CN116355994A

CN116355994A - Method and kit for capturing sequencing range through PCR (polymerase chain reaction) complementary liquid phase hybridization

Info

Publication number: CN116355994A
Application number: CN202310292348.1A
Authority: CN
Inventors: 郑国枭; 颜丙霄; 黄奕孜; 夏金泽; 沈洁; 吴松; 秦杨静; 肖蓁蓁; 刘婉莎; 边佳昕; 陆利; 肖锐
Original assignee: Hangzhou Bosheng Medical Laboratory Co ltd; Zhejiang Bosheng Biological Technology Ltd By Share Ltd
Current assignee: Hangzhou Bosheng Medical Laboratory Co ltd; Zhejiang Bosheng Biological Technology Ltd By Share Ltd
Priority date: 2023-03-23
Filing date: 2023-03-23
Publication date: 2023-06-30

Abstract

The invention discloses a method and a kit for capturing a sequencing range through PCR (polymerase chain reaction) complementary liquid phase hybridization. The method is used for complementarily detecting the gene which cannot be designed by the liquid phase hybridization capture Panel through the PCR complementary liquid phase hybridization capture sequencing, and detecting homologous gene mutation based on the second-generation targeting capture Panel without additional technical means.

Description

Method and kit for capturing sequencing range through PCR (polymerase chain reaction) complementary liquid phase hybridization

Technical Field

The invention relates to the technical field of high-throughput sequencing technology and molecular marking, in particular to a method and a kit for capturing a sequencing range through PCR (polymerase chain reaction) complementary liquid phase hybridization.

Background

Second generation sequencing (Next-generation sequencing, NGS), also known as high throughput sequencing, has two important features: 1. high throughput, second generation sequencing can sequence tens of and millions of DNA molecules in parallel at a time; 2. the length of reading is long, the sequencing process is increased along with the length of reading, and the sequencing quality is reduced. Thus, for genome sequencing, it is necessary to break it into small fragments and re-sequence, splice and align after sequencing.

Many complex structures, such as highly homologous sequences, exist in the sequences of the genome, which can render the results after sequencing ineffective. Many genes have the problem of pseudogenes, the homology of the pseudogenes and the functional genes is generally up to more than 95%, and some can even be up to 99%, and the mutation cannot be detected on true genes in the true and pseudogenes due to the limitation of the technical principle of the second-generation sequencing.

For example: congenital adrenocortical hyperplasia (congenital adrenal hyperplasia, CAH) is a group of diseases caused by enzyme defects in the synthesis pathway of adrenocortical hormone, the 21-hydroxylase defect caused by CYP21A2 gene mutation is one of the most common types, accounts for more than 90% of CAH, and the incidence rate is 1/15000, which is a more common autosomal recessive genetic disease. In the short arm of human chromosome 6, the inactive pseudogene CYP21A1P and the active functional gene CYP21A2 are arranged in series, and the two genes have high homology of 98 percent. Currently, the gold standard for detecting congenital adrenocortical hyperplasia pathogenic gene CYP21A2 mutation is one of the generation sequencing combined with MLPA, and can detect the generated deletion mutation and point mutation. The second generation sequencing Panel cannot design and add congenital adrenocortical hyperplasia pathogenic gene CYP21A2 at present.

The liquid phase hybridization capture sequencing is simply a technology for sequencing a target region after combining a designed nucleic acid probe with the target region by utilizing a base complementary pairing principle. The technique allows for selective targeted sequencing of regions of interest in the genome, such as whole exon sequencing, i.e., detection of the exon regions of nearly all genes. Thousands of target genes of interest can be sequenced at one time by the design of a second generation sequencing Panel. However, also due to technical limitations, highly homologous sequences cannot be designed in.

Similar pathogenic genes such as CYP21A2 gene with pseudogenes cannot be integrated with pathogenic genes of other diseases in a liquid phase hybridization capture sequencing Panel, and additional technical means and equipment support are required, so that the complexity and difficulty of detection are greatly increased. However, it is often necessary to include the homologous gene in the Panel because of its specificity, which is responsible for its pathogenicity. There is an urgent need for a method capable of detecting multiple gene mutations, particularly gene mutations having homologous sequences, in the same system.

Disclosure of Invention

The primary object of the present invention is to provide a method for capturing sequencing scope by PCR-supplemented liquid phase hybridization. The method is used for complementarily detecting the gene which cannot be designed by the liquid phase hybridization capture Panel through the PCR complementary liquid phase hybridization capture sequencing, and detecting homologous gene mutation based on the second-generation targeting capture Panel without additional technical means.

The conventional PCR or special PCR coupled liquid phase hybridization is adopted for capturing and detecting the gene mutation for the first time, including but not limited to a PCR system, a primer design, a coupled detection system, a coupled detection program and a coupled detection data analysis scheme.

A method for capturing sequencing scope by PCR-supplemented liquid phase hybridization, comprising the steps of:

1) Designing primers according to genes with homologous sequences to be detected and performing PCR amplification;

2) Adding the amplified product of the target sequence into genome DNA, and constructing a pre-library;

3) Capturing the pre-library, and sequencing the obtained final library on a machine;

4) And analyzing the sequencing result to determine whether the target gene mutation exists.

The method is specifically one of the following two modes:

mode one: the step 1) adopts common PCR to amplify a target sequence region to obtain a common target product, and the step 3) adopts a probe to capture a pre-library, wherein the probe comprises the target sequence region;

mode two: the step 1) adopts a substance capable of combining with magnetic beads to modify, preferably adopts biotin or ovalbumin modified dNTPs to carry out PCR amplification on a target sequence region, so as to obtain a modified target product, and the modified target product can be captured in the step 3) because of the modified group; the step 3) adopts a probe to capture the pre-library, and the probe does not contain a target sequence region.

The magnetic beads are provided with chains and penicillin which can be combined with biotin or ovalbumin, but the magnetic beads adopt other substances, so long as the magnetic beads can be combined with substances for modifying dNTPs, the reaction of the invention is not influenced, and the magnetic beads are all within the protection scope of the invention.

According to the invention, the conventional PCR (mode one) or the special PCR (mode two) can be selected to amplify the target sequence according to whether the matched liquid phase hybridization capture Panel contains the capture probe of the target sequence.

The conventional PCR is a technical means for amplifying a target sequence through a polymerase chain reaction, and a combined liquid phase hybridization capture Panel designs a probe containing the target sequence and other probes for detecting a target region.

The special PCR is specifically designed and added with special components (namely reagents for modifying dNTPs) compared with the conventional PCR, and the combined liquid phase hybridization capture Panel does not contain probes of a target sequence.

Furthermore, in the step 1), N groups of primers are designed, so that more than N genes with homologous sequences can be detected by the same detection system, and N is more than or equal to 1.

The primers designed in step 1) of the present invention are primers that amplify only true genes, i.e., functional genes, but not homologous pseudogenes. The primer comprises an F primer and an R primer.

The invention is not limited to single-tube single-gene combination single panel, single-tube multi-gene combination single panel and multi-tube multi-gene combination single panel.

Further, at least one of the 4 dNTPs in step 1) is modified with biotin or ovalbumin.

Further, the genomic DNA added in step 2) is used for other gene detection without homologous sequences. Therefore, the invention can realize the purpose of simultaneously detecting multiple genes, including functional genes with pseudogenes, in the same system.

Further, step 4) sequencing result analysis: counting the proportion of mutant forms of mutation sites to all the Reads genotypes of the sites; 0.4 to 0.6 as heterozygous mutations and 0.95 to 1.0 as homozygous mutations.

If the ratio is less than 0.4, it may be caused by the fact that no mutation of the gene occurs, interference of the pseudogene (homologous sequence of true gene), other chromosomal recombination of the target gene or copy number variation occurs; if above 0.6 and below 0.95, it may be caused by interference of pseudogenes (homologous sequences of true genes) or other chromosomal recombination or copy number variation of the gene of interest.

Other chromosomal recombination or copy number variations of the gene of interest are not within the detection range of this method.

The gene with homologous sequence in the invention comprises: at least one of CYP21A2, CFTR, SBDS, PSPH, SLC25A12, SMN1, DCLRE1C, EFL1, CYP11A1, CORO1A, STAT5A, STAT5B, CBS, IGLL1, SLC6A8, ABCD1, SLC16A1, but not limited to the above listed homologous genes.

The gene ID numbers in NCBI are as follows: CYP21A2 Gene ID 1589, CFTR Gene ID 1080, SBDS Gene ID 51119, PSPH Gene ID 5723, SLC25A12 Gene ID 8604, SMN1Gene ID 6606, DCLRE1C Gene ID 64421, EFL 1Gene ID 79631, CYP11A 1Gene ID 1583, CORO1A Gene ID 11151, STAT5A Gene ID 6776, STAT5B Gene ID 6777, CBS Gene ID 875, IGLL 1Gene ID 3543, SLC6A8 Gene ID 6535, ABCD1Gene ID 215, SLC16A 1Gene ID 6566.

The invention provides methods of detection, and types of samples detected include, but are not limited to, human DNA templates from human blood, plasma, cell culture supernatant, saliva, semen, tissue (e.g., amniotic fluid or villus), tissue lysates, bone, or hair.

The detection method provided by the invention is applicable to detection platforms including but not limited to illuminea and bgi.

The second object of the invention is to provide a kit for capturing sequencing scope by PCR (polymerase chain reaction) complementary liquid phase hybridization, which comprises reagents matched with the method.

Further, the kit comprises: the target gene primer, a PCR reagent, a liquid phase hybridization capture reagent and a second generation sequencing reagent.

Still further, the PCR reagents further comprise: biotin or ovalbumin modified dntps.

A third object of the present invention is to provide the use of the reagent used in the above-mentioned method kit for preparing a polygenic sequencing reagent containing homologous sequences.

In conclusion, the detection method and the kit provided by the invention can better solve the problem that the part of the second-generation targeted capture Panel which cannot be detected contains homologous genes, namely true pseudogenes, and achieve the effect of supplementing the detection range through the cooperation of simple PCR and liquid phase hybridization capture without additional complicated technical means. Realizing the purpose that the important genes are not missed in the multi-gene detection; the cost is reduced, and the medical and health value of comprehensive detection is guaranteed.

The method of the invention excludes diagnostic purposes.

Drawings

Fig. 1: example 1 map of the coverage of the CYP21A2 gene zone Reads when detection is carried out using a probe not containing the target gene alone without employing the special PCR coupling liquid phase hybridization capture technique of the invention.

Fig. 2: a: example 1 detection of CYP21A2 Gene mutation Reads coverage map using special PCR coupled liquid phase hybridization Capture according to the invention;

b: example 1 sample DJ21030043 visualizes the results;

c: example 1 sample C21010015 visualizes the results.

Fig. 3: example 2 there is a non-unique alignment Reads overlay of the CYP21A2 gene without PCR.

Fig. 4: example 2 detection of CYP21A2 Gene mutation Reads coverage using conventional PCR coupled liquid phase hybridization Capture according to the invention.

Detailed Description

The following examples are presented to those of ordinary skill in the art to make and evaluate the invention and are merely exemplary of the disclosure and are not intended to limit the scope. Although efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), some errors and deviations should be accounted for. Unless otherwise indicated, temperature is in degrees celsius and pressure is at or near atmospheric pressure.

The invention will be described with respect to the CYP21A2 gene as an example to illustrate the feasibility of the detection method, which includes, but is not limited to, the detection of the gene.

Example 1: detection of CYP21A2 gene mutation by special PCR coupling liquid phase hybridization capture

See SEQ ID NO.1-3.

Step A) PCR amplification:

the PCR system was prepared according to the following standard amplification system:

the preparation steps are as follows: specific components (biotin-modified dNTPs, purchased from Thermo Fisher Scientific) were allowed to stand at room temperature to defrost and vortex to mix, PCR Buffer was vortex to mix, primer and DNA Sample were vortex to mix, then the above reagents were transiently separated, and the PCR Buffer and PCR polymerase were placed on ice and the other were placed at room temperature.

Biotin or ovalbumin modified dNTPs are incorporated into the PCR product, and the product eventually enters the final library during the capture step due to binding to streptavidin magnetic beads, without the need for probes.

Preparing a system: firstly, preparing a premix of other reagents except the DNA Sample in a 200uL PCR tube (on ice), finally, sub-packaging the premix, adding the DNA Sample, blowing to ensure full mixing, and then instantaneously separating and placing on ice.

And (3) PCR amplification: the PCR tube was placed in a pre-programmed PCR instrument and a set cycling program was run.

PCR program

And (3) purifying a PCR product:

VAHTS DNA Clean Beads magnetic beads are balanced at room temperature for half an hour in advance, and 80% ethanol is prepared;

after the PCR is finished, 10 mu L VAHTS DNA clear Beads are added into the sample, and the sample is gently sucked and beaten for 6 times by a pipette and uniformly mixed;

incubating for 5min at room temperature, and placing the PCR tube on a magnetic rack for 3min to clarify the solution;

removing the supernatant, placing the PCR tube on a magnetic rack, adding 200 mu L of 80% ethanol, and standing for 30s;

removing the supernatant, adding 200 mu L of 80% ethanol into the PCR tube, standing for 30s, and thoroughly removing the supernatant (the residual ethanol at the bottom can be removed by using a 10 mu L pipette);

standing at room temperature for 5min to volatilize residual ethanol thoroughly;

adding 25 mu L of Nuclear-free water, taking the PCR tube off the magnetic rack, gently blowing and mixing to re-suspend the magnetic beads, and standing at room temperature for 2min;

placing the PCR tube on a magnetic rack for 2min;

mu.L of the supernatant was pipetted into a 1.5mL centrifuge tube and labeled with sample information.

The purified and recovered product was diluted 100-fold with no nucleic acid water.

Step B): fragmenting, repairing tail ends and adding A:

taking out FEABuffer and FEA Enzyme Mix, thawing on ice, mixing, centrifuging briefly, collecting to bottom, and placing on ice for use.

The PCR instrument was started in advance, and the following procedure was run, keeping the hot lid at 105℃open:

step (a)	Temperature (temperature)	Time
			1	4℃	∞
2	37℃	19min
			3	65℃	30mim
4	4℃	∞

The following reaction system was prepared in a 0.2mL PCR tube:

to each sample, 10. Mu.L of FEA Enzyme Mix was added, and the mixture was blown and mixed with a pipette, and after a short centrifugation, the reaction solution was collected to the bottom of the tube and immediately placed in a PCR machine to start the reaction, and the procedure was followed to step 2.

Immediately after the procedure was completed, the fragmented product was placed on ice.

Joint connection and purification:

rapid Ligation Buffer 3, rapid DNA ligand and DNA Adapters are taken out, thawed and mixed uniformly on ice, collected to the bottom of a tube by short centrifugation and placed on ice for standby;

VAHTS DNA Clean Beads was removed and allowed to equilibrate at room temperature for at least 30min.

The following reaction system was prepared in a 0.2mL PCR tube:

after the system configuration is completed, the reaction solution is collected to the bottom of the tube by using a pipette to gently blow and mix evenly and short centrifugation.

The PCR tube was placed in a PCR apparatus to start the reaction, and the procedure was as follows, keeping the hot lid at 105℃open:

step (a)	Temperature (temperature)	Time
			1	20℃	15min
2	4℃	∞

After the procedure was completed, the PCR tube was centrifuged briefly and the adaptor-ligated product was collected to the bottom of the tube.

Vortex mixing was allowed to equilibrate to room temperature VAHTS DNA Clean Beads and allowed to suspend thoroughly.

mu.L of the magnetic beads were pipetted into 100. Mu.L of the linker ligation product and gently mixed by pipetting. Incubate at room temperature for 5min.

The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, and after the solution was clarified, the supernatant was carefully removed to avoid contact with the beads.

The PCR tube was kept always on a magnetic rack, the beads were rinsed with 200 μl of freshly prepared 80% ethanol, and after 30sec incubation at room temperature the supernatant was carefully removed.

The above rinsing steps were repeated for a total of two rinses.

The PCR tube is kept to be always placed on the magnetic frame, and the magnetic beads are air-dried after being uncapped for 5-10min until no ethanol remains.

The PCR tube was taken out of the magnetic rack, 22.5. Mu.L of nucleic-free water was added, and the mixture was gently stirred and mixed with a pipette, and left at room temperature for 2 minutes.

The PCR tube was briefly centrifuged and placed in a magnetic rack for standing, after the solution was clarified, 20. Mu.L of supernatant was carefully aspirated into the new EP tube, and the beads were not touched.

Library amplification and sorting:

taking out PCR Primer Mix 3for Illumina and VAHTS HiFi Amplification Mix, thawing on ice, mixing, centrifuging briefly, collecting to bottom of tube, and placing on ice for use;

The following reaction system was prepared in a 0.2mL PCR tube:

The PCR tube was placed in a PCR apparatus to start the reaction, the procedure was as follows, keeping the hot lid at 105℃open:

after completion of the PCR reaction, 50. Mu.L of nucleic-free water was added to the amplified product to make up the volume to 100. Mu.L.

Accurately sucking 67 mu L of magnetic beads into 100 mu L of amplified products, and gently beating and uniformly mixing by using a pipette. Incubate at room temperature for 5min.

The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, and after the solution was clarified, the supernatant was carefully transferred to a new PCR tube and the beads were discarded.

20. Mu.L of the magnetic beads were sucked into the supernatant, and gently beaten and mixed by using a pipette. Incubate at room temperature for 5min.

The above rinsing steps were repeated for a total of two rinses.

The PCR tube was removed from the magnetic rack, 22.5. Mu. LNuclease-free water was added, gently mixed by pipetting, and left at room temperature for 2min.

Step C): liquid phase hybridization Capture

Taking out the Hyb Human Block and Universal Block (For Illumina TruSeq Library) from the capture kit preserved at-20deg.C, dissolving in ice box, mixing, and placing on ice or 4deg.C;

500ng of sample pre-library is added into a 1.5mL EP tube, two Block reagents are added according to the following table, and after uniform mixing, the tube is marked as a B tube;

covering a PCR tube of the B tube with a needle cylinder to punch a hole, putting the B tube into a vacuum concentration centrifuge, starting the centrifuge, starting a vacuum pump switch, and starting concentration (concentration time test can be performed by using water with the same volume before concentration, and 40-50 mu L concentration is recommended to 21-23 min);

the B tube was concentrated to a volume of less than 10. Mu.L, then supplemented to 10. Mu.L with Nuclease-free water, gently blotted, mixed, briefly centrifuged and placed on ice for use.

Will be

The One Hyb Buffer is placed in a constant temperature mixer at 50 ℃ for preheating after being melted at room temperature, and 20 mu L of each reaction after complete dissolution (without precipitation and turbidity) is taken>

The One Hyb Buffer is placed in a new 200 mu L PCR tube, a tube cover is covered, and the tube is placed in a PCR instrument for incubation at 50 ℃ for standby, and is marked as a tube A;

placing 5 μl of RNase Block and 2 μl of Probe (for Probe of non-target gene region) into a new 200 μl PCR tube, gently sucking and beating, mixing, centrifuging briefly, placing on ice or 4deg.C for use, and labeling as C tube;

sucking 13 mu L of the tube A and 10 mu L of the tube B from the tube A, adding the mixture into the tube C, vibrating and uniformly mixing, centrifuging briefly, placing the mixture on a PCR instrument, and running the following procedure;

the PCR instrument parameters were set as follows:

Heat lid 85℃
	80℃ for 5min
50℃ hold(16h-24h)

capturing magnetic beads (Cap beads) and taking out from the temperature of 4 ℃, balancing for 30min, and carrying out vortex vibration and resuspension;

placing 50 μl of magnetic beads in a new PCR tube, placing on a magnetic rack for 1min to clarify the solution, and removing supernatant;

taking the PCR tube off the magnetic frame, adding 200 mu L of Binding Buffer, gently sucking and beating for a plurality of times, uniformly mixing, and re-suspending the magnetic beads;

placing on a magnetic rack for 1min, and removing the supernatant;

repeating the steps of re-suspending and removing the supernatant twice, and washing the magnetic beads 3 times;

the PCR tube was removed from the magnetic rack and 180. Mu.L of Binding Buffer was added to gently pipette 6 times to resuspend the beads for use.

Keeping the hybridization product on a PCR instrument, adding 180 mu L Cap beads which are suspended by the magnetic bead counterweight before capturing into the hybridization product, sucking and beating for 6 times by a pipette for uniform mixing, and placing the mixture on a rotary uniform mixer for combining for 30 minutes at room temperature;

placing the PCR tube on a magnetic rack for 2min to clarify the solution, and removing the supernatant;

adding 200 mu L of Wash Buffer 1 into the hybridization product, gently sucking and beating for 6 times, uniformly mixing, placing on a rotary uniformly mixing instrument, cleaning for 15min, then briefly centrifuging, placing a PCR tube on a magnetic rack for 2min, clarifying the solution, and removing the supernatant;

200. Mu.L of preheated at 50℃was added

On Wash Buffer2, gently sucking and beating for 6 times, mixing, placing on a constant-temperature shaking mixer, and incubating at 50deg.C for 10min;

the PCR tube was placed on a magnetic rack for 2min after brief centrifugation and the supernatant removed. Using

One Wash Buffer2 was washed 2 times for a total of 3 times. Finally remove thoroughly->

One Wash Buffer2 (10. Mu.L pipette can be used to remove residue);

keeping the sample on a magnetic rack, adding 200 mu L of 80% ethanol into the PCR tube, standing for 30s, thoroughly removing ethanol solution (residues can be removed by a 10 mu L pipette), and airing at room temperature;

add 30. Mu.L of nucleic-free water to the PCR tube, remove the PCR tube from the magnetic rack, gently pipette 6 times to resuspend the beads for use.

Taking out Post PCR Buffer and Post PCR Primer (25 μm, for ILM) reagent from the kit stored at-20deg.C, dissolving on ice, mixing, and placing on ice for use; the extract Post PCR Polymerase was used immediately after centrifugation.

Enrichment of the DNA library is required after capture, and the reaction system is prepared according to the following table:

the pipettor was adjusted to 40 μl, gently blotted and mixed 6 times, and then immediately placed on a PCR instrument.

Post-PCR experiments

1) Running a PCR instrument program:

the number of Post-PCR cycles at four hours was N-2.

After the PCR is finished, 55 mu L VAHTS DNA clear Beads are added into the sample, and the mixture is gently sucked and beaten for 6 times by a pipette and uniformly mixed;

placing the PCR tube on a magnetic rack for 2min;

pipette 23 μl of supernatant into a 1.5mL centrifuge tube, and mark sample information;

1. Mu.L of library was quantified using Qubit dsDNA HS Assay Kit and library concentrations were recorded, with library concentrations ranging from about 1-20 ng/. Mu.L;

1. Mu.L of library was taken and subjected to fragment length measurement using QIAxcel Advanced System, and the library length was approximately 325bp to 345 bp;

step D) sequencing using a high throughput sequencing platform.

To avoid errors in the results of the Reads alignment to the CYP21A1P gene region, the CYP21A1P gene region is blocked when aligned. At this time, the Reads of the CYP21A2 gene are all sequence information of the CYP21A2 gene introduced by the PCR product.

When a special PCR coupled liquid phase hybridization capture technique is not used, since the probe used does not contain the CYP21A2 gene region, the CYP21A2 gene region is not covered by Reads when the probe is used alone for detection, as shown in FIG. 1.

When the CYP21A2 gene mutation was detected using special PCR-coupled liquid phase hybridization capture, the coverage was as shown in FIG. 2.

Performing conventional SNV detection analysis on the sequencing result, putting the sequencer off the machine, performing fastq on the original data, filtering low-quality bases by using fastp software, removing joints to obtain clean fastq, and comparing the clean fastp with a reference genome by using comparison software bwa to obtain a bam file; then, base correction and repeated reads marking are carried out on the bam by using GATK; finally, the processed bam file is subjected to Variant rolling by GATK HaplotypeCaller to obtain a vcf file. The point mutations detected in the region of interest are selected from the vcf file for analysis.

At this time, the target region coverage Reads are all true genes, and thus are not different from other genes in SNV analysis.

For example, one SNP site, C in the CYP21A2 gene wild type, A in the CYP21A1P gene wild type, and when no special PCR coupled liquid phase hybridization capture technique is used and the hybridization probe does not design the CYP21A2 gene, the region has no Reads in the final sequencing data. After the special PCR coupling liquid phase hybridization capture technology is used, the total Reads is about 200X, and at the moment, the Reads all reflect the mutation condition of the CYP21A2 gene and are not influenced by the mutation of the CYP21A1P gene. At this time, since there is no probe containing the target gene region, i.e., there is no Reads of the CYP21A2 gene and the CYP21A1P gene in the template genomic DNA, if the CYP21A2 gene wild type is C and T heterozygous mutation occurs, the sequencing result should be 100C, 100T, and the T ratio theory is 0.5.

At this time, the mutation of the sample is detected as follows:

/>

EXAMPLE 2 detection of CYP21A2 Gene mutation by ordinary PCR coupled liquid phase hybridization Capture

Primer:

step A): PCR amplification

the preparation steps are as follows: mixing the PCR Buffer with vortex, mixing the primer and the DNA Sample with vortex, then instantly separating the above reagents, placing the PCR Buffer and the PCR polymerase on ice, and placing the other on room temperature.

Preparing a system: firstly, preparing a premix of other reagents except the DNA Sample in a 200 mu L PCR tube (on ice), finally, sub-packaging the premix, adding the DNA Sample, blowing to ensure full mixing, and then instantaneously separating and placing on ice.

PCR program

After the amplification reaction is finished, the whole reaction system is diluted by 1000 times by adding non-nucleic acid water.

Step B): fragmenting, end repair and addition A

And taking out the FEA Buffer and the FEA Enzyme Mix, thawing on ice, uniformly mixing, briefly centrifuging, collecting to the bottom of a tube, and placing on ice for standby.

The following reaction system was prepared in a 0.2mL PCR tube:

because the embodiment has no purification step, the influence of residual enzyme and ions of an amplification system on the library construction step is reduced after dilution; and too high a product concentration will result in too much data in this region in the end, so that 1000-fold dilution is required for recovery of PCR DNA.

Joint connection and purification:

The following reaction system was prepared in a 0.2mL PCR tube:

step (a)	Temperature (temperature)	Time
			1	20℃	15min
2	4℃	∞

The above rinsing was repeated for a total of two times.

The PCR tube was centrifuged briefly and placed in a magnetic rack for standing, after the solution was clarified, 20. Mu.L of supernatant was carefully aspirated into the new EP tube, without touching the beads

Library amplification and sorting:

The following reaction system was prepared in a 0.2mL PCR tube:

The above rinsing was repeated for a total of two times.

Step C): liquid phase hybridization Capture

covering a PCR tube of the B tube with a needle cylinder to punch a hole, putting the B tube into a vacuum concentration centrifuge, starting the centrifuge, starting a vacuum pump switch, and starting concentration (the concentration time test can be carried out by using the same volume of water before concentration, and 40-50ul concentration is recommended to be carried out for 21-23 min);

Will be

placing 5 μl of RNase Block and 2 μl of Probe (containing target gene region and other genes) into a new 200 μl PCR tube, gently sucking and beating, mixing, centrifuging briefly, placing on ice or 4deg.C for use, and marking as C tube;

the PCR instrument parameters were set as follows:

Heat lid 85℃
	80℃ for 5min
50℃ hold(16h-24h)

placing on a magnetic rack for 1min, and removing the supernatant;

200. Mu.L of preheated at 50℃was added

One Wash Buffer2 (10. Mu.L pipette can be used to remove residue);

30 mu LNuclease-free water was added to the PCR tube, the PCR tube was removed from the magnetic rack, and the beads were gently resuspended for 6 times.

Post-PCR experiments

1) Running a PCR instrument program:

the number of Post-PCR cycles at four hours was N-2.

placing the PCR tube on a magnetic rack for 2min;

step D): sequencing was performed using a high throughput sequencing platform.

If PCR is not performed, then, since the probe used contains the CYP21A2 gene region, the CYP21A2 gene region will be covered by a non-unique comparison Reads as shown below when the probe is used alone for detection. Because the CYP21A2 gene and the CYP21A1P gene are highly homologous regions, the designed CYP21A2 capture probe can also capture the DNA fragment of the CYP21A1P gene, and the sequenced ready cannot accurately distinguish whether the CYP21A2 gene or the CYP21A1P gene belongs to. As shown in fig. 3:

when the CYP21A2 gene mutation was detected using conventional PCR-coupled liquid phase hybridization capture, the coverage is as shown in FIG. 4:

at this time, the CYP21A1P gene region is blocked at the time of alignment in order to avoid errors in the results of the Reads alignment to the CYP21A1P gene region. At this time, the Reads of the CYP21A2 gene include the following, 1.pcr products as templates for sequencing Reads;2. the CYP21A2 gene in the template genome DNA is used as a template for sequencing Reads;3. the CYP21A1P gene in the template genome DNA is used as a template for sequencing Reads.

For example, one SNP position, C for the CYP21A2 gene wild type and a for the CYP21A1P gene wild type, total Reads 10000 after common PCR coupled liquid phase hybridization capture technique, wherein 1.pcr products are template sequencing Reads 4900C, 4900T; 2. the CYP21A2 gene in the template genome DNA is used as a template to sequence the Reads into 50C and 50T; 3. the CYP21A1P gene in the template genome DNA is used as a template to sequence the Reads into 50A and 50G. Then, it is found that the theoretical ratio of T is 0.495 and the theoretical ratio of G is 0.005, and thus the SNP of the CYP21A2 gene of the sample is C/T.

The specific sample detection results are as follows:

/>

Claims

1. a method for capturing sequencing scope by PCR-supplemented liquid phase hybridization, comprising the steps of:

2. The method according to claim 1, characterized in that it is in particular any one of the following two ways:

mode two: the step 1) adopts a substance capable of being combined with magnetic beads to modify, preferably biotin or ovalbumin modified dNTPs to carry out PCR amplification on a target sequence region, so as to obtain a modified target product, and the modified target product can be captured in the step 3) because of the modified group; the step 3) adopts a probe to capture the pre-library, and the probe does not contain a target sequence region.

3. The method according to claim 1, wherein in step 1), more than N genes with homologous sequences can be detected by the same detection system by designing N sets of primers, N.gtoreq.1.

4. The method of claim 2, wherein at least one of the 4 dntps in step 1) is modified with biotin or ovalbumin.

5. The method according to claim 1, wherein the genomic DNA added in step 2) is used for the detection of other genes without homologous sequences.

6. The method of claim 1, wherein step 4) sequencing result analysis: counting the proportion of mutant forms of mutation sites to all the Reads genotypes of the sites; 0.4 to 0.6 as heterozygous mutations and 0.95 to 1.0 as homozygous mutations.

7. The method of claim 1, wherein the gene having a homologous sequence comprises: at least one of CYP21A2, CFTR, SBDS, PSPH, SLC25A12, SMN1, DCLRE1C, EFL1, CYP11A1, CORO1A, STAT5A, STAT5B, CBS, IGLL1, SLC6A8, ABCD1, SLC16A 1.

8. A kit for capturing sequencing context by PCR-supplemented liquid phase hybridization, comprising reagents for use with the method of any one of claims 1-7.

9. The kit of claim 8, comprising: the target gene primer, a PCR reagent, a liquid phase hybridization capture reagent and a second generation sequencing reagent.

10. Use of a reagent for use in combination with the method of any one of claims 1 to 7 for the preparation of a polygenic sequencing reagent comprising homologous sequences.