SNP-SNP Genetic Marker Screening Typing Scheme And Population Genetic Characteristics Observation And Investigation

Background: Microhaplotype(MH)is a novel genetic marker with wide application potential,defined as a combination of two or more closely linked Single Nucleotide Polymorphisms(SNPs)within a 200 bp long genomic segment.Compared with traditional Short Tandem Repeats(STR)and SNP genetic markers,microhaplotypes have higher polymorphism than SNPs;lower mutation rate compared with STR;balanced fragment length of loci,less prone to amplification disequilibrium;and no stutter peak generation.All the advantages make microhaplotypes gradually become one of the favored objects of researchers.SNP-SNP genetic marker is a special kind of microhaplotype,which consists of only two SNPs.It also has the advantages and characteristics of microhaplotype,and the fragment length is shorter,which may be more favorable for the application of degradation and micro-inspection materials.Theoretically,without considering recombination and secondary mutations,SNP-SNPs consisting of SNPs of two alleles should have three haplotypes,but previous studies have shown that there are still many SNP-SNPs with four haplotypes.The fourth allele may arise from recombination or secondary mutation in the course of history,or it may arise directly from recombination during meiosis.Therefore,it is important to clarify whether haplotypes arise directly from recombination or are stably inherited.For the screening of microhaplotype loci,there are two main screening methods in today’s research: 1.using public databases as data sources,such as the Thousand Genomes Database,Human Genome Diversity Project(HGDP)database,Hap Map database,etc.,to screen the required loci according to their own research directions;2.2.Follow the loci that have been publicly reported by previous researchers.According to statistics,there are more than 400 microhaplotype loci publicly reported in the literature,which is far from enough for the increasing research demand.Most of the databases currently provide information on individual SNP loci,and for microhaplotype analysis,the genotype and frequency of haplotypes in a population need to be obtained.PHASE software,a statistical tool designed based on Bayesian clustering theory and population genetics,can better meet this need,and it can integrate population genotype data to infer the possible haplotypes in the population and help researchers to perform locus screening.The group has previously established a SNP-SNP detection method based on the Amplification Refractory Mutation System(ARMS)technology and SNa Pshot technology,which is economical,simple,stable and reliable,and can directly obtain single-strand typing data.Objectives: 1.To construct a comprehensive microhaplotype locus screening program,establish a four-allele SNP-SNP microhaplotype locus screening library,and screen for highly polymorphic four-allele SNP-SNP loci.2.Construct a compound amplification system composed of multiple tetraallelic SNPSNPs,initially observe its genetic characteristics,and explore its value in forensic applications;provide some theoretical and technical basis for the future application of microhaplotypes in forensic science.Methods: 1.From the official website of Thousand Genome Database,we downloaded the information of all variant loci of Southern Han(CHS)and Northern Han(CHB) populations.All haplotypes were extracted from the SNP data using the haplotype extraction software developed independently by J.Zhu in the early stage of the group [1].The extraction conditions were:(1)locus length 4-50bp;(2)number of SNPs was 2;(3)indel loci were not included.2.Autonomously write R-script based on R language,correspond the SNP-SNP loci extracted in the first step with the individual typing information of CHB and CHS groups in the VCF file,transform them into input files recognizable by PHASE software,use PHASE software to automatically and batch haplotype imputation,obtain haplotype typing,frequency,as well as locus Ae value,polymorphic information content information,and construct SNP-SNP locus library.We developed a batch primer design tool based on Python language,and used the online tool Primer3 web version4.1.0 to design common primers with the following conditions: the end of the primer is located at the SNP1 position,and the amplicon length is 50-150 bp.The candidate loci were selected from the loci with good polymorphism(high Ae value).4.Specific primer design: Design specific ARMS primers targeting the candidate SNP1.Based on the common preprimers,mismatched bases were introduced at the penultimate third base of the 3’ end of the primers,and the software Oligo7 was used to verify the theoretical hairpin structure formation of the primers and to make adjustments.Depending on the loci fragment length,forward or reverse specific extension primers targeting loci SNP2 were designed using SBE Primer software and verified and adjusted using Oligo7 software.5.10 random samples were selected,and ARMS-PCR and Polyacrylamide Gel Electrophoresis(PAGE)experiments were performed on the candidate loci,and sample SNP1 typing was obtained based on the results of electrophoretic bands.The ARMS primers with good specificity were selected and retained,and the poorly performing loci were discarded.For each candidate locus,2 pure and 1 heterozygous samples were screened.6.Using the same samples from the previous step,SNa Pshot experiments were performed using specific Single Base Extension(SBE)primers,and the samples were typed using the ABI 3130 Genetic Analyzer to obtain sample SNP2 typing.Candidate loci with excellent primer specificity were retained.Combined ARMS-PCR and polyacrylamide gel electrophoresis experiments can be performed to obtain a complete haplotype typing of the sample for this SNP-SNP locus.7.The SNP-SNP loci retained in the previous step were designed with amplicon length of 200-300 bp for sequencing primers.Take 2 separately pure and one heterozygous samples from the previous step,perform PCR using Sanger sequencing primers,cut the products and then perform another PCR,and send them to the sequencing company for Sanger sequencing and sequencing to verify the accuracy of candidate locus typing.8.We added-poly GACT or-poly A tails to some SBE primers,and then divided all the candidate loci into 2 Panels according to the length of each SBE product and the fractionated fluorescence color to construct a composite system.The composite system included the composite of ARMS primers and the composite of SBE extension primers.Firstly,the primer dimer formation of the composite system was evaluated by Auto Dimer software,and after adjustment according to the results,ARMS-PCR and SNa Pshot experiments of the composite system were performed,and the primer concentration ratios were adjusted according to the experimental results to obtain the most balanced,stable and clear typing results as possible.9.147 venous blood samples collected from Han Chinese in Sichuan,China,were subjected to DNA extraction.The samples were typed for DNA using the composite system constructed in this study.The population forensic parameters of 119 unrelated individual samples were calculated using the Modified-Powerstate tool to analyze the candidate SNP-SNP locus haplotype distribution and population genetic characteristics.The paternal power index likelihood ratio was calculated to assess the efficacy of candidate loci for individual identification and parentage determination applications.10.Obtain the genetic distance of SNP-SNP loci using the genetic map provided by Hap Map database.The recombination rate was calculated by the Kosambi function,and the parent-offspring pair sample typing data in the sample were analyzed to observe whether they conformed to the Mendelian inheritance law and whether there was recombination.Results: 1.A four-allele SNP-SNP microhaplotype loci library was successfully constructed based on the Han and Northern Chinese populations,with the following screening criteria: SNP-SNP loci length of 4-50 bp;SNP-SNP loci haplotype number of 4;Hardy-Weinberg equilibrium(HWE)law satisfied;haplotype In all 22 autosomes,there were 1407 SNP-SNP loci that satisfied the above conditions.2.20 SNP-SNP loci meeting the primer design conditions were initially screened,and later verified by ARMS-PCR,polyacrylamide gel electrophoresis,SNa Pshot experiment and Sanger sequencing,a total of 14 SNP-SNP loci with good primer specificity and accurate typing were retained.The site names are: rs4578605-rs2865212,rs9935088-rs11646571,rs2239159-rs2239158,rs4824038-rs7286586,rs2736612-rs10908491,rs6024866-rs8119491,rs11668629-rs2739433,rs1907996-rs2220746,rs1518020-rs2873881,rs832034-rs865559,rs4780314-rs12929277,rs12376066-The polyacrylamide gel electrophoresis and genetic analyzer 3130 typing results of the above loci were all consistent with the Sanger sequencing results.3.10 SNP-SNP loci were selected from the above loci to construct a composite system,and after continuous adjustment of primer concentrations,two stable Panels were finally constructed,each of which was divided into two sub-Panels A and B according to the different primers of ARMS.Panel 1(A&B)contained the following loci: rs4578605-rs2865212 Panel 1(A&B)contains the following loci: rs4578605-rs2865212,rs2239159-rs2239158,rs6024866-rs8119491,rs11668629-rs2739433,rs1907996-rs2220746.rs2736612-rs10908491,rs9935088-rs11646571,rs4824038-rs7286586,rs832034-rs865559.For a random sample typing,then Panel-1A,Panel-1B,Panel-2A,and Panel-2B four tubes of parallel reactions.4.Statistical analysis(1)The recombination rate was calculated and observed.10 SNP-SNP loci had a maximum recombination rate of 0.0037(loci rs9935088-rs11646571),which was close to the mutation rate of STR genetic markers(about 0.002).8 loci had recombination rates between 10-6 and 10-4,which were lower than the mutation rate of STR and higher than the mutation rate of SNP.In the triplicate family samples used in the study,all samples conformed to Mendelian laws of inheritance and no new alleles were generated between parents and offspring.No recombination was observed in the selected loci in the family samples used.(2)Population genetic analysis.DNA samples from 119 unrelated Han Chinese individuals from Sichuan Province,China,were analyzed,and the results showed that the haplotype minimum allele frequencies(MAF)of the 10 SNP-SNP loci ranged from 0.054 to 0.205,with a mean value of 0.141.The mean polymorphism information content(PIC)of the loci was 0.661.The average polymorphism information content(PIC)of the loci was 0.661.As expected from theory,each locus had four alleles.(3)Among the 10 SNP-SNP loci,the Matching Probability(MP)ranged from 0.1140 to 0.1935,with a mean value of 0.1434;the Discrimination Power(DP)ranged from 0.8064 to 0.8859,with a mean value of 0.8565;and the Personal Identification Power(PIP)ranged from 0.8064 to 0.8565.The cumulative probability of matching for the 10 SNP-SNP loci was 0.796397889,and the cumulative Combined Discrimination Power,CDP)was 0.999999996;the cumulative non-parental probability of exclusion(CPE)was 0.999000622.(4)The results showed that all loci were inherited in the family samples according to Mendelian inheritance pattern,and no recombination or mutation was directly observed.The mean value of the Cumulative Paternity Index(CPI)was 4904,the maximum value was 57517,and the minimum value was 77,with three families having a CPI greater than 10000.Conclusion: 1.This study successfully established a genome-wide SNP-SNP genetic marker screening scheme based on 1,000 genomic data combined with PHASE software,which provided a basis for microhaplotype screening.2.This study constructed a compound amplification system based on ARMS-PCR and SNa Pshot technology consisting of 10 SNP-SNP loci,and preliminarily explored the forensic population genetic characteristics of SNP-SNP microhaplotypes,which provided a certain theoretical and technical basis for the future application of microhaplotypes in forensic science.3.This study shows that the typing method based on ARMS and SNa Pshot association can be used to detect SNP-SNP microhaplotypes on the CE platform.4.The SNP-SNP loci selected in this study were highly polymorphic and widely distributed in the Han Chinese population in Sichuan Province,China.These SNPSNP loci have some application potential in the field of forensic personal identification and can be used as a complement to STR markers for forensic personal identification.5.In this study,in the analysis of 28 sets of trios,it was observed that all family lines were inherited in a manner consistent with Mendelian laws of inheritance,and no recombination or mutation was observed.The small sample size of the families used in the study and the lack of polymorphism of SNP-SNP genetic markers for recombination observation are the most important reasons for the non-observation of recombination.