The Contribution of Massively Parallel Sequencing in Disclosing Unusual Tri- and Tetra-Allelic Type Patterns Detected...

Atypical allelic patterns with additional alleles at multicopy Y-short tandem repeats (Y-STRs), such as DYS385a/b and DYF387S1, mainly arise from duplication or gene conversion events occurring in the palindromic regions of the Y chromosome where these markers are located. Although rarely encountered in forensic genetics, these allelic patterns require accurate deconvolution of the single allelic components to ensure correct genotype interpretation. Capillary electrophoresis (CE), the standard method for STR typing, infers multiallelic Y-STR genotypes through intra-locus and intra-color peak height ratios. However, this approach may be insufficient when the pattern includes isoalleles (alleles identical in length but differing in sequence), potentially leading to an underestimation of the number of alleles and therefore the true allelic configuration. Massively parallel sequencing (MPS) technique, through amplicon sequence-based analysis, provides additional information that can overcome ambiguities and interpretative errors arising from CE analysis of complex Y-STR patterns. In this study, analysis of raw MPS sequence data enabled the identification, in two male-derived DNA samples, of complex tri-allelic patterns at DYS385a/b and DYF387S1 loci, classifiable as type 2-B and 2-C, respectively. Furthermore, in a third male-derived DNA sample, a previously undescribed tetra-allelic pattern was detected at DYF387S1, characterized by an isoallele with double read counts.