Patent Application
20230212671
The invention relates to forensic technology, more particular to a primer composition, a kit and a method for detecting microhaplotype (MH) loci based on next generation sequencing technology, and applications thereof. The primer composition is used for amplifying 163 microhaplotype loci covering 22 pairs of autosomes (also referred to as hetero chromosomes).
The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 22033THXT-USP1-US-2022-0034-SL.xml. The XML file is 290,816 bytes; was created on Sep. 28, 2022; contains no new matter; and is being submitted electronically via EFS-Web.
Forensic genetics mainly relies on the detection and analysis of deoxyribonucleic acid (DNA) genetic markers to solve problems related to individual identification and parentage testing in judicial practice. Among many kinds of genetic markers, short tandem repeat (STR) is the most commonly used genetic marker because of its good polymorphism and simple typing method. Biallelic single nucleotide polymorphism (SNP) and insertion-deletion (InDel) markers have the advantages of low mutation rate and short amplification fragment, which can make up for the shortcomings of STR of high mutation rate, large amplification fragment and stutter peaks in typing, and have more advantages in the analysis of degradation samples and biogeographic ancestry inference. However, due to the low polymorphism of a single locus, it is often necessary to increase the number of detection loci to achieve the detection efficiency similar to that of the STR system. Therefore, some scholars proposed the concept of compound genetic markers, including linked genetic markers SNP-STR, InDel-STR, multi-InDel, etc.
In 2014, Professor Kenneth K. Kidd (“Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics”, Forensic Science International: Genetics, 2014, pp 215-224) of Yale University proposed the concept of microhaplotype (MH), which is a locus with two or more SNP sites within a 200-300 base pair (bp) DNA segment. Microhaplotypes composed of SNPs not only have high polymorphism comparable to STR loci and do not produce stutter peaks, but also retain the characteristics of low mutation rate and short fragments of SNPs, which have advantages in forensic community. Some systems including microhaplotype markers (also referred to as microhaplotype loci), such as a compound system with 74 microhaplotype markers constructed by Oldoni et al. (“A sequence-based 74plex microhaplotype assay for analysis of forensic DNA mixtures”, Forensic Science International: Genetics, 2020, page 102367) and a compound system with 118 microhaplotype markers constructed by Maria de la Puente et al. (“Building a custom large-scale panel of novel microhaplotypes for forensic identification using MiSeq and Ion S5 massively parallel sequencing systems”, Forensic Science International: Genetics, 2020, page 102213), have good capabilities of individual identification, parentage testing, and mixture analysis.
For the analysis and detection of mixture samples, the traditional STR typing test often shows multiple allele peaks. It is difficult to distinguish stutter peaks from the allele peaks with a small contribution ratio or noise allele, and the interpretation of the evidence value is quite difficult. MH has no stutter peak interference, advantages of both STR and SNP markers, which is an ideal genetic marker for analysis and detection of the mixture samples.
Due to long-term migration and evolution, the frequency distribution of some SNPs varies greatly among different populations. Screening MH composed of ancestry-informative SNP (AI-SNP) can provide an important basis for research on population structure and ancestry inference in forensic community. Kenneth K. Kidd initially established a system containing 31 MH markers, which can better distinguish the five major geographical regions of Africa, Europe, Southeast Asia, East Asia, America and Pacific islands, showing the superiority of MH as an ancestral information marker.
Next generation sequencing (NGS), also known as massively parallel sequencing, has the advantages of high throughput and high accuracy, which provides a platform for the detection and application of new genetic markers. MH is composed of multiple SNPs, which is essentially sequence polymorphism. The next generation sequencing can obtain all MH typing at one time, realize the parallel analysis and detection of a large number of genetic markers.
In order to overcome the defects in the related art, the invention screens MH loci with forensic application value in ancestry inference, mixture analysis, individual identification and parentage testing in Asian population, and develops and establishes a primer composition and a kit that can simultaneously detect 163 MH loci at a time based on next generation sequencing technology.
To achieve the above purpose, the invention adopts the following technical solutions as follows.
In a first aspect of the invention, a primer composition for detecting MH loci based on the next generation sequencing technology is provided. The primer composition includes one or more pairs of amplification primers of 163 MH loci.
The 163 MH loci consist of mh01CP007, mh01CP008, mh01CP012, mh01CP016, mh01KK001, mh01KK070, mh01KK072, mh01KK106, mh01KK117, mh01KK172, mh01KK205, mh01KK210, mh01KK211, mh02CP004, mh02KK003, mh02KK004, mh02KK073, mh02KK102, mh02KK105, mh02KK131, mh02KK134, mh02KK136, mh02KK138, mh02KK139, mh02KK201, mh02KK202, mh02KK213, mh02KK215, mh03KK006, mh03KK007, mh03KK008, mh03KK009, mh03KK216, mh04CP002, mh04CP003, mh04CP007, mh04KK010, mh04KK011, mh04KK013, mh04KK015, mh04KK016, mh04KK017, mh04KK019, mh04KK028, mh04KK029, mh04KK030, mh04KK074, mh05CP004, mh05CP006, mh05CP010, mh05KK020, mh05KK022, mh05KK062, mh05KK078, mh05KK079, mh05KK122, mh05KK123, mh05KK124, mh05KK170, mh06CP003, mh06CP007, mh06KK026, mh06KK030, mh06KK031, mh06KK080, mh06KK101, mh07KK030, mh07KK031, mh07KK081, mh07KK082, mh08KK032, mh09KK020, mh09KK033, mh09KK034, mh09KK152, mh09KK153, mh09KK157, mh09KK161, mh10CP003, mh10KK083, mh10KK084, mh10KK085, mh10KK086, mh10KK087, mh10KK088, mh10KK101, mh10KK163, mh10KK170, mh11CP003, mh11CP004, mh11CP005, mh11KK036, mh11KK037, mh11KK038, mh11KK039, mh11KK040, mh11KK041, mh11KK089, mh11KK090, mh11KK091, mh11KK180, mh11KK187, mh11KK191, mh12KK042, mh12KK043, mh12KK045, mh12KK046, mh12KK092, mh12KK093, mh12KK202, mh13CP008, mh13KK047, mh13KK213, mh13KK217, mh13KK218, mh13KK225, mh13KK226, mh14CP003, mh14CP004, mh14KK048, mh14KK101, mh15CP001, mh15CP003, mh15CP004, mh15KK066, mh15KK067, mh15KK069, mh15KK095, mh16KK053, mh16KK062, mh16KK096, mh16KK255, mh16KK302, mh17CP001, mh17CP006, mh17KK014, mh17KK052, mh17KK053, mh17KK054, mh17KK055, mh17KK077, mh17KK105, mh17KK110, mh17KK272, mh18CP003, mh18CP005, mh18KK285, mh18KK293, mh19CP007, mh19KK056, mh19KK057, mh19KK299, mh19KK301, mh20KK058, mh20KK059, mh20KK307, mh21KK313, mh21KK315, mh21KK316, mh21KK324, mh22KK060, mh22KK064, and mh22KK303.
In an embodiment, the primer composition includes one or more pairs of the amplification primers with nucleotide sequences respectively shown in SEQ ID NO: 1 through SEQ ID NO: 326.
In an embodiment, the primer composition includes the amplification primers with the nucleotide sequences respectively shown in SEQ ID NO: 1 to SEQ ID NO: 326.
In a second aspect of the invention, a kit for detecting MH loci based on the next generation sequencing technology including the primer composition is provided, and the kit further includes a polymerase chain reaction (PCR) mixed solution and a PCR reaction solution.
In a third aspect of the invention, a method for detecting MR loci based on the next generation sequencing technology using the kit above is provided, including the following steps:
step 1, taking a sample to be tested, extracting a DNA sample, and quantifying extracted DNA sample;
step 2, preparing a multiplex PCR system, and conducting a first round of multiplex PCR; after a reaction of the first round of multiplex PCR is completed, obtaining a product, then adding a purification reaction solution to purify the product, and conducting magnetic bead sorting on the purified product;
step 3, repairing the purified product to make ends equal and adding an adenine base (A) into the ends then ligating sequencing adapters on the ends to obtain a complemented product, and then purifying the complemented product again using purification magnetic beads to obtain a purified elution product;
step 4, conducting a PCR reaction on the purified elution product using a reaction system to construct a library, wherein the reaction system includes the elution purified product, a PCR mixed solution, a QU reagent, a mixed post-P5 primer, and a mixed pre-p7 primer;
step 5, conducting purification and quantification on the library, specifically including: purifying the product by using purification magnetic beads, and conducting quantification and quality control on the library by using Qubit™;
step 6, conducting sequencing and data analysis, specifically including: using the constructed library on a MiSeq FGx™ platform for sequencing to obtain sequencing data; trimming the sequencing adapters of the obtained sequencing data by using a Trimmatic software to obtain sequences, then comparing the sequences with human reference genome hg19 by using a burrows-wheeler aligner (BWA) software, and obtaining MH typing by using a Python tool.
In an embodiment, a concentration of the DNA sample is 5 nanograms per microliter (ng/μL).
In an embodiment, the multiplex PCR system includes 20 μL total reaction volume, specifically including 8 μL of the PCR mixed solution, 2 μL of the PCR reaction solution, 8 μL of primer mixed solution, and 2 μL of the DNA sample.
In an embodiment, a concentration of the primer mixed solution is 0.5 micromoles per liter (μM).
In an embodiment, reaction conditions of the multiplex PCR in the step 2 includes: pre-denaturation at 95° C. for 15 minutes; denaturation at 95° C. for 30 seconds, annealing at 60° C. for 90 seconds, extension at 72° C. for 30 seconds, 24 cycles, heat preservation at 72° C. for 10 minutes.
In an embodiment, a reaction system of the repairing the purified product to make ends equal and adding A into the ends in the step 3 includes 50 μL total reaction volume, specifically including 42 μL of the purified product in the step 2, 6.8 μL of end repair dA-tailing buffer, and 1.2 μL of end repair dA-tailing enzyme.
In an embodiment, reaction conditions of the repairing the purified product to make ends equal and adding A into the ends in the step 3 includes: reaction at 30° C. for 30 minutes, then reaction at 65° C. for 30 minutes, and finally heat preservation at 4° C.
In an embodiment, a reaction system of the ligating sequencing adapters in the step 3 includes 80 μL total reaction volume, specifically including 50 μL of the purified elution product in the step 3, 2.5 μL of adapter mixed solution, 16 μL of ligation buffer, 10 μL of ligase, and 1.5 μL of nuclease-free water.
In an embodiment, reaction conditions of the ligating sequencing adapters in the step 3 includes: reaction at 25° C. for 15 minutes, and heat preservation at 4° C.
In an embodiment, a reaction system of the PCR reaction of the step 4 includes 50 μL total reaction volume, specifically including 14 μL of the elution purified product of the step 3, 25 μL of the PCR mixed solution, 3 μL of the QU reagent, 5μL of the mixed capture post-P5 primer, and 5 μL of the mixed capture pre-p7 primer.
In an embodiment, reaction conditions of the PCR reaction in the step 4 includes: reaction at 37° C. for 15 minutes; pre-denaturation at 98° C. for 45 seconds; denaturation at 98° C. for 15 seconds, annealing at 60° C. for 30 seconds, extension at 72° C. for 30 seconds, 10 cycles, then reaction at 72° C. for 5 minutes, and heat preservation at 4° C.
In a fourth aspect of the invention, an application/use of the primer composition or the kit in individual identification, parentage testing, mixture analysis and ancestry inference.
In an embodiment, the individual identification and parentage testing are mainly based on typing results of 48 MH loci with good polymorphism. The 48 MH loci consist of: mh01CP008, mh01CP012, mh01CP016, mh01KK117, mh01KK205, mh01KK211, mh02KK134, mh02KK136, mh04CP002, mh04CP003, mh04CP007, mh04KK030, mh05CP004, mh05CP006, mh05KK020, mh05KK170, mh06CP003, mh06CP007, mh09KK153, mh10CP003, mh10KK163, mh11CP003, mh11CP005, mh11KK180, mh12KK046, mh12KK202, mh13CP008, mh13KK213, mh13KK217, mh13KK218, mh13KK225, mh14CP003, mh14CP004, mh15CP001, mh15KK066, mh16KK255, mh16KK302, mh17CP001, mh17CP006, mh17KK272, mh18CP003, mh18CP005, mh19CP007, mh19KK299, mh20KK058, mh20KK307, mh21KK315, and mh21KK324.
In an embodiment, the genomic DNA sample extracted from the biological sample or the mixed biological sample is subjected to library construction, purification and quantification by using the primer composition, and the constructed library is placed on a MiSeq FGx™ platform for sequencing analysis, and finally the obtained sequencing data is analyzed to obtain the MH typing.
The invention adopts the above technical solutions and has the following technical effects compared with the related art.
The primer composition for detecting MH loci based on the next generation sequencing technology provided by the invention involves 163 MH loci covering 22 pairs of autosomes, which can provide more new genetic information than the system constructed in the past. In addition, compared with the next generation sequencing kit of STR loci, the kit of the invention has better mixture detection capability. Moreover, the MH loci involved in the invention have high ancestry information content and can distinguish populations in Africa, Europe, South Asia and East Asia.
The invention relates to a primer composition for detecting microhaplotype (MH) loci (also referred to as MH markers or MH) based on a next generation sequencing technology, and the primer composition includes one or more pairs of amplification primers of 163 MH loci.
Specifically, the 163 MHs are all from MH loci included in ALFRED website and MHs published in literature, distributed in intron regions, with good polymorphism in Asian population, and a distribution length being smaller or equal than 300 bp. Names, chromosome information and locus information of the 163 MH loci are shown in Table 1:
Multiplex PCR primers are designed according to physical locations. Design principles include: (1) an optimal melting temperature; (2) avoidance of primer dimers and hairpin structures; (3) guanine and cytosine bases (GC) content between 20% and 80%; (4) off-target analysis to reduce primer off-target hybridization; and (5) overlap analysis to reduce the number of primers. In an embodiment of the invention, the primer composition includes one or more pairs of primers with nucleotide sequences shown in SEQ ID NO: 1 through SEQ ID NO: 326. Specific primer sequence information is shown in Table 2:
In an embodiment of the invention, the primer composition includes primers whose nucleotide sequences are shown in SEQ ID NO: 1 through SEQ ID NO: 326.
The invention also relates to a kit for detecting MH loci based on the next generation sequencing technology, including the primer composition, a PCR mixed solution and a PCR reaction solution.
Hereinafter, the invention is described in detail with reference to specific embodiments and accompanying drawings, so as to better understand the invention, but the following embodiments do not limit the scope of the invention.
In the following embodiments, conventional methods are use unless otherwise specified, and conventional commercially available reagents or reagents prepare according to conventional methods are used unless otherwise specified.
The embodiment provides a method for detecting MH loci based on next generation sequencing technology using a primer composition or a kit, including step (1) through step (7) as follows.
Step (1), a sample to be tested is taken, a DNA sample is extracted, and a quantitative sample concentration is 5 ng/μL.
Step (2), a first round of multiplex PCR is conducted, a PCR amplification system and amplification conditions are shown in Table 3.
PCR reaction conditions includes: pre-denaturation at 95° C. for 15 minutes; denaturation 95° C. at for 30 seconds, annealing at 60° C. for 90 seconds, extension at 72° C. for 30 seconds, 24 cycles; heat preservation at 72° C. for 10 minutes. After reaction, a product is obtained, 1 μL of purification reaction solution is added to purify the product, and the following reactions is completed: 37° C. for 10 minutes; 50° C. for 10 minutes; 65° C. for 10 minutes, and heat preservation at 4° C. Then magnetic beads sorting is conducted.
Step (3), the purified product obtained by the step (2) is repaired make ends equal, and an adenine base (A) is added into the ends, and a reaction system thereof is shown in Table 4:
PCR reaction conditions includes: 30° C. for 30 minutes; 65° C. for 30 minutes, and heat preservation at 4° C.
Step (4), ligating sequencing adapters, and a reaction system thereof is shown in Table 5:
PCR reaction conditions includes: reaction at 25° C. for 15 minutes, and heat preservation at 4° C. Then, the reaction product is purified with purification magnetic beads to obtain a purified elution product.
Step (5), PCR amplification is conducted on the purified elution product again, and a PCR reaction system thereof is shown in Table 6:
PCR reaction conditions includes: reaction at 37° C. for 15 minutes; pre-denaturation at 98° C. for 45 seconds; denaturation at 98° C. for 15 seconds, annealing at 60° C. for 30 seconds, extension at 72° C. for 30 seconds, 10 cycles; reaction at 72° C. for 5 minutes; and heat preservation at 4° C.
Step (6), purification and quantification of the library: the product obtained by the step (5) is purified again by using purification magnetic beads, and Qubit™ is used for library quantification and quality control.
Step (7), sequencing and data analysis: the constructed library is placed on MiSeq FGx™ platform for sequencing analysis to obtain sequencing data. For the obtained sequencing data, Trimmatic software is used to trim the sequencing adapters, and then BWA software is used for sequence alignment to compare the sequence with the human reference genome (hg19), and the Python tool is used to obtain MH typing.
The method can be used for individual identification and parentage testing, specifically to select 48 MH loci with good polymorphism in Asian population consisting of: mh01CP008, mh01CP012, mh01CP016, mh01KK117, mh01KK205, mh01KK211, mh02KK134, mh02KK136, mh04CP002, mh04CP003, mh04CP007, mh04KK030, mh05CP004, mh05CP006, mh05KK020, mh05KK170, mh06CP003, mh06CP007, mh09KK153, mh10CP003, mh10KK163, mh11CP003, mh11CP005, mh11KK180, mh12KK046, mh12KK202, mh13CP008, mh13KK213, mh13KK217, mh13KK218, mh13KK225, mh14CP003, mh14CP004, mh15CP001, mh15KK066, mh16KK255, mh16KK302, mh17CP001, mh17CP006, mh17KK272, mh18CP003, mh18CP005, mh19CP007, mh19KK299, mh20KK058, mh20KK307, mh21KK315, and mh21KK324. The primer sequences in Table 2 are used for detection and analysis according to the above steps.
This method can be used for ancestry inference, specifically based on MEI typing results of all 163 loci.
The embodiment is forensic verification of the method provided in the embodiment 1. The specific experiments and results are as follows.
According to requirements of the Scientific Working Group for DNA Analysis Methods (SWGDAM), the sensitivity, accuracy, repeatability and forensic parameters of the multiplex PCR system constructed in the embodiment 1 are calculated.
The results show that the method constructed in the embodiment 1 (for 163 MEI loci) has high sensitivity, and the complete genotyping of MEI loci can be obtained at all tested concentrations. The data statistics of next generation sequencing of DNA under different concentration gradients is shown in
For 48 MEI loci with good polymorphism, the average heterozygosity of 48 loci reaches 0.7227, the polymorphism information content is greater than 0.60, the average individual identification probability reaches 0.8692, and the cumulative individual identification probability is 1-8.26×10−44, the cumulative probability of exclusion in paternity of dyads and the cumulative probability of exclusion in paternity of the triad are 1-1.26×10−8 and 1-8.27×10−16, respectively.
The embodiment is a comparison between the method provided in the embodiment 1 and the ForenSeg™ DNA Signature Prep Kit of the next generation sequencing platform on the analysis efficiency of mixture samples.
The autosomal STR loci in the ForenSeg™ DNA Signature Prep Kit based on the next generation sequencing platform begin to lose a large number of minor alleles below a mixture ratio of 20:1 due to its high sensitivity and stutter peaks. Samples of DNA mixtures with different mixture ratios are prepared and detected by the method provided in the embodiment 1 (for 163 MH) and ForenSeg™ DNA Signature Prep Kit, respectively, to compare the detection performance of mixtures. Table 7 shows the detection rate of the unique minor alleles in the DNA mixture samples with different mixture ratios. The results show that the detection effect of the method provided in the embodiment 1 is obviously superior to that of the STR kit of the next generation sequencing platform.
The embodiment illustrates an application of the method provided in the embodiment 1 in the ancestry inference. The specific operation steps and results are as follows.
The MH genotyping data of 27 populations including 26 populations in the 1,000 Genomes Project and Han population of China are used to compare the genotype frequency distribution differences among 27 populations. In values of MH loci in 27 populations are calculated, the ancestry information content of the loci is evaluated, and principal component analysis is conducted.
The results show that the In values of 163 MH loci are all greater than 0.185, which had high ancestry information content and could be used for ancestry inference. It can be seen from
It can be seen from the above embodiments that the primer composition, the kit and the method provided by the invention provide a new detection means for individual identification, parentage testing, mixture analysis, ancestry inference, etc. in the field of forensic medicine.
The specific embodiments of the invention are described in detail above, by way of examples only, and the invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions of the invention are also included in the scope of the invention. Therefore, the equivalent changes and modifications made without departing from the spirit and scope of the invention should be included within the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021110077419 | Aug 2021 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/115540 | Aug 2022 | US |
| Child | 18067693 | US |
