Validation of the Microreader 40Y ID System: a Y-STR multiplex for casework and database samples

Y-chromosome-specific short tandem repeat loci (Y-STRs) are commonly analysed in forensic science for paternity testing, familial searches, and, in sexual assault cases, to determine male DNA identity from mixed sources with high background female DNA content. The Microreader 40Y ID System is a six-dye multiplex amplification kit that contains 17 Y-STR loci from the Yfiler Plus PCR Amplification Kit and the powerplex Y23 system (DYS19, DYF385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS549, DYS635(Y GATA C4), DYS643, Y GATA H4, DYS460, DYS481, DYS533, DYF387S1, DYS449, DYS518, DYS570, DYS576, and DYS627), plus six high polymorphic loci (DYS444, DYS447, DYS557, DYS596, DYS527 a/b) as well as 4 additional candidate Y-STR loci (DYS593, DYF404S1, DYS645) and a Y-Indel loci (Rs2032678), thereby providing greater efficiency, compatibility, and accuracy. The Microreader 40Y ID System can directly amplify markers from blood or saliva on filter paper or FTA cards, without template extraction or purification, and can also be used for extracted DNA templates. To verify the efficiency and accuracy of the kit, the Microreader 40Y ID System was validated by investigating sensitivity, amplification conditions, male-male and male-female mixtures, PCR inhibition, species specificity, reproducibility, and efficacy with degraded samples. The Y-STR loci were also tested using 437 male samples from Tibet, Han, and Yi. The Microreader 40Y ID System was able to compensate for some of the shortcomings of Y-STR markers in practical applications, such as cost and profile interpretation, and fully meets the domestic Y chromosome database construction specifications and requirements.