Y chromosome sequencing method based on liquid phase probe capture
阅读说明：本技术 一种基于液相探针捕获的y染色体测序方法 (Y chromosome sequencing method based on liquid phase probe capture ) 是由 文少卿 王凌翔 徐旭鼎 雷波 于 2020-12-22 设计创作，主要内容包括：一种基于液相探针捕获的Y染色体测序方法,包括如下步骤：S1.提取基因组DNA并片段化；S2.对片段化的DNA进行末端修复；S3.在DNA片段两端加上测序接头并纯化连接产物；S4.PCR扩增连有接头的片段DNA进行文库扩增并纯化；S5.文库质量控制；S6.探针区域的挑选及优化,并设计局部密集式探针；S7.将DNA文库和探针进行文库杂交反应,捕获目标DNA片段；S8.将捕获的目标DNA片段进行分离；S9.对分离后的目标DNA片段进行高通量测序。本发明利用液相探针捕获技术,通过对探针区域进行挑选与优化,对Y染色体的测序长度更长,就能够测试到更多的突变,实现了人类Y染色体20M区域的捕获测序。(A method for sequencing Y chromosome based on liquid phase probe capture comprises the following steps: s1, extracting and fragmenting genome DNA; s2, performing end repair on the fragmented DNA; s3, adding sequencing connectors at two ends of the DNA fragment and purifying a connection product; s4, amplifying the fragment DNA connected with the joint by PCR to perform library amplification and purifying; s5, controlling the quality of the library; s6, selecting and optimizing a probe area, and designing a local intensive probe; s7, carrying out library hybridization reaction on the DNA library and the probe to capture a target DNA fragment; s8, separating the captured target DNA fragments; and S9, carrying out high-throughput sequencing on the separated target DNA fragment. The invention utilizes the liquid phase probe capture technology, selects and optimizes the probe region, has longer sequencing length of the Y chromosome, can test more mutations, and realizes the capture sequencing of the 20M region of the human Y chromosome.)
1. A Y chromosome sequencing method based on liquid phase probe capture is characterized by comprising the following steps:
s1, extracting genome DNA, and fragmenting the genome DNA;
s2, performing end repair on the fragmented double-stranded DNA, and adding a base A to the 3' end of the DNA fragment;
s3, adding sequencing adapters at two ends of the DNA fragment and purifying a connection product to remove unconnected adapter sequences;
s4, amplifying the fragment DNA connected with the joint by PCR to perform library amplification and purifying;
s5, controlling the quality of the library;
s6, selecting and optimizing a probe area, and designing a local intensive probe;
s7, carrying out library hybridization reaction on the DNA library obtained in the step S5 and the probe obtained in the step S6 to capture a target DNA fragment;
s8, separating the captured target DNA fragments;
s9, carrying out high-throughput sequencing on the separated target DNA fragment;
selecting and optimizing the probe region in the step S6 to obtain a Y chromosome reference sequence of hg38 version, intercepting 70bp as the length, taking 35bp as a sliding window, obtaining 70bp unit fragments overlapping and covering the whole NRY region, then respectively using BWA-backstrack, BWA-SW, BWA-MEM and bowtie2 to map all the fragments back to the reference genome of hg38, then grading the fragments according to the uniqueness and quality of all the fragments mapping to Y chromosome and the consistency of different mapping methods, finally removing the fragments with worst scores, and splicing the rest fragments to obtain a candidate region for capture sequencing;
the locally dense probes are designed in a staggered mode.
2. The method of claim 1, wherein the genomic DNA of step S1 is derived from a human.
3. The method of claim 1, wherein the DNA fragmentation of step S1 is ultrasonic random break, transposase cleavage or restriction endonuclease cleavage.
4. The method of claim 4, wherein the DNA fragmentation of step S1 is ultrasonic random break.
5. The method of claim 1, wherein the dense probes of step S6 are labeled with biotin.
6. The method of claim 5, wherein the step S7 of capturing the target DNA fragments is to mix streptavidin-bound magnetic beads with the hybridization mixture, and the target fragments to be captured are indirectly bound to the magnetic beads via the probes.
7. The method of claim 6, wherein the separating of step S8 is eluting the target DNA fragment.
8. The method of claim 1, wherein the probe comprises SEQ ID No. 1 to SEQ ID No. 68.
9. The method of claim 1, wherein the step of isolating the target DNA fragments in step S8 further comprises amplifying the captured library and performing high throughput sequencing.
10. The method of claim 1, wherein the high throughput sequencing of step S9 is a DNB sequencing technique.
Technical Field
The invention relates to the technical field of biology, in particular to a Y chromosome sequencing method based on liquid phase probe capture.
Background
The Y chromosome is unique to males and has the characteristic of paternal inheritance. Since the specific region (non-recombinant region) of the Y chromosome is not recombined with the X chromosome, the specific region of the Y chromosome presents stable haplotype paternal inheritance and has high conservation and specificity, and sequence change in the genetic process is only caused by mutation, so that the following information can be obtained by carrying out a complete sequence test of the Y chromosome: 1) discovering a unique Y-SNP marker locus of a paternal family; 2) accurately calculating the accurate differentiation time in the paternal family for hundreds of years; 3) determining the relation of the religion, determining the venation of each branch system in the family, solving the problem of fuzzy or wrong description in the family tree, perfecting family tree information and restoring the history of migration and development in the family history. Therefore, the sequencing of the Y chromosome can be applied to judicial identification related to males, such as family investigation, paternal tracing and the like, and has very important value in forensic medicine application.
Current forensic Y chromosome detection is mainly known STR (short tandem repeat) and SNP (single nucleotide polymorphism) typing: the specific steps comprise (1) STR and SNP typing based on Sanger sequencing, wherein ddNTP is doped in the DNA synthesis process to generate a series of end-terminated DNA chains, then fragments with different sizes are separated through gel electrophoresis, and finally, the fragments can be detected by X-ray film autoradiography or non-isotopic labeling. (2) STR and SNP typing based on second-generation sequencing has the main principle that: STR and SNP locus capture. At present, the next generation sequencing method mainly adopts a multiple PCR method to capture a plurality of sites simultaneously, and simultaneously carries out typing on the sites less than or equal to 1000, thereby realizing the specific capture of target STR and SNP sites. And adding a sample label. After the first round of multi-site capture amplification is completed in the multiplex PCR, a sample tag and a sequencing adapter are added on the basis of the first round of PCR products, and then the second round of PCR is performed. Sequencing and result interpretation. After the sequencing is carried out on the computer, hundreds of reads can be read from the same STR and SNP position of each sample, the specific information of each sequence can be clearly seen, and the judgment of the locus genotype is realized through the cluster analysis of the locus. There are also several Y chromosome commercialization services based on next generation sequencing. The 'Big Y' test service provided by Family Tree DNA (ftDNA) detects a region of 11-14M bp of Y chromosome, and the test depth is 55-80X. FGC provides the "Y-Elite" test service, testing a region of about 12-16 Mbp, with a test depth of 40-80X.
Because the current forensic Y chromosome detection is mainly based on known STR and SNP typing, the accuracy requirement of actual detection cannot be met. Has the following disadvantages: the unknown information can not be searched without considering other STR and SNP loci with distinguishing capability. Although the detection result can give a known judgment of the upstream haplotype group, it cannot know more detailed information of the downstream unknown locus, and therefore only very rough information of the paternal type can be obtained. Secondly, new sites are continuously discovered along with academic and commercial researches, the results and information obtained based on the previous detection need to be updated again, and the chips need to be continuously updated again; and thirdly, in the detection process, too many designed points may interfere with each other, so that the test results are contradictory. For example, patent CN106399543A discloses a forensic second-generation sequencing kit based on 74Y chromosome SNP genetic markers, patent CN110846420A discloses a rapid mutation Y chromosome STR typing system, a next-generation sequencing typing kit, a typing method, applications, and the like, and the Y chromosome sequencing is mainly based on known STR and SNP typing, has a small region included in sequencing, cannot achieve the purpose of forensic fine pedigree research, and has high detection cost. The current commercialized kit based on the second generation sequencing has the following defects: the sequencing region is small, more unique private sites belonging to the family can not be found, different branches in the family can not be distinguished, the error of generation estimation is large, and the actual precision requirement can not be met. Secondly, the detection cost is high, the method highly depends on imported sequencing platforms and related reagents, and the price is high.
Therefore, it is highly desirable to develop a method for sequencing Y chromosome, which has a longer sequencing length for Y chromosome, can find more STR and Y-SNP sites with distinguishing capability, and has a larger sequencing region, so as to meet the requirement of forensic fine pedigree research.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide a Y chromosome sequencing method based on liquid phase probe capture.
The above object of the present invention is achieved by the following technical solutions:
a method for sequencing Y chromosome based on liquid phase probe capture comprises the following steps:
s1, extracting genome DNA, and fragmenting the genome DNA;
s2, performing end repair on the fragmented double-stranded DNA, and adding a base A to the 3' end of the DNA fragment;
s3, adding sequencing adapters at two ends of the DNA fragment and purifying a connection product to remove unconnected adapter sequences;
s4, amplifying the fragment DNA connected with the joint by PCR to perform library amplification and purifying;
s5, controlling the quality of the library;
s6, selecting and optimizing a probe area, and designing a local intensive probe;
s7, carrying out library hybridization reaction on the DNA library obtained in the step S5 and the probe obtained in the step S6 to capture a target DNA fragment;
s8, separating the captured target DNA fragments;
s9, carrying out high-throughput sequencing on the separated target DNA fragment;
selecting and optimizing the probe region in the step S6 to obtain a Y chromosome reference sequence of hg38 version, intercepting 70bp as the length, taking 35bp as a sliding window, obtaining 70bp unit fragments overlapping and covering the whole NRY region, then respectively using BWA-backstrack, BWA-SW, BWA-MEM and bowtie2 to map all the fragments back to the reference genome of hg38, then grading the fragments according to the uniqueness and quality of all the fragments mapping to Y chromosome and the consistency of different mapping methods, finally removing the fragments with worst scores, and splicing the rest fragments to obtain a candidate region for capture sequencing;
the locally dense probes are designed in a staggered mode.
The invention firstly selects and optimizes the probe region, the design of the probe region removes the recombination region of the Y chromosome, but for the remaining 20M region, some regions are high GC, self-repeat and palindrome, insertion and deletion regions, the traditional probe design adopts the shingled design and is tiled 2X probe tiling, the invention adopts the staggered design of the probe, the probe density is pertinently increased for the complex structures, so that the uniformity and the coverage of the final captured product are optimal, then the probe and the DNA library are hybridized, in the hybridization process, the probe can be combined with the target DNA fragment by the complementary combination principle of the nucleic acid sequence, finally the captured target DNA fragment is separated out for sequencing, and the Y chromosome region with longer sequencing length and near 20M is obtained. The length of the obtained sequence is close to the limit of the available region on the Y chromosome, and more mutations can be tested, so that the differentiation time between different branches can be calculated more accurately. Roughly estimated, mutations occurring between 2 to 3 generations of humans can be detected, corresponding to mutations occurring every 70 years or so, which is already near the limit of the Y chromosome mutation rate. The key point of the invention is to obtain a candidate region for capture sequencing, and after the candidate region is obtained, the probe is designed according to the principle of staggered design of the probe, and the specific probe sequence is not limited.
Specifically, the genomic DNA obtained in step S1 is derived from a human.
Preferably, the DNA fragmentation in step S1 is ultrasonic random break, transposase cleavage or restriction endonuclease cleavage.
Further preferably, the DNA fragmentation is ultrasound random breaks.
Preferably, the dense probes of step S6 are labeled with biotin.
Preferably, in step S7, the target DNA fragment to be captured is obtained by mixing magnetic beads to which streptavidin is bound with a hybridization mixture, and the target fragment to be captured is indirectly bound to the magnetic beads via a probe.
Preferably, the separation in step S8 is to elute the target DNA fragment.
Preferably, after the target DNA fragment is isolated, the method further comprises amplifying the captured library and performing high-throughput sequencing.
Preferably, the probe comprises SEQ ID NO 1 to SEQ ID NO 68.
Preferably, the high throughput sequencing of step S9 is a DNB sequencing technique.
Compared with the prior art, the invention has the following beneficial effects:
the invention is based on the liquid phase probe capture technology, firstly selects and optimizes the probe area, and then designs the capture probe by adopting a new probe primer design mode, thereby realizing the capture sequencing of the near 20M area of the human Y chromosome, the sequencing length of the invention is close to the limit of the available area on the Y chromosome, more mutations can be tested, more STR and Y-SNP loci with distinguishing capability can be found, thereby more accurately calculating the differentiation time between different branches, and providing a new thought and scheme for new genetic marker screening and generation calculation between samples in the forensic research.
Drawings
FIG. 1 is a schematic flow chart of a Y chromosome liquid phase probe capture sequencing method based on DNB technology.
FIG. 2 is a schematic diagram of the probe design of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1
A liquid phase probe capture sequencing method for Y chromosome based on DNB technology is disclosed, and its operation flow is shown in FIG. 1. The method mainly comprises the following steps:
method and device
1) Genome DNA fragmentation-Covaris interruption instrument
1. Check the level of fresh deionized water in the Covaris tank at 12-point level to ensure that the water level has not broken the glass portion of the tube.
2. The cooling temperature is set between 2 ℃ and 5 ℃ to ensure that the temperature display water temperature is 5 ℃ when the water heater is used.
3. The air exhaust button on the control panel is opened for a minimum of 30min before use, and experimental operation is carried out by referring to the usage instruction of the Covaris instrument.
4. Samples were diluted to 35 μ L (200ng) in 1.5mL PCR tubes using a1 XLow TE Buffer.
5. The diluted sample is carefully added into the breaking tube to avoid bubbles in the process, the breaking tube is placed into the breaking instrument, and recommended parameters are set as shown in the following table (or can be adjusted according to the model of the instrument).
6. A sample with the main peak of detection of the sample after normal fragmentation of 1 μ L of the sample was taken and subjected to fragment detection by Agilent 2100(DNA1000 Assay) to be approximately 150bp-200 bp.
2) DNA library construction
Step1 end repair/dA tail addition
1. After thawing the reagents in the following table, the reagents were mixed by inversion and placed on ice for use.
2. The following reaction system was prepared in a sterile PCR tube:
3. gently flick or shake the mixture by using a pipettor, and centrifuge the reaction solution to the bottom of the tube by short-time centrifugation.
4. The PCR tube was set in a PCR apparatus, and the reaction program shown in Table 5 was set to carry out a terminal repair/dA tail addition reaction (hot lid 105 ℃ C.).
Step2 linker ligation
1. The Adapter was diluted to the appropriate concentration based on the amount of Input DNA.
2. The reagents in the following table were thawed, mixed by inversion, and placed on ice for use.
3. The reaction systems shown in the following table were prepared in a Step1 product PCR tube.
4. Gently flick or shake the mixture by using a pipette, and then, briefly centrifuge the mixture to collect the reaction solution to the bottom of the tube.
5. The PCR tube was placed in a PCR apparatus, and the reaction program shown in the following table was set to perform a linker reaction (hot lid 105 ℃ C.).
Purification of Step3 ligation product
Note that: before starting the procedure, please confirm that the Agencourt AMPure XP beads had equilibrated to room temperature.
1. And a 0.8X magnetic bead purification system is configured.
2. Gently sucking, stirring uniformly, standing and incubating at room temperature for 5min for 6 times, and placing the PCR tube on a magnetic frame for 3min to clarify the solution.
3. The supernatant was removed, the PCR tube was placed on a magnetic stand, 200. mu.L of 80% ethanol solution was added to the PCR tube, and the tube was allowed to stand for 30 seconds.
4. The supernatant was removed, 200. mu.L of 80% ethanol solution was added to the PCR tube, and the supernatant was removed thoroughly after standing for 30s (it was recommended to remove the residual ethanol solution at the bottom using a 10. mu.L pipette).
5. Standing at room temperature for 35min to completely volatilize residual ethanol.
6. Add 22. mu.L of nucleic-free water to remove the PCR tube from the magnetic frame, gently pipette the resuspended beads to avoid air bubbles, and let stand at room temperature for 2 min.
7. The PCR tube was placed on a magnetic stand for 2min to clarify the solution.
8. Pipette 20. mu.L of the supernatant, transfer to a new PCR tube (on an ice box), mark the reaction tube with the sample number, and prepare for the next reaction.
Step 4 library amplification
1. Is configured as the following reaction system
2. And gently beating the reaction solution by using a pipette 10 times, uniformly mixing the reaction solution, and carrying out transient instantaneous centrifugation to collect the reaction solution to the bottom of the tube.
3. The PCR reaction was carried out (hot lid 105 ℃ C.) according to the procedure shown in the following table.
Magnetic bead purification of Step 5 amplification product
Note that: before starting the procedure, please confirm that the Agencourt AMPure XP beads had equilibrated to room temperature.
1. And a 0.9X magnetic bead purification system is configured.
2. Vortex well and mix the 0.2mL centrifuge tube with ligation product and magnetic beads and incubate for 5min at room temperature.
3. A 0.2mL centrifuge tube was placed on a magnetic rack for incubation until the solution was clear and the supernatant carefully removed.
4. The 0.2mL centrifuge tube was kept in the magnetic rack, 100. mu.L of 80% ethanol was added to rinse the beads, and after incubation at room temperature for 30sec, 80% ethanol was carefully removed.
5. The magnetic beads were washed 1 time with 80% ethanol.
6. And keeping the 0.2mL centrifuge tube in the magnetic frame all the time, and drying the magnetic beads at room temperature for 3-5 min.
7. The 0.2mL centrifuge tube was removed from the magnetic frame, 22. mu.L of nucleic-Free Water was added to resuspend the beads thoroughly, and the mixture was allowed to stand at room temperature for 5 min.
8. Place 0.2mL centrifuge tube in magnetic rack and after the solution cleared, carefully remove 20. mu.L of supernatant into a new 0.2mL centrifuge tube. The purified DNA library can be stored at 4 ℃ for one week or stored at-20 ℃ for a long time.
Step 6 library quality control
In general, the quality of the constructed library can be evaluated by concentration detection and length distribution detection.
3) DNA library hybrid Capture
Selection and optimization of Step1 probe region
In order to obtain an optimal sequencing region, the present invention re-evaluates the non-recombinant region of the Y chromosome (NRY region). First, a Y chromosome reference sequence of hg38 version was obtained, using 70bp as the truncation length and 35bp as the sliding window, to obtain a 70bp unit fragment overlapping the entire NRY region. Then, all fragments were mapped back to the reference genome of hg38 using the four methods BWA-backprack, BWA-SW, BWA-MEM and bowtie2, respectively. The fragments are then scored according to their uniqueness to the Y chromosome, quality and consistency using different mapping methods. And finally, removing the fragments with the worst scores, and splicing the rest fragments to obtain a candidate region for capture sequencing.
Step2 local dense probe design scheme: liquid phase probe capture technology
Compared with the traditional probe design which is a imbricated design, the invention adopts a staggered design, as shown in figure 2. And designing probes according to the obtained candidate regions, and intensively increasing the probes according to regions with high GC, high repetition, palindromic structure and the like to improve the capture efficiency. Wherein the probe comprises SEQ ID NO 1-68;
step3 library hybridization reaction
1. The following reagents were mixed in a 0.2mL centrifuge tube.
2. And (4) mixing the closed reaction system by vortex, centrifuging instantaneously, and collecting all names to the bottom of the tube.
3. The reaction mixture was concentrated by freezing the centrifuge tube and evaporated to dryness.
4. To the tube containing the dried DNA, the following reagents were added in order.
5. Mixing with vortex oscillator, incubating at room temperature for 5-10min, mixing with vortex oscillator, and centrifuging instantly.
6. 0.2mL centrifuge tubes were placed on a PCR instrument and incubated according to the following procedure.
7. Immediately place 0.2mL centrifuge tubes in a PCR apparatus with a hot lid that has reached 75 ℃ for 4h or overnight hybridization.
Step 4 washing streptavidin magnetic bead
Note that: confirmation of Dynabeads before washing of magnetic beadsTMM-270Streptavidin (ProbeCap SA beads) beads had equilibrated to room temperature.
1. The beads were mixed well by vortexing for 15 sec.
2. Transfer 100. mu.L of ProbeCap SA Beads to a new 1.5mL centrifuge tube.
3. The centrifuge tube was placed on a magnetic stand to thoroughly separate the magnetic beads from the solution.
4. The supernatant was removed and the beads were retained.
5. The following cleaning was performed:
add 150. mu.L of 1 XB to the beads and vortex for 10 sec.
And transferring the centrifugal tube to a magnetic frame, and completely separating the magnetic beads from the solution.
③ carefully remove the supernatant.
6. Repeating the step 5, and cleaning twice.
7. Then 100. mu.L of 1 XBWB solution was added and vortexed for 30 seconds.
8. The magnetic beads and 1 × BWB mixture were transferred to a new 0.2mL centrifuge tube.
9. And adsorbing the magnetic beads by using a magnetic rack, removing the supernatant, keeping the magnetic beads, and immediately performing Step3 to avoid the magnetic beads from being excessively exposed in the air.
Step 5 magnetic bead capture
1. A0.2 mL centrifuge tube from Step2.9 was placed in the PCR apparatus which had been preheated to 65 ℃.
2. 16. mu.L of the hybridization solution hybridized at 65 degrees in Step1.8 was transferred to a 0.2mL centrifuge tube prepared in Step2.9.
3. Repeatedly pipetting the solution 10 times by using a pipette, and fully and uniformly mixing.
4. The incubation was performed as follows.
5. Take out 0.2mL centrifuge tube from PCR instrument every 10min, shake for 5sec, gently resuspend reaction solution, and put into PCR instrument again for reaction.
Step 6 washing
Note that: ensure that sufficient 1 xwi, 1 xwii, 1 xwiii has equilibrated to room temperature.
1. The PCR instrument was heated to 65 ℃ at least 30min in advance, 300. mu.L/reaction of 1X S-W and 125. mu.L/reaction of 1 XWI were loaded into new 0.2mL centrifuge tubes, respectively, and these two washes were placed on the 65 ℃ PCR instrument for at least 10 min.
2. To a 0.2mL centrifuge tube of Step3.5 was added 120. mu.L of 1 XWI preheated at 65 ℃ and vortexed for 3 sec.
3. The 0.2mL centrifuge tube was placed on a magnetic rack and the beads were completely separated from the solution.
4. Put 0.2mL centrifuge tube into PCR instrument to preheat: 10-20 sec at 65 ℃.
5. Quickly putting back the magnetic frame, and removing the supernatant as soon as possible after clarification.
6. And (4) carrying out hot cleaning.
Adding 140 mu L of 1X S-W preheated at 65 ℃ into a 0.2mL centrifuge tube, and slowly sucking for 10 times to avoid generating bubbles.
Placing the 0.2mL centrifuge tube in a PCR instrument for warm bath and accurately timing: 65 ℃ for 5 min.
③ put 0.2mL centrifuge tube on magnetic frame for 2min, and remove the supernatant rapidly with pipettor.
7. Repeat step 6 once, heat wash beads twice in total.
8. And (5) cleaning at room temperature.
140 μ L of room temperature 1 XWI was added to a 0.2mL centrifuge tube. The vortex was followed for 30s with 4 oscillations for 2min to fully suspend the beads.
② placing 0.2mL centrifuge tube on a magnetic frame for 1min, and quickly removing supernatant by a pipettor.
③ adding 140. mu.L of room temperature 1 XWII, adding into a 0.2mL centrifuge tube, whirling for 30s, oscillating for 1min at 2 intervals, and fully suspending the magnetic beads.
And fourthly, placing the 0.2mL centrifuge tube on a magnetic frame for 2min, and quickly removing the supernatant.
Fifthly, adding 140 mu L of room temperature 1X WIII, adding into a 0.2mL centrifuge tube, and carrying out vortex oscillation for 30s to fully suspend the magnetic beads.
Sixthly, transferring the mixed suspension into a new 0.2mL centrifuge tube, placing the centrifuge tube on a magnetic rack to completely separate the magnetic beads from the solution, and removing the supernatant.
9. Resuspension magnetic bead
Add 25. mu.L of clean-Free Water and remove the 0.2mL centrifuge tube from the magnetic stand.
And repeatedly sucking and beating for 10 times by using a pipettor to ensure that all the magnetic beads are fully resuspended.
post-Step 7 capture library amplification
1. The PCR reaction system was prepared as shown in the following table.
2. Vortex for 3sec, and gently throw or flash centrifuge to ensure that the beads remain suspended in solution.
3. A0.2 mL centrifuge tube was placed in a PCR instrument with a hot lid temperature of 105 ℃ and PCR amplification was performed according to the following procedure.
Step 8 purification of post-Capture PCR products
1. And configuring a 1.0X magnetic bead purification system.
2. Vortex well and mix 0.2mL centrifuge tube mixed with PCR product and magnetic beads, incubate 5min at room temperature.
3. A 0.2mL centrifuge tube was placed on a magnetic rack for incubation until the solution was clear and the supernatant carefully removed.
4. The 0.2mL centrifuge tube was kept in the magnetic rack, 100. mu.L of 80% ethanol was added to rinse the beads, and after incubation at room temperature for 30sec, 80% ethanol was carefully removed.
5. And keeping the 0.2mL centrifuge tube in the magnetic frame all the time, and drying the magnetic beads at room temperature for 3-5 min.
6. Remove 0.2mL centrifuge tube from the magnetic frame, add 30. mu.L Nuclear-Free Water to resuspend the beads thoroughly, and let stand at room temperature for 5 min.
7. Place 0.2mL centrifuge tube in magnetic rack and after the solution cleared, carefully remove 28. mu.L of supernatant into a new 0.2mL centrifuge tube. The purified DNA library can be stored at 4 ℃ for one week or stored at-20 ℃ for a long time.
4) Sequencing by using a second-generation sequencing (DNB sequencing technology of Huada second-generation sequencer) platform, and analyzing results.
Second, result in
According to the method, the probe region of the Y chromosome is selected and optimized, and the capture probe is designed by adopting a new probe primer design mode, so that capture sequencing of the near 20M region of the human Y chromosome is realized, wherein the positions and sequencing results of the probe are shown in Table 1:
the length of the sequencing method is close to the limit of the available region on the Y chromosome, more mutations can be tested, and more STR and Y-SNP loci with distinguishing capability can be found.
TABLE 1
Sequence listing
<110> Guangzhou deep dawn Gene science and technology Co., Ltd
<120> Y chromosome sequencing method based on liquid phase probe capture
<160> 68
<170> SIPOSequenceListing 1.0
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<211> 75
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 1
gttggacatt taggttggtt ccaagtcttt gctattgtga ataatgccgc aataaacata 60
catgtgcatg tgtct 75
<210> 2
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 2
ttgggcagta tggccatttt cacgatattg attcttccta cccatgagca tggaatgttc 60
ttccatttgt ttgtatcctc ttttatttcc ttgagcagtc gtttgtagtt ctccttgaag 120
<210> 3
<211> 89
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 3
ttccatttgt ttgtatcctc ttttatttcc ttgagcagtc gtttgtagtt ctccttgaag 60
aggtccttca catctcttgt aagttggat 89
<210> 4
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 4
gaataccctt tatttccttc tcctgcctaa ttgccctggc cagaacttcc aacactatgt 60
tgaataggag tggtgagaga gggcatccct gtcttttgcc agttttcaaa gggaatgctt 120
<210> 5
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 5
tgaataggag tggtgagaga gggcatccct gtcttttgcc agttttcaaa gggaatgctt 60
ccagtttttg cccattcagt atgatattgg ctgtgggttt gtaatagata gctcttatta 120
<210> 6
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 6
ccagtttttg cccattcagt atgatattgg ctgtgggttt gtaatagata gctcttatta 60
ttttgaaata cgtcccatca atacctaatt tattgagagt ttttagcata aagtgctgtt 120
<210> 7
<211> 99
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 7
ttttgaaata cgtcccatca atacctaatt tattgagagt ttttagcata aagtgctgtt 60
gaattttgtc aaaggccttt tctgcatcta ttgagataa 99
<210> 8
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 8
attcaggaaa tacagagaac gccacaaaga tactgctcga gaagagcaac tccaagacac 60
ataattgtca gattcaccaa agttgagatg atgttactct ttttccttgg tgttttccta 120
<210> 9
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 9
ataattgtca gattcaccaa agttgagatg atgttactct ttttccttgg tgttttccta 60
aggaagcctt gtgacatatc tcaggaccca gcaccgaggt gatgtggccc ttttgcctgg 120
<210> 10
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 10
aggaagcctt gtgacatatc tcaggaccca gcaccgaggt gatgtggccc ttttgcctgg 60
tttcttccca tgtgttagac tgtgacatat acctaaagaa acacctaggt gatgccactc 120
<210> 11
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 11
tttcttccca tgtgttagac tgtgacatat acctaaagaa acacctaggt gatgccactc 60
tcttcttctt cttgagtcct gcttcaggac atagtgactc tatatacaca cctaggtaac 120
<210> 12
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 12
tcttcttctt cttgagtcct gcttcaggac atagtgactc tatatacaca cctaggtaac 60
agttaaaggt gccaccctca aagatgagca gattgtgtca tatcactggg cctagtaccc 120
<210> 13
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 13
agttaaaggt gccaccctca aagatgagca gattgtgtca tatcactggg cctagtaccc 60
agttgttaaa acttttgctt aaattatttc ttgtgtgtat tgtgacatat catgtcagaa 120
<210> 14
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 14
agttgttaaa acttttgctt aaattatttc ttgtgtgtat tgtgacatat catgtcagaa 60
tcataataat gtgacacttt tgccctggcc ctgccaacaa gagatatcat cacatatctc 120
<210> 15
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 15
tcataataat gtgacacttt tgccctggcc ctgccaacaa gagatatcat cacatatctc 60
tgagcctaac tggtaggtga tttgtctttt tcttgtgctt tgcccccaag aaatattgtg 120
<210> 16
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 16
tgagcctaac tggtaggtga tttgtctttt tcttgtgctt tgcccccaag aaatattgtg 60
atatttttgt atgtagcatc taggaaatgt atctcttctc tcttgcctag gacctcccta 120
<210> 17
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 17
atatttttgt atgtagcatc taggaaatgt atctcttctc tcttgcctag gacctcccta 60
ctaaaggaat tgtgccatac agctgagtgc aaaaccttgg tagtgcaact ttccccttta 120
<210> 18
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 18
ctaaaggaat tgtgccatac agctgagtgc aaaaccttgg tagtgcaact ttccccttta 60
ttctggagtt tgttacgaga ggggattatg acatattgct gagcccagca cctaggtgat 120
<210> 19
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 19
ttctggagtt tgttacgaga ggggattatg acatattgct gagcccagca cctaggtgat 60
gtgagtttcc tgtttttttc aaccctgtct acagtggaca tggtgccata ttacttgtgg 120
<210> 20
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 20
gtgagtttcc tgtttttttc aaccctgtct acagtggaca tggtgccata ttacttgtgg 60
ctgtatccag gtgatgtgac tctccttgct tgcccctgcc agcaaaggag ttgatagtgt 120
<210> 21
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 21
ctgtatccag gtgatgtgac tctccttgct tgcccctgcc agcaaaggag ttgatagtgt 60
atcaggagct cagcatcaag gtgatgagac tctccttcct tgtttttgcc cacaggtgaa 120
<210> 22
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 22
atcaggagct cagcatcaag gtgatgagac tctccttcct tgtttttgcc cacaggtgaa 60
attatgtcat atgcctggga tcatctcaca tgcacaatta taaccatcaa actgggaccc 120
<210> 23
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 23
attatgtcat atgcctggga tcatctcaca tgcacaatta taaccatcaa actgggaccc 60
agaaaggaga gagattttga ttcttatagc tagtcttatt gccataagta aaataatggg 120
<210> 24
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 24
agaaaggaga gagattttga ttcttatagc tagtcttatt gccataagta aaataatggg 60
tctcctaatt gtataaagtt cacagaggat tatgacactc agacatatca tataaagcct 120
<210> 25
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 25
tctcctaatt gtataaagtt cacagaggat tatgacactc agacatatca tataaagcct 60
cagtggtaaa aagagtgtca tgatagggaa gaacaacctg gggtgattgc aaatcatgga 120
<210> 26
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 26
cagtggtaaa aagagtgtca tgatagggaa gaacaacctg gggtgattgc aaatcatgga 60
ggcacaccca gctagcttga ttgtcattct tacacaagaa catggcctac aagtagggta 120
<210> 27
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 27
ggcacaccca gctagcttga ttgtcattct tacacaagaa catggcctac aagtagggta 60
ctaaatttca cacaaaagag cagtcaaagg ttgaaatttt tcccctcata cacggttcaa 120
<210> 28
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 28
ctaaatttca cacaaaagag cagtcaaagg ttgaaatttt tcccctcata cacggttcaa 60
ccccatgggt agtgtggtga tgcaatattc agcccaactg tgagattgtg actttcctac 120
<210> 29
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 29
ccccatgggt agtgtggtga tgcaatattc agcccaactg tgagattgtg actttcctac 60
tggaacataa tcttcaagtg gaactggaca acttatgcat ggatcctgtt aagactgtga 120
<210> 30
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 30
tggaacataa tcttcaagtg gaactggaca acttatgcat ggatcctgtt aagactgtga 60
gtcctcagct ttgactcaac tcacaggaga tgttgactca catgcacaaa gccaggactt 120
<210> 31
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 31
gtcctcagct ttgactcaac tcacaggaga tgttgactca catgcacaaa gccaggactt 60
gtgtagggct gtggaactta tttctgaata tttccaagtg tgtgattaga atgtagaagt 120
<210> 32
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 32
gtgtagggct gtggaactta tttctgaata tttccaagtg tgtgattaga atgtagaagt 60
tagcccagct cctgaataat ttgactctcc tttttaggcc atgaccacag ataaaattgt 120
<210> 33
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 33
tagcccagct cctgaataat ttgactctcc tttttaggcc atgaccacag ataaaattgt 60
gacatatgtg gacagtacac ctaagcaaag cttcctgggc ctgactacca aggatacttt 120
<210> 34
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 34
gacatatgtg gacagtacac ctaagcaaag cttcctgggc ctgactacca aggatacttt 60
tacatatcat tgggactagc atcaaggtga ggtgaattct ttgtcttttc cctgcctaca 120
<210> 35
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 35
tacatatcat tgggactagc atcaaggtga ggtgaattct ttgtcttttc cctgcctaca 60
aaaggcattg tggcttacag ttaagtccat catataagtt catataagtg atgtcactcc 120
<210> 36
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 36
aaaggcattg tggcttacag ttaagtccat catataagtt catataagtg atgtcactcc 60
cttctagtgc cttggccctg ctatttcagt tcattgtgac acataactgg gtactgcacc 120
<210> 37
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 37
cttctagtgc cttggccctg ctatttcagt tcattgtgac acataactgg gtactgcacc 60
caggtgatat gactctctgt ttagggttct gccagcagga agctttgtaa catatcactt 120
<210> 38
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 38
caggtgatat gactctctgt ttagggttct gccagcagga agctttgtaa catatcactt 60
agctcagcac ctagctaatg cttcttctct cttgccttgc tgtgatcaga ggggagactg 120
<210> 39
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 39
agctcagcac ctagctaatg cttcttctct cttgccttgc tgtgatcaga ggggagactg 60
tttcatattg ctaaccctag tgctgaagtc atgtcacttt cataccgtgg ttctgaacat 120
<210> 40
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 40
tttcatattg ctaaccctag tgctgaagtc atgtcacttt cataccgtgg ttctgaacat 60
agtgaacatt gtgacatgtc taggccaatt ccttaggtaa agtgagtctc ctcatcctct 120
<210> 41
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 41
agtgaacatt gtgacatgtc taggccaatt ccttaggtaa agtgagtctc ctcatcctct 60
taagatttgc tcacaggggg aattttgatt tatcactaaa accagtatcc agctgatgtg 120
<210> 42
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 42
taagatttgc tcacaggggg aattttgatt tatcactaaa accagtatcc agctgatgtg 60
agtcttattc cagggtcctg cccacaagaa agattgtgac atctcactgg accagcaccc 120
<210> 43
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 43
agtcttattc cagggtcctg cccacaagaa agattgtgac atctcactgg accagcaccc 60
acccaaatga tgtgacattt ctgtttgttc tctgcccaca ggtcatattg tgccatatac 120
<210> 44
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 44
acccaaatga tgtgacattt ctgtttgttc tctgcccaca ggtcatattg tgccatatac 60
ctaacaccac ttaagatgac taatcatgaa tttttttttt tttttttttt tttttttttt 120
<210> 45
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 45
ctaacaccac ttaagatgac taatcatgaa tttttttttt tttttttttt tttttttttt 60
ttttttttga gacggagtct cgctctgtcg cccaggctgg agtgcagtgg cgggatctcg 120
<210> 46
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 46
ttttttttga gacggagtct cgctctgtcg cccaggctgg agtgcagtgg cgggatctcg 60
gctcactgca agctccgcct cccgggttca cgccattctc ctgcctcagc ctcccgagta 120
<210> 47
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 47
gctcactgca agctccgcct cccgggttca cgccattctc ctgcctcagc ctcccgagta 60
gctgggacta caggcacccg ctaccacgcc cggctaattt tttgtatttt tagtagagac 120
<210> 48
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 48
gctgggacta caggcacccg ctaccacgcc cggctaattt tttgtatttt tagtagagac 60
ggggtttcac cgtgttagcc aggatggtct cgatctcctg acctcgtgat ccgcccgcct 120
<210> 49
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 49
ggggtttcac cgtgttagcc aggatggtct cgatctcctg acctcgtgat ccgcccgcct 60
cggcctccca aagtgctggg attacaggca tgagccactg cgcccagcct aatcatgact 120
<210> 50
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 50
cggcctccca aagtgctggg attacaggca tgagccactg cgcccagcct aatcatgact 60
tttaaacctg gatcctggtc atatgcaaga tggaaactcc cattcctgga actttccacc 120
<210> 51
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 51
tttaaacctg gatcctggtc atatgcaaga tggaaactcc cattcctgga actttccacc 60
agtgttattg tgacatatac ctttgcccgg ctcctgagtg atttaataat actgcctaat 120
<210> 52
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 52
agtgttattg tgacatatac ctttgcccgg ctcctgagtg atttaataat actgcctaat 60
tgtagccctc aaattcgatt tagacatata gttcatgtga tcacctttat gatttcactc 120
<210> 53
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 53
tgtagccctc aaattcgatt tagacatata gttcatgtga tcacctttat gatttcactc 60
tcctgtttta acaatatctt gcagaaggat tgtaacagat ttctggaccc aacatctagt 120
<210> 54
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 54
tcctgtttta acaatatctt gcagaaggat tgtaacagat ttctggaccc aacatctagt 60
tacctgacac tcctctctta cctggaccct gcttccacta tggattgtag catttctaag 120
<210> 55
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 55
tacctgacac tcctctctta cctggaccct gcttccacta tggattgtag catttctaag 60
cactacctcc aaattatatg aatctcttgc ctggtccttt caagaggaga ccttgtgaca 120
<210> 56
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 56
cactacctcc aaattatatg aatctcttgc ctggtccttt caagaggaga ccttgtgaca 60
tatctctggg cttaccattt aggtgatatg agtctcctct tctgcctgga cactgcctac 120
<210> 57
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 57
tatctctggg cttaccattt aggtgatatg agtctcctct tctgcctgga cactgcctac 60
aaggggtatt gtgttataca tgttgatgta acccctgagt tatgcaactt ttctgcccag 120
<210> 58
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 58
aaggggtatt gtgttataca tgttgatgta acccctgagt tatgcaactt ttctgcccag 60
aacatgccta caaggagaat attggaaaat ttctggctca gtatttaggt gacttgtctg 120
<210> 59
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 59
aacatgccta caaggagaat attggaaaat ttctggctca gtatttaggt gacttgtctg 60
tcatgcctgt ttcattacca cagactgaat tgtgatatat acccaagcac agctcacagg 120
<210> 60
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 60
tcatgcctgt ttcattacca cagactgaat tgtgatatat acccaagcac agctcacagg 60
catgataatg actttcttat gtggatccca caaatagaag taattttgac tctcataact 120
<210> 61
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 61
catgataatg actttcttat gtggatccca caaatagaag taattttgac tctcataact 60
tgctttagag acatgagtga ttaaatctct atctggcacc aaaaaaaaaa aaaaaaaaca 120
<210> 62
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 62
tgctttagag acatgagtga ttaaatctct atctggcacc aaaaaaaaaa aaaaaaaaca 60
aacaaaaggt caaagaagat tataatggcc tcacatattt tataaagcct ttggcttgta 120
<210> 63
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 63
aacaaaaggt caaagaagat tataatggcc tcacatattt tataaagcct ttggcttgta 60
cagagtgtca taacagaacc cagcagagag gtgaaattgt gagtctcaaa tgcacaccca 120
<210> 64
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 64
cagagtgtca taacagaacc cagcagagag gtgaaattgt gagtctcaaa tgcacaccca 60
gctgtcacta aggtctaatc accatctcac ctatatgaag ctaactgtca ctaatgaaaa 120
<210> 65
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 65
gctgtcacta aggtctaatc accatctcac ctatatgaag ctaactgtca ctaatgaaaa 60
cagcacacac gtgatactgt acacctcatc aggggaattt tctgatagtg ttattgtgat 120
<210> 66
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 66
cagcacacac gtgatactgt acacctcatc aggggaattt tctgatagtg ttattgtgat 60
ataaatcttt gttgagcacc tgtgtgattt aattctccat aattgttcca gcccacatat 120
<210> 67
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 67
ataaatcttt gttgagcacc tgtgtgattt aattctccat aattgttcca gcccacatat 60
gttattgtca tatctacctg ggccaaactc tacatgatgt gactctcctg cctaggccct 120
<210> 68
<211> 120
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 68
gttattgtca tatctacctg ggccaaactc tacatgatgt gactctcctg cctaggccct 60
gctctcagta agaatcatga cttattacta catccagcac ctaggtcatg ttacatcttt 120