Microhaplotype And Y-SNP/STR(MY):A Novel MPS-based System For Genotype Pattern Recognition In Two-person DNA Mixtures

Y-chromosomal haplotypes based on Y-short tandem repeats(STRs)and Y-DNA haplogroups are used to characterize paternal lineages of unknown male trace donors.However,Y-chromosomal genetic markers are not currently sufficient for precise individual identification.Microhaplotype analysis was recently introduced in forensic genetics with the development of massive parallel sequencing technology and may facilitate identification and DNA mixture deconvolution.Therefore,using both genetic markers is beneficial in many forensic scenarios,especially crime scenes with male suspects,such as sexual assault cases.We developed a novel MPS-based panel,Microhaplotype and Y-SNP/STR(MY),by multiplex PCR and 150 bp paired-end sequencing,including 114 Y-SNPs(twelve dominant Y-DNA haplogroups),45 Y-STRs(N-1 Stutter<0.09;estimated mutation rate<5 × 10-3),and 22 microhaplotypes(allele coverage ratio>0.91;pairwise distance>10 Mb).The effective number of alleles(Ae)ranged from 3.62 to 14.72,with an average of 7.17,in 100 Chinese Guangdong Han individuals.The cumulative discrimination power was 1-5.00 × 10-31,and the cumulative power of exclusion was 1-5.00 × 10-8 and 1-4.85 × 10-12 for duo and trio paternity testing,respectively.Based on the forensic evaluations and population genetics analyses of populations from 1000 Genomes Project,the system efficiency of the 22 microhaplotypes was equivalent to that of 2628 forensic CODIS and non-CODIS STRs,demonstrating that 22 microhaplotypes in MY system could be regarded as a complement to conventional STRs for forensic application(human individual identification,paternity testing,DNA mixture deconvolution,and biogeographic ancestry inference,etc).Additionally,MY system-based genotype pattern recognition(GPR)of twoperson DNA mixtures is proposed.We integrated 26 two-person genotype combinations into nine genotype patterns and validated the application range of GPR based on DNA profiles of ten sets of simulated male-male DNA mixtures(1:10-1:2).Furthermore,the actual mixing ratio-depth of coverage(DoC)ratio(RDoc)regression relationships were established for different genetic markers and genotype patterns.In five overlapping areas,genotype differentiation of the major and minor contributors required likelihood ratio methods.In nonoverlapping areas,the genotype pattern could be recognized by comparing the observed RDoC and RDoC ranges.In conclusion,GPR can be used to deconvolute two-person DNA mixtures(application range:1:10-1:2)for individual identification.