Developmental validation of the ForenSeq® Kintelligence kit, MiSeq FGx® sequencing system and ForenSeq Universal Analysis Software.

Forensic Investigative Genetic Genealogy, a recent sub discipline of forensic genomics, leverages the high throughput and sensitivity of detection of next generation sequencing and established genetic and genealogical approaches to support the identification of human remains from missing persons investigations and investigative lead generation in violent crimes. To facilitate forensic DNA evidence analysis, the ForenSeq® Kintelligence multiplex, consisting of 10,230 SNPs, was developed. Design of the ForenSeq Kintelligence Kit, the MiSeq FGx® Sequencing System and the ForenSeq Universal Analysis Software is described. Developmental validation in accordance with SWGDAM guidelines and forensic quality assurance standards, using single source samples, is reported for the end-to-end workflow from library preparation to data interpretation. Performance metrics support the conclusion that more genetic information can be obtained from challenging samples compared to other commercially available forensic targeted DNA assays developed for capillary electrophoresis (CE) or other current next generation sequencing (NGS) kits due to the higher number of markers, the overall shorter amplicon sizes (97.8% <150 bp), and kit design. Data indicate that the multiplex is robust and fit for purpose for a wide range of quantity and quality samples. The ForenSeq Kintelligence Kit and the Universal Analysis Software allow transfer of the genetic component of forensic investigative genetic genealogy to the operational forensic laboratory.

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.

Classification of STR allelic variation using massively parallel sequencing and assessment of flanking region power.

The application of massively parallel sequencing (MPS) to forensic genetics has led to improvements in multiple aspects of DNA analysis, however, additional complexities are concurrently associated with these advances. In relation to short tandem repeat (STR) typing, the move to sequence rather than length-based methodologies has highlighted the extent to which previous allelic variation was masked - both within and outside of the repeat regions (the flanking regions). In order to fully implement MPS for autosomal STR analysis, sequence-based allelic frequencies must be available, and concordance with previous typing techniques needs to be assessed. In this work, a series of samples (n = 1007) from five different population groups were genotyped using the MiSeq FGx™ Forensic Genomics System. Results were compared to those obtained using capillary electrophoresis (CE), and sequence variation has been characterised both within and outside STR repeat regions, with allelic frequencies provided for all variants observed within this database. Analysing and characterising flanking region sequence is currently less straightforward than studying repeat region variation alone, and the added value of doing so remains largely unexplored - this paper provides data to show that the gain in polymorphism achieved when analysing flanking regions is less than might be expected. In the White British population for example, including the sequence variation within repeat regions of 26 autosomal STRs made the average combined random match probability (RMP) over 700 times lower than with length-based alleles alone. Including the sequence variation within the flanking regions only resulted in a combined RMP that was a further 4 times lower.

Global patterns of STR sequence variation: Sequencing the CEPH human genome diversity panel for 58 forensic STRs using the Illumina ForenSeq DNA Signature Prep Kit.

The 944 individuals of the CEPH human genome diversity panel (HGDP-CEPH), a standard sample set of 51 globally distributed populations, were sequenced using the Illumina ForenSeq™ DNA Signature Prep Kit. The ForenSeq™ system is a single multiplex for the MiSeq/FGx™ massively parallel sequencing instrument, comprising: amelogenin, 27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 SNPforID+Kiddlab autosomal ID-SNPs (plus optionally detected ancestry and phenotyping SNP sets). We report in detail the patterns of sequence variation observed in the repeat regions of the 58 forensic STR loci typed by the ForenSeq™ system. Sequence alleles were characterized and repeat region structures annotated by aligning the ForenSeq™ sequence output to the latest GRCh38 human reference sequence, necessitating the reversal and re-alignment of STR allele sequences reported by the Forenseq™ system in 20 of 58 STRs (plus the reverse alleles in two Y-STRs with duplicated-inverted repeat regions). Individual population sample sizes of the HGDP-CEPH panel do not allow reliable inferences to be made about levels of genetic variability in low frequency STR alleles-where particular sequence variants are found in only a few individuals; but we assessed the occurrence of both population-specific sequence variants and singleton observations; finding each of these in a sizeable proportion of HGDP-CEPH samples, with consequences for planning the co-ordinated compilation of sequence variation on a much larger scale than was required before by forensic laboratories now adopting massively parallel sequencing.

Concordance of the ForenSeq™ system and characterisation of sequence-specific autosomal STR alleles across two major population groups.

By using sequencing technology to genotype loci of forensic interest it is possible to simultaneously target autosomal, X and Y STRs as well as identity, ancestry and phenotypic informative SNPs, resulting in a breadth of data obtained from a single run that is considerable when compared to that generated with standard technologies. It is important however that this information aligns with the genotype data currently obtained using commercially available kits for CE-based investigations such that results are compatible with existing databases and hence can be of use to the forensic community. In this work, 400 samples were typed using commercially available STR kits and CE, as well as using the Ilumina ForenSeq™ DNA Signature Prep Kit and MiSeq® FGx to assess concordance of autosomal STRs and population variability. Results show a concordance rate between the two technologies exceeding 99.98% while numerous novel sequence based alleles are described. In order to make use of the sequence variation observed, sequence specific allele frequencies were generated for White British and British Chinese populations.

STRSeq: A catalog of sequence diversity at human identification Short Tandem Repeat loci.

The STR Sequencing Project (STRSeq) was initiated to facilitate the description of sequence-based alleles at the Short Tandem Repeat (STR) loci targeted in human identification assays. This international collaborative effort, which has been endorsed by the ISFG DNA Commission, provides a framework for communication among laboratories. The initial data used to populate the project are the aggregate alleles observed in targeted sequencing studies across four laboratories: National Institute of Standards and Technology (N=1786), Kings College London (N=1043), University of North Texas Health Sciences Center (N=839), and University of Santiago de Compostela (N=944), for a total of 4612 individuals. STRSeq data are maintained as GenBank records at the U.S. National Center for Biotechnology Information (NCBI), which participates in a daily data exchange with the DNA DataBank of Japan (DDBJ) and the European Nucleotide Archive (ENA). Each GenBank record contains the observed sequence of a STR region, annotation ("bracketing") of the repeat region and flanking region polymorphisms, information regarding the sequencing assay and data quality, and backward compatible length-based allele designation. STRSeq GenBank records are organized within a BioProject at NCBI (https://www.ncbi.nlm.nih.gov/bioproject/380127), which is sub-divided into: commonly used autosomal STRs, alternate autosomal STRs, Y-chromosomal STRs, and X-chromosomal STRs. Each of these categories is further divided into locus-specific BioProjects. The BioProject hierarchy facilitates access to the GenBank records by browsing, BLAST searching, or ftp download. Future plans include user interface tools at strseq.nist.gov, a pathway for submission of additional allele records by laboratories performing population sample sequencing and interaction with the STRidER web portal for quality control (http://strider.online).

Determining the authenticity of athlete urine in doping control by DNA analysis.

The integrity of urine samples collected from athletes for doping control is essential. The authenticity of samples may be contested, leading to the need for a robust sample identification method. DNA typing using short tandem repeats (STR) can be used for identification purposes, but its application to cellular DNA in urine has so far been limited. Here, a reliable and accurate method is reported for the successful identification of urine samples, using reduced final extraction volumes and the STR multiplex kit, Promega® PowerPlex ESI 17, with capillary electrophoretic characterisation of the alleles. Full DNA profiles were obtained for all samples (n = 20) stored for less than 2 days at 4 °C. The effect of different storage conditions on yield of cellular DNA and probability of obtaining a full profile were also investigated. Storage for 21 days at 4 °C resulted in allelic drop-out in some samples, but the random match probabilities obtained demonstrate the high power of discrimination achieved through targeting a large number of STRs. The best solution for long-term storage was centrifugation and removal of supernatant prior to freezing at -20 °C. The method is robust enough for incorporation into current anti-doping protocols, and was successfully applied to 44 athlete samples for anti-doping testing with 100% concordant typing.

Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR-type transcriptional regulators.

Activation of LysR-type transcription factors (LTTRs) is thought to result from conformational changes that occur when inducer molecules bind to their Inducer Binding Domains (IBDs). However, the exact nature of these changes remains to be fully elucidated. We present the crystal structures of two truncated constructs of the LTTR DntR in their apo- forms and in complex with its natural inducer molecule, salicylate. These provide a fuller picture of the conformational changes that can occur in LTTR IBDs and offer insights that may be relevant when considering the mechanism of activation of LTTRs. Two of the crystal structures show that DntR IBDs can bind up to two inducer molecules. The full extent of conformational changes observed is achieved only when inducer molecules are bound in both binding sites identified. Point mutations disrupting the putative secondary binding site produce DntR variants with a reduced response to salicylate in a whole cell system, suggesting that this site is functionally relevant.