CN112746096A - Human Y-STR detection method based on next-generation sequencing and application thereof - Google Patents
Human Y-STR detection method based on next-generation sequencing and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of forensic medicine application, and particularly relates to a human Y-STR detection method based on second-generation sequencing and application thereof. The method provided by the invention is that 88Y chromosome loci including 88Y chromosome STR loci are finally screened out according to the analysis result of the STR population genetic polymorphism of Chinese population, and primers are further designed for the Y chromosome STR loci to complete detection through library construction, purification, sequencing, data analysis and the like. The detection method provided by the invention has the advantages of high sensitivity, good sequencing performance and the like.
Description
Technical Field
The invention belongs to the technical field of forensic medicine application, and particularly relates to a human Y-STR detection method based on second-generation sequencing and application thereof.
Background
The human Y chromosome short tandem repeat (Y-STR) has the characteristics of paternal inheritance, lack of recombination, simple typing, large information amount, high polymorphism and the like, and is a powerful tool for researching the origin evolution, ethnic difference, population and regional distribution and the like of human beings. At present, family investigation by applying the Y-STR becomes an important way for criminal investigation, and the investigation work in criminal case handling can be effectively reduced. However, the number of Y-STR sites in the current commercial kit is insufficient, so that the number of individuals actually returning results is too large, and the workload of criminal investigation cannot be effectively reduced.
Currently, fluorescence labeling multiplex amplification combined with capillary electrophoresis (PCR-CE) is the mainstream technology of STR typing. However, when typing is carried out by PCR-CE, there is a case where the sequence structures of the repetitive regions are not identical among alleles having the same fragment length. In addition, PCR-CE technology typing multi-copy Y-STR can only detect length polymorphism.
The second generation sequencing technology (NGS) has the advantages of high sequencing flux and high sequencing speed. NGS can not only analyze STR loci from length polymorphism, but also detect sequence information of multiple copies of Y-STR loci, and has higher gene diversity and individual identification capability. At present, the research and development of a second-generation sequencing commercialized Y-STR typing kit are in the initial stage, and mainly comprise
Y23 System(Promega)、ForenSeqTM DNA Signature Prep Kit(Illumina)、YfilerTMPlus PCR Amplification Kit (Thermol Fisher) and GlobalFiler PCR Amplification Kit (Thermol Fisher). However, the Y-STR related to the kit is relatively few, and the four kits respectively comprise 23Y-STR loci, 24Y-STR loci, 27Y-STR loci and 2Y-STR loci. Meanwhile, the price of the kit is high, the matched data analysis software only can be used for developing the kit respectively, the setting of parameters is relatively fixed, and only sequencing data of the inherent locus can be analyzed, so that the kit is not beneficial to the actual application of forensic medicine.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention creatively provides a human Y-STR detection method based on next generation sequencing. The method provided by the invention has the advantages of high accuracy, good sensitivity and better sequencing performance, and can be applied to the research in the forensic field.
In order to achieve the purpose, the invention adopts the technical scheme that:
a human Y-STR detection method based on next generation sequencing comprises the following steps:
s1, finally screening 88Y chromosome loci, namely DYS19, DYF371a/b/c/d, DYS385a/b, DYF387S1a/b, DYS388, DYS389-I, DYS-II, DYS390, DYS391, DYS392, DYS393, DYF397S1a/b, DYF399S1a/b/c, DYF403S1a1/a2/b1/b2, DYF404S1a/b, DYS425a/b/c/d, DYS437, DYS439, DYS438, DYS443, DYS446, DYS447, DYS449, DYS458, DYS456, DYS526, DYS481, DYS520, DYS481, DYS520, DYS 320, DYS520, DYS 320, DYS520, DYS481, DYS520, DYS720, DYS722, Y-GATA-A10, Y-GATA-H4;
s2, designing primers according to the sequence information of the gene locus, and amplifying the sequence of the gene locus;
s3, constructing a gene library according to the primer designed in the step S2;
s4, sequencing the gene library constructed in the step S3, and analyzing sequencing data.
Preferably, when the primers and each primer described in step S2 are used for PCR amplification, the sequences of each primer are shown in the sequence table, and the concentrations of each primer are shown in the following table.
Preferably, the specific process for constructing the gene library in step S3 is:
(1) preparing a multiplex PCR premix and carrying out PCR amplification: 10 mu L of primer mixed solution, 4 mu L of multiplex PCR amplification buffer solution, 1-100 ng of template DNA, adding sterile ultrapure water to a total reaction system of 20 mu L, and carrying out PCR reaction according to the following procedures: at 95 ℃ for 3 min; 15s at 95 ℃, 90s at 72 ℃ and 28 cycles; 10min at 72 ℃; storing at 4 ℃ to prepare a PCR product;
(2) digesting the PCR product prepared in the step (1): mu.L of the PCR product obtained in step S1 was taken, 2. mu.L of digestion buffer was added thereto, and digestion was carried out according to the following procedure: storing at 50 deg.C for 10min, 55 deg.C for 10min, 60 deg.C for 20min, and 10 deg.C to obtain digested product;
(3) connecting a joint: the reaction mixture was prepared as follows: 22 mu L of digestion product prepared in the step (2), 6 mu L of connection buffer solution, 1 mu L of linker and 1 mu L of ligase, wherein the total amount is 30 mu L, and the ligation is carried out according to the following reaction: storing at 22 deg.C for 30min, 72 deg.C for 10min, and 10 deg.C to obtain ligation product;
(4) purifying the ligation product prepared in the step (3), and amplifying the purified library again, wherein the amplification system is as follows: 20 mu L of purified library, 25 mu L of HiFi library amplification buffer solution and 5 mu L of PCR primer mixed solution; the amplification reaction conditions are as follows: 3min at 95 ℃; 20s at 98 ℃, 15s at 60 ℃, 30s at 72 ℃ and 8 cycles; preserving at 72 ℃ for 10min and 4 ℃ to prepare an amplification library;
(5) and (4) further purifying the amplified library prepared in the step (4), performing quality inspection, and quantifying each library by using a qubit to obtain the target product.
Preferably, the primer mixture in step (1) mainly comprises oligonucleotide and 1 XTE Buffer, and the multiplex PCR amplification Buffer mainly comprises DNA polymerase with 5-20U enzyme activity, dNTP with 0.1-0.5mmol/L final concentration, MgCl with 1.5-6mmol/L final concentration2The digestion buffer solution in the step (2) mainly comprises Tris-HCl with the final concentration of 50-100mmol/L and MgCl with the final concentration of 1.5-6mmol/L2ATP with final concentration of 0.1-0.5mmol/L, dNTP with final concentration of 0.1-0.5mmol/L, and the connection buffer solution in the step (3) mainly comprisesTris-HCl with final concentration of 50-100mmol/L, MgCl with final concentration of 1.5-6mmol/L2DTT with the final concentration of 0.2-1mmol/L and ATP with the final concentration of 0.1-0.5 mmol/L.
Preferably, the HiFi library amplification buffer in step (4) mainly comprises DNA polymerase, dNTP with final concentration of 0.15-0.35mmol/L, and MgCl with final concentration of 2-5mmol/L2The PCR premix comprises oligonucleotide and 1 × TE Buffer.
Preferably, the library purification process in step (4) comprises the steps of:
a. preparation before purification: magnetic beads VAHTSTMUniformly mixing DNA Clean Beads by shaking, balancing to room temperature of 25 ℃, preparing enough fresh ethanol with volume fraction of 80%, wherein each sample needs about 400 mu L, and before the purification step is carried out, the sample needs to be filled with sterilized water to 60 mu L;
b. vortexing the magnetic beads to fully mix the magnetic beads, adding 60 mu L (1X) of magnetic beads into the PCR reaction system, gently blowing and beating the mixture for 10 times by using a pipettor to ensure that the whole system is uniform, and incubating the mixture at room temperature for 8min to enable the library to be combined on the magnetic beads;
c. centrifuging the reaction tube at the rotating speed of 1000rpm for 20s, and placing the reaction tube on a magnetic frame to separate magnetic beads and liquid;
d. keeping the PCR tube on a magnetic frame, after 5min, clarifying the solution, carefully discarding the supernatant, and paying attention to not disturb the magnetic beads;
e. keeping the PCR tube on a magnetic frame, adding 200. mu.L of freshly prepared 80% ethanol, taking care not to disturb the magnetic beads when adding ethanol, and carefully removing the supernatant after incubating for 30 s;
f. repeating the step e, and rinsing twice;
g. centrifuging at 1000rpm for 20s, collecting the sample to the bottom of the PCR tube, placing on a magnetic frame for 30s, sucking off all residual ethanol with a pipette, and uncovering and air drying for 3-5 min.
h. After the magnetic beads are dried in the air, taking down the PCR tube from the magnetic frame, adding 22 mu L of enzyme-free water to cover the magnetic beads, and blowing and uniformly mixing the magnetic beads by using a pipettor;
i. incubating at room temperature for 2-3min, centrifuging the PCR tube at 1000rpm for 20s, collecting PCR products, placing the PCR products in a magnetic frame, and separating magnetic beads from liquid until the solution is clear;
k. carefully pipette 20. mu.L of the supernatant into a new EP tube.
Preferably, the purification process of step (5) is to add 120. mu.L (1.2X) of magnetic beads in step b, and the rest of the purification process is the same as described above.
Preferably, the process of analyzing the sequencing data in step S4 is:
(1) analysis threshold value: expressed as a percentage of the set threshold for filtering the Noise sequence, the depth of sequence sequencing below the set analysis threshold multiplied by the total depth of sequencing for that locus is considered to be the Noise sequence and is not further analyzed;
(2) locus sequence composition ratio: the locus sequence composition ratio is the ratio of Allle, Stutter and Noise in each locus in the total coverage of the locus (the sequence of each locus is divided into 3 types, namely Allle, Stutter and Noise sequence, Stutter is defined as a sequence which is +/-4 bp longer than the corresponding Allle length, Noise is defined as a sequence which is neither Allle nor Stutter);
(3) depth of locus coverage: expressed as the sequencing depth of a locus, i.e., the total reads number for that locus;
(4) sample coverage depth: representing the average sequencing depth of each sample in one sequencing;
(5) reading frequency estimation of alleles: the reading obtained for the reference allele is divided by the sum of the readings for the reference allele and the alternate allele.
Preferably, the ratio of each type of locus sequence in step (2) is calculated by: all% ═ all reads/Total reads; stuffer% ═ stuffer reads/Total reads; and 3, 1-allele% -stuffer%.
The invention also provides an application of the detection method in forensic medicine identification.
The invention is based on the second generation sequencing technology, combines with the MiSeq FGx TM system of Illumina company which is most applied in the court science field, constructs a composite amplification system which is based on the second generation sequencing technology and can accommodate more Y-STR loci, and constructs a high-sensitivity STR detection method. On the basis of the family investigation and case-solving of the Y-STR, more Y-STR sites are provided, and the method has important significance for the construction of nationwide Y databases. Meanwhile, a reference scheme is provided for the research and development of the domestic multi-site STR reagent.
The invention aims to select the loci which accord with the genetic polymorphism of STR population of Chinese population, and adopts multi-copy loci to combine with low-frequency mutation loci on the selection of the loci to prepare the kit capable of detecting more Y chromosome STR loci. This study analyzed and evaluated 88Y chromosome STR genetic markers. The DNA input was reduced to 125pg, and this experiment also gave a complete genotype. When the DNA input amount was reduced to 50pg, 92.67% of the markers were correctly genotyped; the present detection method provides a well balanced result in most markers, and estimates the allele reading frequency for the remaining number of DNA inputs in the dilution series of the gradient, and heterozygote STRs remain well balanced even at low DNA inputs (0.125 ng). Furthermore, the method is compatible with the MiSeq FGx TM system of Illumina company which is most applied in the forensic science field, and provides important reference significance for the research and development of domestic reagents in the forensic medicine field in the future.
The inventor of the invention obtains a large number of candidate Y chromosome STR loci by consulting documents, the existing database and the existing second-generation sequencing STR typing product, and finally obtains 88Y chromosome STR loci by screening according to the analysis result of the STR population genetic polymorphism of Chinese population.
The 88Y-STR loci screened finally contain 12 multicopy STRs, namely DYS385, DYF371, DYF387S1, DYF397, DYF399S1, DYF403S1, DYF404S1, DYS425, DYS459, DYS464, DYS526 and DYS 527; and contains multicopy loci, DYS385a/b, DYF387S1a/b, DYF397S1a/b, DYF404S1a/b, DYS459a/b, DYS526a/b and DYS527a/b respectively comprise two typing fragments, DYF399S1a/b/c comprises three typing fragments, DYF371a/b/c/d, DYF403S1a1/a2/b1/b2, DYS425a/b/c/d and DYS464a/b/c/d comprise four typing fragments; meanwhile, the selected loci contain 20 core sites and 15 preferred sites of the department of public Security; also included are 12 rapid mutation loci: DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS570, DYS576, DYS612, DYS626, DYS 627.
Moreover, when designing a primer according to a locus sequence, the primer is designed according to the following ideas and steps: (1) the primers are not bound to each other nor to regions other than the target fragment on the template DNA. (2) The combination of the primers needs to solve the problem that different amplification fragments compete with each other, and the high-abundance template is prevented from being completely covered, so that the low-abundance template falls into the background completely. (3) The collection of locus sequence information comes from various authoritative databases and the search of large amounts of literature, as well as the mature commercial kits (databases: GeneBank database of NCBI; YHDR-Y chromosome haplotype reference database https:// yhrd.org/; GRCh38 human genome SNP database https:// www.snpedia.com/index.php/GRCh 38; and STRBase database https:// strbase.nist.gov /), primers were designed using primer5 software, primer amplification specificity was considered by BLAST function in GenBank, species specificity of these designed locus primers was considered by other species (4) genome data in the database, multiplex PCR amplification systems were used, and some interference between primers in mixed systems per set of PCRs existed, so that experimental screening of large amounts of primers had to be designed according to the needs of the detection method, and the interference and balance among multiple groups of primers of the multiplex PCR are considered through professional primer design software, and the primers are adjusted. (5) Then PCR amplification is carried out to obtain specific amplification products of each gene locus. And (3) combining the amplification conditions of the single gene locus, selecting a proper amplification program, and carrying out composite amplification. Because the number of complex loci is large and the mutual inhibition condition among primers is complex, the loci need to be eliminated one by one, found out, redesigned and synthesized. (6) In addition, primer dimers are formed between primers at different loci, which reduces the amplification efficiency of the primers and requires redesign and synthesis of primers. (7) During primary composite amplification, the concentration of each primer in a reaction system is 0.3 mu M; then, the concentration of the primers is adjusted to obtain the optimal concentration of each primer during PCR amplification, 88 pairs of primers are adjusted according to the amount, and finally mixed and adjusted to the concentration (adjusted according to the corresponding concentration of each primer in table 1) to prepare a primer mixture, namely, the oligonucleotide in the PCR amplification process.
Compared with the prior art, the detection method provided by the invention has the following advantages:
(1) the kit provided by the invention can detect loci of up to 88Y chromosomes (compared with more than 20Y-STR loci on the market, the kit contains more locus information and provides more practical information for the construction of Y databases in China) and evaluates the amplification and sequencing performances of the loci by comparing the sequencing depth obtained by each locus, and the analysis result shows that 88Y-STR loci in the method are well balanced, the value is close to 1 and accounts for 95.2 percent, in addition, 40 heterozygote Y-STR loci are analyzed, each heterozygote locus has good balanced alleles, and the average reading frequency is 0.4-0.6;
(2) the results of the invention show that the mean read depth per marker obtained with NTC is 269.93 times lower than the value observed for the sample with 0.01ng of DNA (lowest input tested here); it is further shown that in most Y-STR markers, the present detection method also exhibits very low baseline noise;
(3) the detection method of the invention obtains complete 88Y-STP genotypes in all the repeats as low as 125pg DNA, and the 50pg fragment repeat generates 92.67% correct genotypes, and the detection method has higher sensitivity and good sequencing performance.
Drawings
FIG. 1 is the average sequencing depth statistics of 8 male DNA samples;
FIG. 2 is a statistical result of sequencing depth when a locus is balanced;
FIG. 3 is a graph showing the equilibrium results of 88Y-STR loci;
FIG. 4 is a graph of mean allele read frequency results for the equilibrium alleles for each heterozygote locus.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The linker is VAHTSTM AmpSeq Adapters for Illumina, available from Illumina Inc., cat # NA111/NA 121; the magnetic Beads are VAHTSTM DNA Clean Beads, a cargo number # N411; the Qubit dsDNA HS Assay Kit is a product of Thermo Fisher company, and the product number is Q32854; the Miseq reagent kit v3-600cycles sequencing reagent is a product of Illumina company, and the product number is MS-102-3003; the second generation sequencer is a product of Illumina company, and the product model is as follows: miseq FGxTM.
Example 1 evaluation of the sequencing Performance of 8 Male DNA samples Using the detection method of the present invention
(1) Preparation of DNA samples: blood card samples of 8 men were collected and nucleic acid DNA was extracted by the chelex method according to the standard in GA/T383-2014.
2. Library construction: the concrete implementation steps of the library construction method are consistent with the construction processes of the step S2 and the step S3 in the specification;
3. sequencing and data analysis: by applying the sequencing method disclosed by the invention, sequencing performance analysis is carried out on the constructed 8 male DNA samples and the negative control sample by utilizing the NGS platform, and the specific implementation steps are as described in the step S4 of the invention.
4. And (4) analyzing results:
(1) by analyzing NTC samples, baseline noise generated by sequencing results was studied.
As a result of the analysis, 884.3 reads were obtained in 88Y-STRs, and the average read depth of each marker was 13.6. + -. 64.1reads (Mean. + -. SD). Of these 2 (DYS448 and DYS626) show more than 100 reads (248 and 466 reads, respectively). The mean read depth per marker obtained with NTC was 269.93 times lower than the value obtained for the sample with 0.01ng of DNA (lowest input tested here).
Statistical analysis of data quality parameters shows that the average sequencing depth of 8 male DNA samples is 5675 +/-546 reads (mean +/-SD), and the average sequencing depth of a gene locus is 5570 +/-945 reads; when the threshold value was set to 5%, the mean coverage depth of allel sequence, stutter sequence and noise sequence in the locus formation ratio was 97.96%, 1.73% and 0.31%, respectively, and the gene structure of each locus was as shown in table 2.
TABLE 2 Gene constitution at each locus
Example 2 analysis of the sensitivity of 1 Male gradient diluted DNA samples Using the detection method of the present invention
(1) Preparation of DNA samples: after the quantification of the Qubit using the DNA sample of example 1, 1 of the male DNA samples was diluted in a gradient from 1ng to 10 pg. The input amount of the library construction DNA is 1ng, 0.5ng, 0.25ng, 0.125ng, 0.05ng and 0.01ng respectively.
(2) Library construction: the specific implementation steps of the library construction method are shown in the library construction link implementation method in the specific technical scheme.
(3) Sequencing and data analysis: the sequencing method provided by the invention is applied, the NGS platform is utilized to analyze the repeatability and the sensitivity of the constructed gradient dilution sample, and the specific implementation steps are shown in the sequencing link implementation method in the specific technical scheme.
(4) And (4) analyzing results: specific results as shown in fig. 3, balanced sequencing depth was obtained for each locus in all replicates with sample input as low as 125pg DNA; 1ng of locus sequencing coverage is 4160 + -3078 reads; sequencing the locus at 0.5ng with a coverage depth of 4037 + -3026 reads; 0.25ng of locus sequencing coverage was 3973 ± 2974 reads; 0.125ng of locus sequencing coverage is 3875 +/-2868 reads; sequencing for 50pg locus coverage depth 3517 ± 2713 reads; locus sequencing at 10pg coverage depth 2998 ± 1768 reads. At a loading of 10pg, one third of the loci were lost repeatedly to yield more than 900 reads. This indicates that the amount of template DNA was too low during PCR, which may create an imbalance in allele amplification.
Example 3 Performance analysis of sequencing of the results of examples 1 and 2
The performance of locus amplification and sequencing was evaluated by comparing the locus balance of the system.
FIG. 3 shows the distribution of locus equilibria, which is calculated from the ratio of reads obtained for each locus to the average read for each locus. Most loci are well balanced, with values close to 1 (95.2%). The average reading for only 7 loci (7.8%) per locus was more than doubled over the population, with 4 loci (4.54%) reading less than half the average reading per locus.
For the 40Y-STR heterozygotes observed for the 8 male DNA samples analyzed, the allele reading frequency was estimated as the reading obtained for the reference allele (ref) divided by the sum of the readings for the reference allele and the alternate allele (ref + alt). Each heterozygote locus had well balanced alleles with average read frequencies between 0.4 and 0.6 (FIG. 4). The mean of 75% of loci is between 0.5. + -. 0.03. In one iteration, the allele read frequency for only 6 loci exceeded the threshold for the above-described heterozygotes, 3 of which exceeded 0.6(DYS458, DYS448, and DYS393), and the remaining 3 were below 0.4(DYS635, DYS630, and DYS 627).
In conclusion, the invention is based on the second generation sequencing technology and combines MiSeq FGx of Illumina company which is most applied in the court science fieldTMThe system constructs a multiplex amplification system which is based on the second-generation sequencing technology and can accommodate more Y-STR loci, and constructs a high-sensitivity STR detection method. This study analyzed and evaluated 88Y chromosome STR genetic markers. The DNA input was reduced to 125pg, and this experiment also gave a complete genotype. When the amount of DNA added is reduced to 50pg,92.67% of the markers were correctly genotyped; the performance of locus amplification and sequencing was evaluated by comparing the locus balance of the system, the test method provided good balance results in most markers, allele read frequency was estimated for the remaining number of DNA inputs in the dilution series of the gradient, and heterozygote Y-STR remained well balanced even at low DNA inputs (0.125 ng).
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Zhengzhou high and New Biotechnology Co., Ltd
<120> human Y-STR detection method based on next generation sequencing and application thereof
<130> 2020.12.29
<160> 132
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> DYS19-F
<400> 1
tgccctgggc tctgtaa 17
<210> 2
<211> 24
<212> DNA
<213> DYS19-R
<400> 2
gaggccaacc atcagagctt aaac 24
<210> 3
<211> 24
<212> DNA
<213> DYF371a/b/c/d-F
<400> 3
aggacaagga gtccatctgg gtta 24
<210> 4
<211> 27
<212> DNA
<213> DYF371a/b/c/d-R
<400> 4
gatttcacta tgactactga gtttctg 27
<210> 5
<211> 22
<212> DNA
<213> DYS385a/b-F
<400> 5
agtaattctg gaggtaaaat gg 22
<210> 6
<211> 21
<212> DNA
<213> DYS385a/b-R
<400> 6
tggagagaag aagagagaaa c 21
<210> 7
<211> 31
<212> DNA
<213> DYF387SIa/b-F
<400> 7
aaatatttta aaaaataatc tatctattcc a 31
<210> 8
<211> 25
<212> DNA
<213> DYF387SIa/b-R
<400> 8
gggtgacaga gctagacacc atgcc 25
<210> 9
<211> 28
<212> DNA
<213> DYS388-F
<400> 9
aaagcagaac atctgtgtat cagtgctg 28
<210> 10
<211> 30
<212> DNA
<213> DYS388-R
<400> 10
tgactgcact ccagcctggg tgacagagct 30
<210> 11
<211> 29
<212> DNA
<213> DYS389 I /II-F
<400> 11
tagccgttta gcgatatata catattatg 29
<210> 12
<211> 20
<212> DNA
<213> DYS389 I /II-R
<400> 12
cgcaaccact gcgctccagc 20
<210> 13
<211> 25
<212> DNA
<213> DYS390-F
<400> 13
gaagaatgtc atagatagat gatgg 25
<210> 14
<211> 23
<212> DNA
<213> DYS390-R
<400> 14
ctctcatctg tattatctat gtg 23
<210> 15
<211> 26
<212> DNA
<213> DYS391-F
<400> 15
gtatactcag aaacaaggaa agatag 26
<210> 16
<211> 23
<212> DNA
<213> DYS391-R
<400> 16
gccctgcatt ttggtacccc ata 23
<210> 17
<211> 24
<212> DNA
<213> DYS392-F
<400> 17
tcattcaatc atacacccat atct 24
<210> 18
<211> 21
<212> DNA
<213> DYS392-R
<400> 18
ctccctggtt gcaagcaatt g 21
<210> 19
<211> 23
<212> DNA
<213> DYS393-F
<400> 19
agaagtcaaa acagagggat cat 23
<210> 20
<211> 25
<212> DNA
<213> DYS393-R
<400> 20
caactaattt gatttcaagt gtttg 25
<210> 21
<211> 28
<212> DNA
<213> DYS397S1a/b-F
<400> 21
tgtcattcct aatgtggtct tctacttg 28
<210> 22
<211> 27
<212> DNA
<213> DYS397S1a/b-R
<400> 22
ctcaagtcca aaaaatgagg tatgtct 27
<210> 23
<211> 22
<212> DNA
<213> DYF399S1a/b/c-F
<400> 23
aagcttgttc tgctaatcca gc 22
<210> 24
<211> 19
<212> DNA
<213> DYF399S1a/b/c-R
<400> 24
tttcaagctg tttgcctcg 19
<210> 25
<211> 24
<212> DNA
<213> DYF403Sla1/a2/b1/b2-F
<400> 25
agtctctcaa gcctgttcta tgaa 24
<210> 26
<211> 24
<212> DNA
<213> DYF403Sla1/a2/b1/b2-R
<400> 26
attagctgga agtggagttt gctg 24
<210> 27
<211> 21
<212> DNA
<213> DYF404Sla/b-F
<400> 27
ccatgttact gcaaaataca c 21
<210> 28
<211> 19
<212> DNA
<213> DYF404Sla/b-R
<400> 28
tgacagagca taaacgtgt 19
<210> 29
<211> 24
<212> DNA
<213> DYS425a/b/c/d-F
<400> 29
gactgcagtg agccatgatg gaac 24
<210> 30
<211> 31
<212> DNA
<213> DYS425a/b/c/d-R
<400> 30
ttaaacaata ctgaagttta tcaaagggct t 31
<210> 31
<211> 20
<212> DNA
<213> DYS437-F
<400> 31
ggagagaaga agagagaaat 20
<210> 32
<211> 22
<212> DNA
<213> DYS437-R
<400> 32
agtaattctg gaggtaaaat gg 22
<210> 33
<211> 26
<212> DNA
<213> DYS438-F
<400> 33
cagtctcctg agtagctggg actatg 26
<210> 34
<211> 27
<212> DNA
<213> DYS438-R
<400> 34
atagataagt agatagacat cattcac 27
<210> 35
<211> 25
<212> DNA
<213> DYS439-F
<400> 35
aattagtggg gaatagttga acggt 25
<210> 36
<211> 27
<212> DNA
<213> DYS439-R
<400> 36
gagatcacac cattgcattt cagcctg 27
<210> 37
<211> 28
<212> DNA
<213> DYS443-F
<400> 37
attcaaggtg atagatatac agatagat 28
<210> 38
<211> 28
<212> DNA
<213> DYS443-R
<400> 38
attacaggca taatccacca tgcctggc 28
<210> 39
<211> 20
<212> DNA
<213> DYS444-F
<400> 39
ccttccattt acacttcctg 20
<210> 40
<211> 24
<212> DNA
<213> DYS444-R
<400> 40
tttttcattg gccacctgac atta 24
<210> 41
<211> 26
<212> DNA
<213> DYS446-F
<400> 41
gtgcaataga tatataggta ggtaag 26
<210> 42
<211> 30
<212> DNA
<213> DYS446-R
<400> 42
gaaggaaatc tatatataag tgagcccatg 30
<210> 43
<211> 18
<212> DNA
<213> DYS447-F
<400> 43
atagtctatg gtccatta 18
<210> 44
<211> 18
<212> DNA
<213> DYS447-R
<400> 44
tacgcttcgt tgcactcc 18
<210> 45
<211> 25
<212> DNA
<213> DYS448-F
<400> 45
ggttttatac attttaggga gacat 25
<210> 46
<211> 23
<212> DNA
<213> DYS448-R
<400> 46
ctttgcgtta tctctgcctt tct 23
<210> 47
<211> 22
<212> DNA
<213> DYS449-F
<400> 47
agattagaaa tagagatcgc ga 22
<210> 48
<211> 26
<212> DNA
<213> DYS449-R
<400> 48
cctcatattt ctggccggtc tggaaa 26
<210> 49
<211> 20
<212> DNA
<213> DYS456-F
<400> 49
ttgggctgag gagttcaatc 20
<210> 50
<211> 19
<212> DNA
<213> DYS456-R
<400> 50
gcagtctcat ttcctggag 19
<210> 51
<211> 27
<212> DNA
<213> DYS458-F
<400> 51
ctcggactgg ctcatcttgc tcctcag 27
<210> 52
<211> 29
<212> DNA
<213> DYS458-R
<400> 52
ccaaaacttc ttaaactgat gtattaggg 29
<210> 53
<211> 21
<212> DNA
<213> DYS459a/b-F
<400> 53
ctgagcaaca ggaatgaaac t 21
<210> 54
<211> 28
<212> DNA
<213> DYS459a/b-R
<400> 54
cagccacctc ggcctcccaa agttctgg 28
<210> 55
<211> 20
<212> DNA
<213> DYS460-F
<400> 55
gattttcttg gtaagccggt 20
<210> 56
<211> 20
<212> DNA
<213> DYS460-R
<400> 56
acgtagcacc atggttttct 20
<210> 57
<211> 31
<212> DNA
<213> DYS464a/b/c/d-F
<400> 57
tccagtagtg atgctgtgtc actatatttc t 31
<210> 58
<211> 28
<212> DNA
<213> DYS464a/b/c/d-R
<400> 58
cacaagaata ccagaggaat ctgacacc 28
<210> 59
<211> 19
<212> DNA
<213> DYS481-F
<400> 59
cttagcacac aaagctatt 19
<210> 60
<211> 20
<212> DNA
<213> DYS481-R
<400> 60
gataatggcc gtcctacgat 20
<210> 61
<211> 26
<212> DNA
<213> DYS485-F
<400> 61
gaatgtggct aacgctgttc agcatg 26
<210> 62
<211> 30
<212> DNA
<213> DYS485-R
<400> 62
cagcatgtct tggcatactt aacaattcat 30
<210> 63
<211> 24
<212> DNA
<213> DYS504-F
<400> 63
cctgggtgac aagagttata ctct 24
<210> 64
<211> 21
<212> DNA
<213> DYS504-R
<400> 64
cagacttcgc cactacataa t 21
<210> 65
<211> 20
<212> DNA
<213> DYS505-F
<400> 65
tctacaccac tgtgccaagc 20
<210> 66
<211> 18
<212> DNA
<213> DYS505-R
<400> 66
gcaacagagc aaccctct 18
<210> 67
<211> 20
<212> DNA
<213> DYS508-F
<400> 67
ttagaacgca cgttctgcat 20
<210> 68
<211> 20
<212> DNA
<213> DYS508-R
<400> 68
tggtgccagc cacgtttgta 20
<210> 69
<211> 21
<212> DNA
<213> DYS510-F
<400> 69
gttagtattg tcatctagtt a 21
<210> 70
<211> 20
<212> DNA
<213> DYS510-R
<400> 70
taggtccgtg acaggcatcg 20
<210> 71
<211> 20
<212> DNA
<213> DYS518-F
<400> 71
ggactttccg attcgtacca 20
<210> 72
<211> 20
<212> DNA
<213> DYS518-R
<400> 72
gagagttgtt taactctgaa 20
<210> 73
<211> 31
<212> DNA
<213> DYS520-F
<400> 73
actccagcct gggcaacaca agtgaaactg c 31
<210> 74
<211> 25
<212> DNA
<213> DYS520-R
<400> 74
ggacttagtt ttctaatcac atctt 25
<210> 75
<211> 19
<212> DNA
<213> DYS522-F
<400> 75
ctgtggatag ctcggacta 19
<210> 76
<211> 19
<212> DNA
<213> DYS522-R
<400> 76
cgagctaaga tgcatccgc 19
<210> 77
<211> 19
<212> DNA
<213> DYS526a/b-F
<400> 77
aaatctgtgc atggggaag 19
<210> 78
<211> 19
<212> DNA
<213> DYS526a/b-R
<400> 78
cccagtcagc cctgctac 18
<210> 79
<211> 19
<212> DNA
<213> DYS527a/b-F
<400> 79
cctcccttct ccctgtctt 19
<210> 80
<211> 18
<212> DNA
<213> DYS527a/b-R
<400> 80
cagagcagga taccatct 18
<210> 81
<211> 27
<212> DNA
<213> DYS531-F
<400> 81
gcccagacaa cagagcaaaa ctctatc 27
<210> 82
<211> 29
<212> DNA
<213> DYS531-R
<400> 82
acaacataag taaggtagtt ttcttttca 29
<210> 83
<211> 22
<212> DNA
<213> DYS533-F
<400> 83
cggtcaccaa cttgatttga ct 22
<210> 84
<211> 21
<212> DNA
<213> DYS533-R
<400> 84
ctctgtcact tctacagggt c 21
<210> 85
<211> 28
<212> DNA
<213> DYS549-F
<400> 85
atgtattatc tatcaatctt ctacctat 28
<210> 86
<211> 28
<212> DNA
<213> DYS549-R
<400> 86
aaatgtattt attcatgatc agttctta 28
<210> 87
<211> 25
<212> DNA
<213> DYS552-F
<400> 87
aataaggtag acatagcaat taggt 25
<210> 88
<211> 25
<212> DNA
<213> DYS552-R
<400> 88
gtaatgtccc cttttccatt tgtga 25
<210> 89
<211> 19
<212> DNA
<213> DYS557-F
<400> 89
gttatggttt atcggttcc 19
<210> 90
<211> 21
<212> DNA
<213> DYS557-R
<400> 90
actcatccgt tgcccgtcgg a 21
<210> 91
<211> 28
<212> DNA
<213> DYS570-F
<400> 91
gtgccaagcc tacatataat attttgac 28
<210> 92
<211> 28
<212> DNA
<213> DYS570-R
<400> 92
ggtcctgtag gcagggttaa gacagaag 28
<210> 93
<211> 24
<212> DNA
<213> DYS576-F
<400> 93
tgaatgatga ctaggtagaa atcc 24
<210> 94
<211> 31
<212> DNA
<213> DYS576-R
<400> 94
gacaactggt ggcaacctaa gctgaaatgc a 31
<210> 95
<211> 25
<212> DNA
<213> DYS587-F
<400> 95
aggagttcaa tctcagccaa gcaac 25
<210> 96
<211> 24
<212> DNA
<213> DYS587-R
<400> 96
tggagatgaa ggaggagatg ggag 24
<210> 97
<211> 21
<212> DNA
<213> DYS593-F
<400> 97
gtgctttagt cggaatctaa c 21
<210> 98
<211> 20
<212> DNA
<213> DYS593-R
<400> 98
ttatgatccc gtatccaagt 20
<210> 99
<211> 27
<212> DNA
<213> DYS596-F
<400> 99
atagaagatc tcaccagtgg actccag 27
<210> 100
<211> 26
<212> DNA
<213> DYS596-R
<400> 100
tttatgccca agtgacactg ctgatt 26
<210> 101
<211> 28
<212> DNA
<213> DYS612-F
<400> 101
ccgtgccctt tactgcataa atgacatg 28
<210> 102
<211> 30
<212> DNA
<213> DYS612-R
<400> 102
ctattactac tgagtttctg atatagtgtt 30
<210> 103
<211> 19
<212> DNA
<213> DYS617-F
<400> 103
ggctttcacc agtttgcat 19
<210> 104
<211> 20
<212> DNA
<213> DYS617-R
<400> 104
ccatgtttag ggacattcct 20
<210> 105
<211> 19
<212> DNA
<213> DYS622-F
<400> 105
gtcaatgcca gcaggtttc 19
<210> 106
<211> 19
<212> DNA
<213> DYS622-R
<400> 106
tggcttacgt ggtgagcta 19
<210> 107
<211> 20
<212> DNA
<213> DYS626-F
<400> 107
ctatccccga tcatacggct 20
<210> 108
<211> 20
<212> DNA
<213> DYS626-R
<400> 108
caatgcagta tctccattag 20
<210> 109
<211> 21
<212> DNA
<213> DYS627-F
<400> 109
tctggtgaac tgatccaatc c 21
<210> 110
<211> 20
<212> DNA
<213> DYS627-R
<400> 110
gtttgggtta cttcgccaga 20
<210> 111
<211> 24
<212> DNA
<213> DYS630-F
<400> 111
ctccacccta ggtgacagcg cagg 24
<210> 112
<211> 22
<212> DNA
<213> DYS630-R
<400> 112
ttctttcctt ccttacttcc at 22
<210> 113
<211> 23
<212> DNA
<213> DYS635-F
<400> 113
attctaccaa gattgtgagg act 23
<210> 114
<211> 24
<212> DNA
<213> DYS635-R
<400> 114
tcttttcttt tgaggtggag tctt 24
<210> 115
<211> 30
<212> DNA
<213> DYS641-F
<400> 115
ggaaccagcc caaatatcca tcaatcaatg 30
<210> 116
<211> 27
<212> DNA
<213> DYS641-R
<400> 116
ctgctgaatg ggagcagaaa tgcccaa 27
<210> 117
<211> 18
<212> DNA
<213> DYS643-F
<400> 117
ttgagcccag gaagcata 18
<210> 118
<211> 20
<212> DNA
<213> DYS643-R
<400> 118
ccacacgatg caattttgtc 20
<210> 119
<211> 26
<212> DNA
<213> DYS644-F
<400> 119
aggtgttcac tgcaagccat gcctgg 26
<210> 120
<211> 29
<212> DNA
<213> DYS644-R
<400> 120
agaacttgtt catgtaacca aacaccacc 29
<210> 121
<211> 22
<212> DNA
<213> DYS645-F
<400> 121
ggaagaagct gatttcaatc tc 22
<210> 122
<211> 21
<212> DNA
<213> DYS645-R
<400> 122
aggagactga ggcagaaagt c 21
<210> 123
<211> 28
<212> DNA
<213> DYS710-F
<400> 123
ttactacctt ccacacacgt ccactcaa 28
<210> 124
<211> 28
<212> DNA
<213> DYS710-R
<400> 124
taattacagc taagttaatt atatggac 28
<210> 125
<211> 23
<212> DNA
<213> DYS720-F
<400> 125
caggtcaagg ctgcaagaat cta 23
<210> 126
<211> 20
<212> DNA
<213> DYS720-R
<400> 126
atactctccc tccctctctt 20
<210> 127
<211> 21
<212> DNA
<213> DYS722-F
<400> 127
tggtgataga gggaggcttc t 21
<210> 128
<211> 19
<212> DNA
<213> DYS722-R
<400> 128
cggcatgagc tattgagtc 19
<210> 129
<211> 19
<212> DNA
<213> Y-GATA-A10-F
<400> 129
ctgtgtctca catcggact 19
<210> 130
<211> 19
<212> DNA
<213> Y-GATA-A10-R
<400> 130
cttaacctgc ttcagataa 19
<210> 131
<211> 27
<212> DNA
<213> Y-GATA-H4-F
<400> 131
caggataaat cacctatcta tgtatct 27
<210> 132
<211> 28
<212> DNA
<213> Y-GATA-H4-R
<400> 132
tcctaggaat catcattaaa atgttatg 28
Claims (10)
1. A human Y-STR detection method based on next generation sequencing is characterized by comprising the following steps:
s1, finally screening 88Y chromosome loci, namely DYS19, DYF371a/b/c/d, DYS385a/b, DYF387S1a/b, DYS388, DYS389-I, DYS-II, DYS390, DYS391, DYS392, DYS393, DYF397S1a/b, DYF399S1a/b/c, DYF403S1a1/a2/b1/b2, DYF404S1a/b, DYS425a/b/c/d, DYS437, DYS439, DYS438, DYS443, DYS446, DYS447, DYS449, DYS458, DYS456, DYS526, DYS481, DYS520, DYS481, DYS520, DYS 320, DYS520, DYS 320, DYS520, DYS481, DYS520, DYS720, DYS722, Y-GATA-A10, Y-GATA-H4;
s2, designing primers according to the sequence information of the gene locus, and amplifying the sequence of the gene locus;
s3, constructing a gene library according to the primer designed in the step S2;
s4, sequencing the gene library constructed in the step S3, and analyzing sequencing data.
2. The detection method according to claim 1, wherein the primers and the primer concentrations of the primers in the PCR amplification in step S2 are shown in Table 1 below.
TABLE 1 primer sequences and concentrations for different loci
3. The method according to claim 1, wherein the specific process of constructing the gene library in step S3 is:
(1) preparing a multiplex PCR premix and carrying out PCR amplification: 10 mu L of primer mixed solution, 4 mu L of multiplex PCR amplification buffer solution, 1-100 ng of template DNA, adding sterile ultrapure water to a total reaction system of 20 mu L, and carrying out PCR reaction according to the following procedures: at 95 ℃ for 3 min; 15s at 95 ℃, 90s at 72 ℃ and 28 cycles; 10min at 72 ℃; storing at 4 ℃ to prepare a PCR product;
(2) digesting the PCR product prepared in the step (1): mu.L of the PCR product obtained in step S1 was taken, 2. mu.L of digestion buffer was added thereto, and digestion was carried out according to the following procedure: storing at 50 deg.C for 10min, 55 deg.C for 10min, 60 deg.C for 20min, and 10 deg.C to obtain digested product;
(3) connecting a joint: the reaction mixture was prepared as follows: 22 mu L of digestion product prepared in the step (2), 6 mu L of connection buffer solution, 1 mu L of linker and 1 mu L of ligase, wherein the total amount is 30 mu L, and the ligation is carried out according to the following reaction: storing at 22 deg.C for 30min, 72 deg.C for 10min, and 10 deg.C to obtain ligation product;
(4) purifying the ligation product prepared in the step (3), and amplifying the purified library again, wherein the amplification system is as follows: 20 mu L of purified library, 25 mu L of HiFi library amplification buffer solution and 5 mu L of PCR primer mixed solution; the amplification reaction conditions are as follows: 3min at 95 ℃; 20s at 98 ℃, 15s at 60 ℃, 30s at 72 ℃ and 8 cycles; preserving at 72 ℃ for 10min and 4 ℃ to prepare an amplification library;
(5) and (4) further purifying the amplified library prepared in the step (4), performing quality inspection, and quantifying each library by using a qubit to obtain the target product.
4. The detection method according to claim 3, wherein the primer mixture in step (1) mainly comprises oligonucleotide and 1 XTE Buffer, and the multiplex PCR amplification Buffer mainly comprises DNA polymerase with 5-20U enzyme activity, 0.1-0.5mmol/L dNTP, and 1.5-6mmol/L MgCl2The digestion buffer solution in the step (2) mainly comprises Tris-HCl with the final concentration of 50-100mmol/L and MgCl with the final concentration of 1.5-6mmol/L2ATP with final concentration of 0.1-0.5mmol/L, dNTP with final concentration of 0.1-0.5mmol/L, and the connection buffer solution in the step (3) mainly comprises Tris-HCl with final concentration of 50-100mmol/L and MgCl with final concentration of 1.5-6mmol/L2DTT with the final concentration of 0.2-1mmol/L and ATP with the final concentration of 0.1-0.5 mmol/L.
5. The detection method according to claim 3, wherein the HiFi library amplification buffer in step (4) mainly comprises DNA polymerase with enzyme activity of 3-6U, dNTP with final concentration of 0.15-0.35mmol/L, MgCl with final concentration of 2-5mmol/L2The PCR premix comprises oligonucleotide and 1 × TE Buffer.
6. The assay of claim 3, wherein the library purification process of step (4) comprises the steps of:
a. preparation before purification: magnetic beads VAHTSTMUniformly mixing DNA Clean Beads in a shaking way, balancing the mixture to the room temperature of 25 ℃, preparing enough fresh ethanol with the volume fraction of 80%, wherein each sample needs 400 mu L, and before the purification step is carried out, the sample needs to be filled with sterilized water to 60 mu L;
b. vortexing the magnetic beads to fully mix the magnetic beads, adding 60 mu L (1X) of magnetic beads into the PCR reaction system, gently blowing and beating the mixture for 10 times by using a pipettor to ensure that the whole system is uniform, and incubating the mixture at room temperature for 8min to enable the library to be combined on the magnetic beads;
c. centrifuging the reaction tube at the rotating speed of 1000rpm for 20s, and placing the reaction tube on a magnetic frame to separate magnetic beads and liquid;
d. keeping the PCR tube on a magnetic frame, after 5min, clarifying the solution, carefully discarding the supernatant, and paying attention to not disturb the magnetic beads;
e. keeping the PCR tube on a magnetic frame, adding 200. mu.L of freshly prepared 80% ethanol, taking care not to disturb the magnetic beads when adding ethanol, and carefully removing the supernatant after incubating for 30 s;
f. repeating the step e, and rinsing twice;
g. centrifuging at 1000rpm for 20s, collecting the sample to the bottom of the PCR tube, placing on a magnetic frame for 30s, sucking off all residual ethanol with a pipette, and uncovering and air drying for 3-5 min.
h. After the magnetic beads are dried in the air, taking down the PCR tube from the magnetic frame, adding 22 mu L of enzyme-free water to cover the magnetic beads, and blowing and uniformly mixing the magnetic beads by using a pipettor;
i. incubating at room temperature for 2-3min, centrifuging the PCR tube at 1000rpm for 20s, collecting PCR products, placing the PCR products in a magnetic frame, and separating magnetic beads from liquid until the solution is clear;
k. carefully pipette 20. mu.L of the supernatant into a new EP tube.
7. The detection method according to claim 3, wherein the purification process in step (5) is performed by adding 120. mu.L (1.2X) of magnetic beads in step b, and the remaining purification process is the same as in claim 6.
8. The detection method according to claim 1, wherein the step of analyzing the sequencing data in step S4 comprises:
(1) analysis threshold value: expressed as a percentage of the set threshold for filtering the Noise sequence, the depth of sequence sequencing below the set analysis threshold multiplied by the total depth of sequencing for that locus is considered to be the Noise sequence and is not further analyzed;
(2) locus sequence composition ratio: the locus sequence composition ratio is the ratio of Allle, Stutter and Noise in each locus in the total coverage in that locus;
(3) depth of locus coverage: expressed as the sequencing depth of a locus, i.e., the total reads number for that locus;
(4) sample coverage depth: representing the average sequencing depth of each sample in one sequencing;
(5) reading frequency estimation of alleles: the reading obtained for the reference allele is divided by the sum of the readings for the reference allele and the alternate allele.
9. The detection method according to claim 8, wherein the proportion of each type of locus sequence in the step (2) is calculated by: all% ═ all reads/Total reads; stuffer% ═ stuffer reads/Total reads; and 3, 1-allele% -stuffer%.
10. Use of the detection method of claim 1 in forensic identification.
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