Development, validation and applications of a novel multiplex assay RM-Yplex amplifying 13 rapidly mutating Y chromosome short tandem repeat regions

A polymerase chain reaction (PCR) multiplex assay capable of amplifying 13 rapidly mutating Y chromosome short tandem repeats (RM Y-STRs) simultaneously was developed and optimised. This multiplex assay which was termed RM-Yplex is the first to include all 13 RM Y-STRs including DYF387S1, DYF399S1, DYF403S1a/b, DYF404S1, DYS449, DYS518, DYS526a/b DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. A developmental validation was performed following the Scientific Working Group for DNA Analysis Methods (SWGDAM) revised guidelines. Robustness and limitations of the assay were demonstrated through a range of studies including reproducibility, sensitivity, specificity, stability and mixture studies. Appropriate controls were used during the studies that included a number of male and female commercial controls including, 2800M, 9948 and Taqman male controls and 9947A female control. An allelic ladder was developed for the assignment of the alleles. This was done by choosing samples with different alleles, amplifying them and then adjusting the volumes of amplified products in a mixture. The developed mixtures were used to balance the composite ladder. Multiple alleles of the various loci included in the ladder were sequenced. Reference haplotypes were developed for the 5 male samples included in the Y chromosome Standard Reference Material 2395 (SRM2395) using RM-Yplex. The International Society of Forensic Genetics (ISFG) recommendations were followed for adopting allele nomenclature. As part of developmental validation, the assay was included in an external proficiency trial which was concluded successfully. An internal validation of RM-Yplex was carried out at the Department of Forensic Sciences and Criminology Laboratory, Dubai where apart from other studies; application of the assay was demonstrated using non-probative forensic casework samples. The value of RM-Yplex was demonstrated for differentiating close male relatives in a case where a previously used Y-STR multiplex assay had shown identical haplotypes for those individuals. 1160 male individual samples were analysed in this study including UAE, other Arabian Peninsula populations as well as two South Asian populations residing in United Arab Emirates. RM-Yplex haplotypes have extremely high power of discrimination. The haplotype diversity for RM-Yplex haplotype is much more than the existing commercial Y-STR assays. Population studies have been carried out for the Arab, Indian and Pakistani populations. AMOVA was conducted for determining the apportionment of diversity and pairwise FST’s were estimated between populations. These have shown a marked homogeneity within the UAE Arab sub-populations. MDS plots of pairwise FST’s indicated that populations were not grouped significantly in accordance with the geographical locations. A network analysis showed the extent of distribution of haplotypes of various populations and their relationships. A highly sensitive and reliable RM-Yplex multiplex assay has been thus developed, which is expected to help genetic populations studies and forensic casework.