Internal validation of an improved system for forensic application: a 41-plex Y-STR panel

Abstract

Y-chromosome short tandem repeats (Y-STRs) have a unique role in forensic investigation. However, low-medium mutating Y-STRs cannot meet the requirements for male lineage differentiation in inbred populations, while rapidly mutating (RM) high-resolution Y-STRs might cause unexpected exclusion of paternal lineages. Thus, combining Y-STRs with low and high mutation rates help to distinguish male individuals and lineages in family screening and analysis of genetic relationships. In this study, a novel 6-dye, 41-plex Y-STR panel was developed and validated, which included 17 loci from the Yfiler kit, 9 RM Y-STR loci, 15 low-medium mutating Y-STR loci, and three Y-InDels. Developmental validation was performed for this panel, including size precision testing, stutter analysis, species specificity analysis, male specificity testing, sensitivity testing, concordance evaluation, PCR inhibitors analysis, and DNA mixture examination. The results demonstrated that the novel 41-plex Y-STR panel, developed in-house, was time efficient, accurate and reliable. It showed good adaptability to directly amplify a variety of case-type samples. Further, adding multiple Y-STR loci significantly improved the system’s ability to distinguish related males, making it highly informative for forensic applications. In addition, the data obtained was compatible with the widely used Y-STR kits, facilitating the search and construction of population databases. Moreover, the addition of Y-Indels with short amplicons improves analyses of degraded samples.