA

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ggctcgtgcg tcgatgaaga acgcagctag ctgcgag 37

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ggatcactcg gctcgtgcgt cgatgaagaa cgcagctagc tgcgag 46

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<212> DNA

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ggtggatcac tcggctcgtg cgtcgatgaa gaacgcagct agctgcgag 49

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<212> DNA

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gcggtggatc actcggctcg tgcgtcgatg aagaacgcag ctagctgcga g 51

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<212> RNA

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guagccccgg gaggaacccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

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<212> DNA

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ctcttagcgg tggatcactc ggctcgtgcg tcgatgaaga acgcagctag ctgcgagaat 60

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<212> DNA

<213> Artifical sequence

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<212> DNA

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gagacggagt ctctctctgt cacctaggct ggagcgtag 39

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<212> DNA

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ccacctgcct cggcctcctg gagtactggg attacaggcg tgag 44

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<212> DNA

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gggcaggcca ggcacagtgg ctcaagcctg taactgcagc ac 42

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<212> DNA

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gctctcatag agctcatatt ctagtgtggt agacagttga tac 43

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<212> DNA

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gtggctcacg cctgtaatcc cagcatttgg gaggccgagg 40

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<212> DNA

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gatcgagacc atccyggcta amamggtgaa accccgtctc 40

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<212> DNA

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<212> DNA

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gcaagtgcgt tcgaagtgtc 20

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cctagtgggc cacttttggt 20

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ggctagttga ttcggcaggt 20

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ccttccagca gatgtggatc a 21

<210> 25

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<212> DNA

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ttctgcgcaa gttaggtttt gtc 23