The design, optimization and testing of Y chromosome short tandem repeat megaplexes

A multiplex polymerase chain reaction (PCR) assay capable of the simultaneous amplifying 20 Y chromosome short tandem repeat (STR) markers has been developed and tested to aid human testing and population studies. These markers include all of the Y-STR markers that make up the "extended haplotype" used in Europe (DYS19, DYS385 a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, and YCAII a/b) plus the additional polymorphic Y-STR markers (DYS437, DYS438, DYS439, DYS447, DYS448, DYS388, DYS460, and GATA H4). The Y-STR 20plex is the first to include a simultaneous amplification of all the markers within the European "minimal" and "extended haplotype." A subset of the Y-STR 20plex primers, the Y-STR 9plex was also developed and tested. The Y-STR 9plex contains only the markers within the European minimal haplotype. Lastly, a Y-STR 11plex was designed and tested. The markers within the Y-STR 11plex are DYS385 a/b, DYS447, DYS448, DYS450, DYS456, DYS458 and DYS 464 a/b/c/d. Validation experiments were performed in order to assess the reliability of the haplotypes generated by these newly designed Y-STR multiplexes. The validation experiments included concordance, precision, specificity and sensitivity studies. Additionally, a total of 647 male samples from three different U.S. populations were analyzed for all the loci included in the Y-STR 20plex and Y-STR 11plex. Allelic frequencies for all of the Y-STR markers were tabulated as well as haplotype diversity data for various combinations of markers. Y-STR multiplexes were tested against samples from forensic cases that have already been closed. Analysis of these samples demonstrate that the Y-STR multiplexes developed here can handle real casework samples. Finally, the prototype Y Standard Reference Material (SRM) 2395 was characterized. SRMs are often used to validate a laboratory's measurement capability. SRM 2395 is a Y-STR profiling standard that consists of five components, four male (A-D) and one female (E). The characterization included the DNA sequencing of 18 different Y-STR loci for each component (including all of the markers in the European minimal haplotype), presenting a suggested nomenclature for allele designations, and running these components against commercially available Y-STR kits and the Y-STR multiplexes designed in this study.