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

A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other applications to improve differentiating unrelated males and allow separating related males. The 13 RM Y-STR markers included in the multiplex are: DYF387S1, DYF399S1, DYF403S1ab, DYF404S1, DYS449, DYS518, DYS526ab, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. This study reflects the proof of concept to analyse all currently known RM Y-STRs simultaneously and describes the optimization of the multiplex assay. The RM-Yplex assay generated complete RM Y-STR profiles down to 62.5 pg of male template DNA, and from male–female DNA mixtures at all ratios tested. We herewith introduce and make available for widespread use in forensic and anthropological studies, an effective and sensitive single multiplex assay for simultaneous genotyping of 13 RM Y-STRs

Rapidly mutating Y-STRs multiplex genotyping panel to investigate UAE population

Development of a new multiplex comprising of 13 Rapidly Mutating Y STRs (RM Y-STR) is presented. The multiplex will aid investigating the human genetic structure of United Arab Emirates (UAE) populations and would also be used to investigate unresolved forensic cases in Dubai Forensic Laboratory. Thirteen markers have been included in the multiplex. A mini validation of the new multiplex has been carried out including specificity, sensitivity and mixture studies. These markers have been analysed in 600 male samples from UAE population. Allelic frequencies, haplotype diversity and discrimination capacity were determined for the 13 RM Y-STRs. Mutations pattern analysis of the RM Y-STR loci in a typical UAE family has also been carried ou

Development and validation of an allelic frequency database for Qatari population using 13 rapidly mutating Y-STRs multiplex assay

Differentiating male lineages using non-recombining Y-chromosomal genetic markers is highly informative for tracing human migration and for forensic studies. Recently, it has been shown that the level of male lineage resolution can be enhanced by analysing rapidly mutating (RM) Y-STRs. The aim of this study was to develop an allelic frequency database for Qatari population to evaluate the resolution power of 13 RM Y-STRs. The overall haplotype diversity (HD) was 100%. It was found that the markers which contributed the most toward high HD were DYF399S1 and DYF403S1a/b. Together with their value for paternal male relative differentiation, these RM Y-STRs will be a valuable asset for forensic casework. AMOVA test was performed between Qatari population in comparison to Gulf countries, Middle East, and several worldwide population data sets. FST values were also calculated. Geography was found to account considerably for the pattern of population sub structuring. The RM Y-STR markers showed remarkable haplotype resolution power in the Qatari population, high gene diversity and sufficient robustness for a diverse range of applications