Discriminatory power of the Precision ID GlobalFiler™ NGS STR panel v2 in monozygotic twins for forensic applications

Short Tandem Repeats (STRs) are the standard technique used in forensic genetics for individual identification due to their high polymorphism and robustness. Although Capillary Electrophoresis (CE) enables the analysis of many STRs, Next-Generation Sequencing (NGS) offers enhanced resolution and the ability to detect STRs' isoalleles and their flanking regions, enhancing the discrimination power of this analysis. Despite the fact that STR kits for NGS are well standardized for evaluating forensic samples, there is no data on their effectiveness in differentiating monozygotic (MZ) twins, which are indistinguishable by CE. This study aimed to assess the efficacy of Thermo Fisher Scientific's Precision ID GlobalFiler™ NGS STR Panel v2 in discriminating monozygotic twins by analyzing STRs and their flanking regions. Peripheral blood samples from thirty-two pairs of monozygotic twins were collected and CE and NGS profiles were compared. Then, NGS data were analyzed using Converge, IGV and STRait Razor software. The results showed agreement between the profiles generated by CE and NGS, with the exception of individual G037B, which presented a dropout in allele 13 of the Penta D marker in the Converge analysis. Using IGV, it was possible to note that this result was probably due to sequencing failure in different parts of the reads. On the other hand, STRait Razor was successful in detecting allele 13 in this individual, even with its low coverage (19 reads). Isoalleles were observed in 8 STRs markers, in both individuals of the 10-pair MZ, however, it was not possible to differentiate identical twins. Next, by analyzing the flanking region of the markers, the Converge software pointed out two SNPs in the regions adjacent to the STRs. For individual G016A, rs560609904 was detected in the TPOX marker and for individual G027B, the SNP rs569521603 in D6S1043. But after analysis by STRait Razor and Sanger sequencing to validate these results, these findings were not confirmed. The NGS error rate was analyzed, showing that the SNPs previously pointed out by Converge were in fact sequencing errors and not somatic mutations. In summary, in spite of not differentiating MZ twins, we concluded that the Precision ID GlobalFiler^TM NGS STR Panel v2 kit is an effective tool at identifying isoalleles, which increases the discrimination power for forensic analysis. We also strongly recommend the use of more than one genotype calling software to analyze NGS data in order to confirm the results and to tell apart sequencing errors from actual genetic variability.