Developmental validation of the Yfiler^® Plus PCR Amplification Kit: An enhanced Y-STR multiplex for casework and database applications

Y-chromosomal loci have proven useful in solving investigations where low levels of male DNA are present in a high female DNA background. An intrinsic limitation of Y-STRs compared with autosomal STRs is a reduced power of discrimination due to a lack of recombination throughout most of the Y-chromosome. Thus, in an effort to increase the power of discrimination we have developed a new 6-dye, 27-plex Y-STR system that includes the 17 loci from the Yfiler^® and Yfiler^® Direct kits (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 (Y GATA C4), and Y GATA H4) plus three highly polymorphic Y-STR loci (DYS460, DYS481, and DYS533), and seven rapidly mutating Y-STR loci (DYF387S1a/b, DYS449, DYS518, DYS570, DYS576, DYS627) which allow for improved discrimination of related individuals. The Yfiler^® Plus PCR Amplification Kit is a dual application assay designed to amplify DNA from extracted casework and database samples from storage cards and swab lysates via direct amplification. Compared to the Yfiler PCR Amplification Kit, the new multiplex shows increased discrimination of male lineages and also improved performance in inhibited samples, improved balance in male DNA samples mixed with female DNA at ratios >1:1000, and faster time to results. The Yfiler Plus Kit shows very high concordance to the Yfiler Kit but discordance with the PowerPlex^® Y23 Kit at the DYS481 locus was observed in 2 out of 30 samples tested. This developmental validation work follows the SWGDAM guidelines and demonstrates that the assay is robust and suitable for use on forensic casework and database samples.