A Forensic Genetic Study of Qatari Population Using Massively Parallel Sequencing Methods

Abstract

For the last three decades, Short Tandem Repeat (STR) markers and capillary electrophoresis-based DNA sequencers have been the gold standard technology for human identification testing in the forensic field. However, Massively Parallel Sequencing (MPS) has the potential to replace the current CE-based technology. All laboratories globally, including the Qatar Forensic Laboratory, have stringent strategies and regulations in improving and processing high numbers of reference and casework samples, using instrumentation and technologies to maximize output using new STR kits consisting of larger number of loci. In this project, the highly sensitive GlobalFilerTM PCR amplification kit was validated as per Scientific Working Group in DNA Analysis (SWGDAM), recommendations. This kit was demonstrated to be highly sensitive for all types of samples included degraded bone samples and it had an extremely high power of discrimination. This was compared to MPS based on Illumina ForenSeq™ DNA Signature kit. MPS technology has enabled sequencing of several types of genetic loci in one multiplex including single nucleotide polymorphisms (SNPs) and STRs. The Illumina ForenSeq™ DNA Signature kit includes autosomal/Y/XSTRs and Ancestry/Identity/Phenotype SNP (AISNPS, iSNPs & pSNPs) loci.
One hundred and fifty (150) reference samples and 30 bone samples were profiled using the ForenSeqTM DNA Signature kit. PCR Primer Mix B for this kit was used containing autosomal/Y/X STRs and AISNPS, iSNPs and pSNPs loci. A high percentage of alleles from all loci included in the kit could be obtained when 50 or fewer sample libraries were pooled per run. The average read depth was 20,000 reads for all sequencing runs. This thesis also focused on studying the efficacy of MPS technology for severely degraded bone samples. The results demonstrated the effectiveness of MPS methods due to the small size of the PCR amplicons that can help in amplifying low template/ degraded DNA samples. Samples were profiled successfully for most loci from an input DNA of up to 10 pg, which showed that the MPS methods were highly sensitive. The data generated through MPS were analysed using ForenSeqTM Universal software and STRait Razor V3 for the primary, secondary and tertiary analyses. The ForenSeq™ DNA Signature kit had a much higher discrimination power than the GlobalFiler™ kit. The analyses of the sequence of STR alleles in STRait Razor software were able to determine novel alleles in the autosomal, Y and X STR as well as SNPs loci. This increased number of alleles had a clear advantage in forensic casework. Several of these sequenced-based alleles had not been reported earlier in the literature. This study also confirmed some of the previously determined alleles in several loci. The MPS kit included 55 Ancestry Informative Marker SNPs (AISNPs). The Qatari population has been a melting pot of various populations and this forensic study was the first of its kind to generate new data on the genetics of Qatari population. The 55 ancestry marker allele frequency data for 150 samples were analysed and compared to 139 world populations using STRUCTURE software. The Qatari population data for the Kidd AISNPs were found to be similar in patterns when compared to other Middle Eastern populations. FROG-kb analysis led to all Qatari samples being correctly assigned to Middle Eastern populations showing that the Illumina software needs to be enhanced by adding more databases from the Middle East. In conclusion, the results have clearly demonstrated the potential use of MPS methods to study the genetics of Qatari population.