An overview of autosomal STRs and identity SNPs in a Norwegian population using massively parallel sequencing.

In recent years, probabilistic genotyping software has been adapted for the analysis of massively parallel sequencing (MPS) forensic data. Likelihood ratios (LR) are based on allele frequencies selected from populations of interest. This study provides an outline of sequence-based (SB) allele frequencies for autosomal short tandem repeats (aSTRs) and identity single nucleotide polymorphisms (iSNPs) in 371 individuals from Southern Norway. 27 aSTRs and 94 iSNPs were previously analysed with the ForenSeq™ DNA Signature Prep Kit (Verogen). The number of alleles with frequencies less than 0.05 for sequenced-based alleles was 4.6 times higher than for length-based alleles. Consistent with previous studies, it was observed that sequence-based data (both with and without flanks) exhibited higher allele diversity compared to length-based (LB) data; random match probabilities were lower for SB alleles confirming their advantage to discriminate between individuals. Two alleles in markers D22S1045 and Penta D were observed with SNPs in the 3´ flanking region, which have not been reported before. Also, a novel SNP with a minor allele frequency (MAF) of 0.001, was found in marker TH01. The impact of the sample size on minor allele frequency (MAF) values was studied in 88 iSNPs from Southern Norway (n = 371). The findings were then compared to a larger Norwegian population dataset (n = 15,769). The results showed that the smaller Southern Norway dataset provided similar results, and it was a representative sample. Population structure was analyzed for regions within Southern Norway; FST estimates for aSTR and iSNPs did not indicate any genetic structure. Finally, we investigated the genetic differences between Southern Norway and two other populations: Northern Norway and Denmark. Allele frequencies between these populations were compared, and we found no significant frequency differences (p-values > 0.0001). We also calculated the pairwise FST values per marker and comparisons between Southern and Northern Norway showed small differences. In contrast, the comparisons between Southern Norway and Denmark showed higher FST values for some markers, possibly driven by distinct alleles that were present in only one of the populations. In summary, we propose that allele frequencies from each population considered in this study could be used interchangeably to calculate genotype probabilities.

Forensic identity SNPs: Characterisation of flanking region variation using massively parallel sequencing.

Single nucleotide polymorphisms (SNPs) can be analysed for identity or kinship applications in forensic genetics to either provide an adjunct to traditional STR typing or as a stand-alone approach. The advent of massively parallel sequencing technology (MPS) has provided a useful opportunity to more easily deploy SNP typing in a forensic context, given the ability to simultaneously amplify a large number of markers. Furthermore, MPS also provides valuable sequence data for the targeted regions, which enables the detection of any additional variation seen in the flanking regions of amplicons. In this study we genotyped 977 samples across five UK-relevant population groups (White British, East Asian, South Asian, North-East African and West African) for 94 identity-informative SNP markers using the ForenSeq DNA Signature Prep Kit. Examination of flanking region variation allowed for the identification of 158 additional alleles across all populations studied. Here we present allele frequencies for all 94 identity-informative SNPs, both including and excluding the flanking region sequence of these markers. We also present information on the configuration of these SNPs in the ForenSeq DNA Signature Prep Kit, including performance metrics for the markers and investigation of bioinformatic and chemistry-based discordances. Overall, the inclusion of flanking region variation in the analysing workflow for these markers reduced the average combined match probability 2175 times across all populations, with a maximum reduction of 675,000-fold in the West African population. The gain due to flanking region-based discrimination increased the heterozygosity of some loci above that of some of the least useful forensic STR loci; thus demonstrating the benefit of enhanced analysis of currently targeted SNP markers for forensic applications.

An alternate workflow for preparing Precision ID Ancestry and Identity Panel libraries for Illumina sequencing.

Single nucleotide polymorphisms (SNPs) are well-established for forensic applications. Although they are not compatible with existing criminal databases, they offer some advantages over short tandem repeat (STR) markers including smaller amplicons, no stutter artifacts, and biogeographic ancestry and phenotype predictions. The Precision ID NGS System, a commercial workflow by Thermo Fisher Scientific, offers a streamlined solution for genotyping forensically relevant SNPs using next-generation sequencing. The Precision ID Ancestry and Identity Panels combined target 289 SNPs, and their sensitivity, reproducibility, and accuracy have been evaluated by the forensic community. The aim of this study was to develop an alternative workflow to genotype these SNP panels using Illumina chemistry. Commercial genomic DNAs (gDNAs) (n, 3) were amplified using three uracil-tolerant polymerase master mixes. Resulting amplicons were prepared into libraries using the KAPA Hyper Prep Kit (KAPA Biosystems) and sequenced via Illumina's MiniSeq. Reads were analyzed using a published analysis pipeline to compile final genotypes with read depth information. Phusion U Multiplex PCR Master Mix (Thermo Fisher Scientific) statistically outperformed the other master mixes tested (P <0.0001), with respect to the number of SNPs genotyped. To ensure a workflow using Phusion U would be compatible across diverse samples, we optimized PCR cycle number using the same commercial gDNAs (n, 3), reference buccal swabs (n, 3), and environmental (household dust) samples (n, 6). Using the developed workflow, 93.9% of all SNPs were successfully genotyped across sample types. Implementation of the developed workflow should be straightforward for forensic laboratories and suitable for processing reference and casework samples.

Sequenced-based French population data from 169 unrelated individuals with Verogen's ForenSeq DNA signature prep kit.

Massively Parallel Sequencing (MPS) applied to forensic genetics allows the simultaneous analysis of hundreds of genetic markers and the access to full amplicon sequences which help to increase available allele diversity. Meanwhile, sequence variation within the repeat regions represents the majority of the allele diversity, flanking regions adjacent to the repeat core provide an additional degree of variation. The forensic genetics community needs access to population data, from relevant parts of the world that contain this new sequence diversity in order to perform statistical calculations. In this study, we report sequence-based Short Tandem Repeat (STR) and identity Single Nucleotide Polymorphism (iSNPs) allele data for 169 French individuals across 58 STRs and 92 SNPs included in the Verogen ForenSeq DNA Signature Prep kit. 42 STRs out of 58 showed an increased number of alleles due to sequence variation in the repeat motif and/or the flanking regions. D9S1122 showed the largest overall gain with an increase of observed heterozygosities of almost 25 %. The combined match probability combining 27 autosomal STRs and 91 identity SNPs was 1.11E-69. Sequence-based allele frequencies included in this publication will help forensic laboratories to increase the power of discrimination for identification, kinship analysis and mixture interpretation.

Reverse Complement PCR: A novel one-step PCR system for typing highly degraded DNA for human identification.

Reverse Complement PCR (RC-PCR) is an innovative, one-step PCR target enrichment technology adapted for the amplification of highly degraded (fragmented) DNA. It provides simultaneous amplification and tagging of a targeted sequence construct in a single, closed-tube assay. A human identification (HID) RC-PCR panel was designed targeting 27 identity single nucleotide polymorphisms (SNPs) generating targets only 50 base pairs in length. In a single reaction, the complete sequencing construct is produced which is essential for massively parallel sequencing (MPS) library preparation, thus reducing time and labor as well as minimizing the risk of sample carry-over or other forms of contamination. The RC-PCR system was evaluated and found to produce reliable and concordant variant calls. Also, the RC-PCR system demonstrated to have substantial sensitivity of detection with a majority of alleles detected at 60 pg of input DNA and robustness in tolerating known PCR inhibitors. The RC-PCR system may be an effective alternative to current forensic genetic methods in the analysis of highly degraded DNA.