Next Generation Sequencing On Y Chromosomes Of 120 Male Henan Hans And Genetic Diversity Analysis On Their Y-SNPs

Background and purposeHuman genome is composed of nuclear genome and mitochondrial genome.The nuclear genome is composed of 22 pairs of autosomes,and two sex chromosomes: X and Y.About 95% of Y chromosome sequences can neither be matched nor be recombinated with other chromosomes,thus it is called the non-recombination regions of Y chromosome,or NRY.Because Y chromosome can only be passed from father to son,as it follows the strict paternal genetic law,Y chromosome has been accumulating accidental genetic mutations,and passed them down from father to son during the long process of human evolution.For these reasons,Y chromosome has become a valuable collection of genetic markers.These genetic markers include satellite DNA with various lengths,In Del sites,and single nucleotide polymorphism(SNP).SNP sites have many advantages over other markers,such as wide distribution,easy typing,and no reversion mutation;so,it has great values for the researches on human origin,migration,evolution and related historical events.The Y-DNA haplogroup phylogenetic tree(ISOGG Y-DNA Haplogroup Tree,http://www.isogg.org/tree)based on Y-SNPs has become an important reference in the field of anthropology and population genetics.In recent ten years,the next generation sequencing(NGS)has been greatly developed.It directly presents genomic sequences and other detailed information to researchers,demonstrating its advantages such as high throughput,low cost and high speed.Especially in recent years,new SNPs found by NGS increase exponentially,providing new possibilities for related researches and applications.In this study,we sequenced the whole Y chromosomes of 120 male Hans in Henan Province by NGS technology.Based on the sequencing data,we searched and selected Y-SNPs for forensic application purpose.At the same time,the ISOGG Y-DNA phylogenetic tree of male Han population in Henan Province was drawn,the evolution history of male Henan Hans was explored,with their characteristics and branch structures being studied,which finally provided vital reference to certain forensic applications and anthropological/genetic studies.MaterialsAfter informed consent,120 unrelated male individuals of the Han,aged not less than 18 years old,with ancestral origin in Henan and having lived in Henan Province for at least three generations,were selected.2ml peripheral blood was taken from each person and treated with sodium citrate anticoagulant or EDTA anticoagulant.The blood was cryopreserved at-80? for use.Methods1.Genomic DNA was extracted from peripheral blood of the 120 male Henan Hans by using the 2ml blood genomic DNA extraction kit of Shanghai Laifeng company.2.The Precision Medicine Center of Zhengzhou University and Shanghai Yinglaidun Biotechnology Co.,Ltd.were entrusted to complete Y chromosome sequencing with their own next generation sequencing platform.Sixty samples for each.3.The NGS data were evaluated on Linux cluster of National Supercomputing Zhengzhou Center and Zhengzhou University Supercomputing platform according to the standard bioinformatics analysis process(BWA + samtools),with sequences compared to human reference genome 19(hg19).4.Extraction of Y-SNPs from sequencing results: using GATK software and referring to the db SNP database of UCSC,all known Y-SNP loci in our NGS results among the 120 samples were extracted and genotyped.Then they were statistically analyzed and screened with population genetics method,and their haplotype diversities(HD)were calculated to evaluate the forensic application value.5.Y-DNA haplogroup determination: inputting the BAM file into Yleaf and running the program.Referring to the ISOGG Y-haplogroup evolutionary tree to get the Y-DNA haplogroups for the 120 samples,and determining their branch attributions of all samples in the Y-haplogroup evolutionary tree.The proportion of each haplogroup among the 120 male Henan Hans was calculated.6.Differentiation time analysis: Beast2 software was used to calculate the differentiation time of the 120 samples through Y-SNP analysis,to reconstruct the mutation accumulation process of Y-SNP on the time scale,and try to analyze the origin and migration history of male Henan Han population.Results1.Sixty samples were successfully sequenced in the Precision Medicine Center of Zhengzhou University;effective data was obtained from each sample.The average sequencing depth was 15.01 ×,the base quality was stable and reliable,and the sequencing quality was good.Sixty samples were also successfully sequenced in Shanghai Yinglaidun Biotechnology Co.,Ltd.,effective data was also obtained from each sample.Its average depth was 74.30×,the base quality was stable and reliable,and its sequencing quality was good.2.A total of 71543 known Y-SNPs were obtained by comparing the 120 Ychromosome NGS data with db SNP,among which 66290 were bi-allelic Y-SNPs.3.After screening,36 bi-allelic Y-SNPs with base calling rate higher than 95%and wild-type allele frequency in the range of 0.4-0.6 were obtained from our 120 male Henan Hans.Among which 35 were: rs374887753,rs79917345,rs76000750,rs13305177,rs76204317,rs935098066,rs113547797,rs12164443,rs370594792,rs1200009271,rs911641840,rs200510226,rs144763445,rs200346965,rs796107181,rs60001530,rs200839143,rs2267801,rs77684578,rs76047574,rs1021199046,rs2527475,rs77081563,rs377448563 Rs199777144,rs764534147,rs1179626263,rs879181519,rs925064378,rs6568353,rs73628906,rs9285384,rs369323647 and rs74747930.When their allele combination among the 120 individuals were evaluated as haplotypes,119 haplotypes were observed among the 120 samples,and the haplotype diversity(HD)was 0.99986.4.With reference to the ISOGG 2019 Y-DNA haplogroup tree,a total of 78 different Y-DNA haplogroups were listed by the y-leaf software.103 samples were identified as O haplogroup,accounting for 85.83% of 120,among which 25 samples were classified as O1 haplogroup,accounting for 20.83% of 120,78 samples were classified as O2 haplogroup,accounting for 65%.Besides,there were 8 samples classified as C haplogroup,accounting for 6.67%;one sample was classified as D haplogroup,accounting for 0.83%;six samples were classified as N haplogroup,accounting for 5%.Still there were 2 samples classified as R haplogroup,accounting for 1.67%.5.When using BEAST2 software to estimate the differentiation time,the phylogenetic tree obtained by BEAST2 is similar to that by Yleaf software.The outbreak time of haplogroup differentiation was about 2000-5000 years ago.Conclusion1.There are some differences in terms of sequencing coverage and SNP calling when different sequencing platforms and batches are used,which may affect the subsequent analysis process.2.The acquisition efficiency of Y-SNPs can be greatly improved by NGS technology.However,due to the large scale raw data generated by NGS,large amount of computing resources will be needed for operations like data quality control,comparison,typing and other bioinformatic analysis.This will be a big challenge for the current forensic DNA labs.3.After screening,thirty-five Y-SNPs with allele frequencies at 0.4-0.6 were obtained.The combination use of these 35 Y-SNPs,as a haplotype,is also highly diversified.119 different haplotypes were observed among the 120 samples.This haplotype diversity value was high,demonstrating that such Y-SNP haplotypes had a good application prospect in forensic medicine.4.The Y-DNA haplogroups of the 120 samples include 78 haplogroups,among which 5 major haplogroups(C,D,N,O,R)were detected,indicating that the structure of male Han population in Henan Province was highly mixed,and the structure of male Han population was mainly characterized by O haplogroups(85.83%).Non-O haplogroups,including C,D,N and R,exist,with their proportions relatively small.This indicates that not only the sources of modern male Henan Hans is diversified,but also several haplogroups have very big contributions to the formation of modern male Henan Hans.Thus the formation of modern male Henan Hans is rather complex in the process of beginning,migration and fusion.5.According to the analysis on differentiation time,it can be inferred that the formation time of modern male Henan Y haplogroup was mainly located between 5000 to 2000 years ago,which is consistent with archaeology and historical data,reflecting a large-scale population expansion in late Neolithic and Bronze Age,and is consistent with records about population migration and ethnic fusion with surrounding ethnic groups during that period.