Evaluation of DNA Polymorphisms for Kinship Testing in the Population of Saudi Arabia

Short Tandem Repeats (STRs) have been the standard DNA marker employed in forensic laboratories for more than two decades. Due to the advances in the kit chemistries and separation technologies (capillary electrophoresis (CE) systems), the number of STRs that can be simultaneously typed has grown to 21-26; this provides sufficient confidence in the conclusions of most kinship cases. However, more complex cases (e.g. testing distant relatives, potential mutations, deficient cases or incest cases) or when the target population shows an increased level of consanguinity, the genetic evidence may prove inconclusive. This necessitates testing additional STRs included in supplementary STR kits. Another option is by using Massively Parallel Sequencing (MPS) systems that allow simultaneous sequencing of additional DNA markers. A total 500 samples from the population of Saudi Arabia were collected. Two CE-based STR kits were used: Globalfiler™ PCR amplification kit (AB, USA) and SureID® 23 comp Human Identification kit (Health Gene Technologies, China) that together allowed 38 aSTRs to be analysed. In addition, as the SureID® 23 comp kit has not been validated either by an independent laboratory or by the manufacturer, the kit was validated following the minimum criteria of the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM). Moreover, the ForenSeq™ DNA Signature Prep Kit (Verogen) was used to sequence 87 samples and to generate sequence-based data for 122 autosomal markers included in the kit. The project allowed, in total, obtaining size-based data for 136 autosomal markers (42 aSTRs and 94 iiSNPs) and sequence-based data for 122 autosomal markers (28 aSTRs including SE33 and 94 iiSNPs). The data were evaluated for human identification and kinship testing in Saudi Arabia Although Globalfiler™ kit provided combined match probability (CMP) of 1.42E-26 that is much higher than the kit currently used in Saudi Arabia that has a CMP of 2.23E-18 (Identifiler plus kit), the availability of data for 42 aSTRs allowed other commercially available kits to be evaluated (based on the loci they contain). The study suggests adopting VeriFilerTM Plus (AB) or PowerPlex Fusion 6C system (Promega Corporation, USA) as a standard STR kit that would provide the lowest CMPs (9.26E-29 and 1.03E-29, respectively). Adopting any of the three kits would provide sufficient confidence in most parent-child cases (trio or duo). The validation of the SureID® 23 comp has shown that the kit met the criteria commonly used in forensic genetics laboratories. In addition, the kit can benefit from some developments that were identified by the validation, in particular the addition of extra alleles in the allelic ladder and also to increase the amount of input DNA that can be added to an amplification. The kit can be used if any kinship cases showed inconclusive results with GlobalFilerTM, VeriFilerTM Plus or PowerPlex Fusion 6C allowing 38-40 aSTRs to be analysed. The ForenSeq™ DNA Signature Prep Kit provided CMP of 1.97E-68 and 3.65E-77 for the size and sequencebased data respectively, where 1.24E-37 (size-based data) and 5.6E-41 (sequence-based data) were provided from the iiSNPs alone. The kit can be used when two or three mismatches were suspected to be mutations or when testing distant relationships. The study highlighted 220 syntenic pairs, 46 of which would have significant impact on LR estimation due to lower RFs (< 0.12). The case-specific impact of linkage should be included in the estimation of LRs by using the RFs values estimated in this project

Sequence‐Based Saudi Population Data for The SE33 Locus

A set of 87 reference samples collected from the population of Saudi Arabia were sequenced using the ForenSeqTM DNA Signature Prep Kit on a MiSeq FGxTM. The FASTQ files contain the sequences of the SE33 STR, but are not reported by the ForenSeqTM Universal Analysis Software (UAS). The STRait Razor software was used to recover and to report SE33 sequence‐based data for the Saudi population. Ninety-six sequence-based alleles were recovered, most of which had previously reported motif patterns. Two unreported motif patterns found in three alleles and seven novel allele sequences were reported. We also reported a single discordance between the sequence-based data and the CE data that was due to the presence of a common TTTT deletion. SE33 had 130% more sequence-based alleles; the highest number of observed sequence variants were in alleles 27.2 and 30.2, which each had 7 sequence variants. The statistical parameters emphasize the usefulness of using the sequence-based data

Sequence data of six unusual alleles at SE33 and D1S1656 STR Loci

When profiling a reference dataset of 500 DNA samples for the population of Saudi Arabia, using the GlobalFiler® PCR amplification kit, six unusual alleles were detected. At the SE33 locus, four novel alleles were found: 2, 14.3, 20.3, and 38; two alleles, at the D1S1656 locus: 7 and 8, had been previously reported, but no published sequence data was available. The D1S1656 alleles were sequenced using ForenSeq™ DNA Signature Prep with the MiSeq FGx System (Illumina, USA). As the SE33 is not reported by available Massively Parallel Sequencing (MPS) systems, samples that exhibited the unreported alleles were sequenced using BigDye™ Terminator v3.1 Cycle Sequencing Kit. Here we present the sequence and structure of the previously uncharacterized alleles

An evaluation of the SureID 23comp Human Identification Kit for kinship testing

Short tandem repeat (STR) profiling has been routinely used in kinship testing since the introduction of commercial kits in the mid-1990s. While 15 to 23 STR loci normally give definitive results in simple kinship testing, additional loci are sometimes required to resolve complex cases. The SureID 23comp Human Identification Kit, recently released by Health Gene Technologies (China), multiplexes amelogenin and 22 autosomal STRs, 17 of which are non-CODIS STRs. This enables the profiling of 38–40 loci when used in conjunction with widely used commercial kits. In this study, the kit was evaluated for kinship applications as a supplementary STR kit following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples. Performance was comparable with other commercial kits demonstrating: repeatability and reproducibility; precision (maximum s.d. 0.1048 nt); accuracy, all alleles were within ±0.41 nt compared to the actual sizes; heterozygous peak balances at all loci >68%; stutter ratios ranged from 3.8% to 16.15%; full profiles were generated with 125 pg DNA (95.12% of alleles at 62 pg),; and we found 100% concordance over 5 common STRs with the GlobalFiler kit

Population genetic data for 17 non-CODIS STR loci for the Saudi Arabian population using the SureID®23comp Human Identification Kit

Our previous work focused on validation the SureID 23comp Human Identification Kit (Health Gene Technologies, China), following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples from the population of Saudi Arabia. The kit genotypes 22 STRs, 17 of which are non-CODIS, and Amelogenin. The validation tests showed that it has the potential to increase the power of testing in complex cases. In this paper, the allele frequency data, common forensic parameters for the 17 non-CODIS STR loci are presented. We found the majority of loci had an excess of homozygosity in the data set, which is most likely explained by the relatively high levels of consanguinity in the population of Saudi Arabia

Population genetic data for 17 non-CODIS STR loci for the Saudi Arabian population using the SureID®23comp Human Identification Kit

Our previous work focused on validation the SureID 23comp Human Identification Kit (Health Gene Technologies, China), following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples from the population of Saudi Arabia. The kit genotypes 22 STRs, 17 of which are non-CODIS, and Amelogenin. The validation tests showed that it has the potential to increase the level of confidence in conclusions in complex cases especially in a country like Saudi Arabia where consanguineous marriage is more common. Previous studies on the population of Saudi Arabia, either by questionnaires or by genetic testing using 21 STRs included in the GlobalFiler kit, suggested an elevated rate of consanguinity in the population of Saudi Arabia. In this paper, the allele frequency data, common forensic parameters for the 17 non-CODIS STR loci are presented. We examined whether the loci would exhibit excess of homozygosity in the data set