Operationalisation of the ForenSeq® Kintelligence Kit for Australian unidentified and missing persons casework

Single nucleotide polymorphism (SNP) genotyping technologies can generate investigative leads for human remains identification, including estimation of biological sex, biogeographical ancestry (BGA), externally visible characteristics (EVCs), identity, uniparental lineage and extended kinship. The ForenSeq® Kintelligence Kit provides forensic laboratories with the ability to apply this suite of genetic tools to forensic samples using one panel targeting 10,230 SNPs (including 56 ancestry-informative, 24 phenotype-informative, 94 identity-informative, 106 X chromosome, 85 Y chromosome and 9867 kinship-informative SNPs) sequenced on the MiSeq FGx® Sequencing System. The ForenSeq® Kintelligence Kit has been internally validated, optimised and operationalised by the Australian Federal Police National DNA Program for Unidentified and Missing Persons (AFP Program) for coronial casework. The internal validation was conducted according to the Scientific Working Group on DNA Analysis Methods guidelines (excluding mixture analysis), focussing on sample types typically encountered in human remains identification casework, such as bones, teeth, nail, blood and hair. The workflow was optimised for a high throughput library preparation and sequencing workflow, and additional analytical thresholds were developed to improve genotyping accuracy for low DNA input samples. Additionally, the genetic intelligence generated from the kit was compared to the self-declared biological sex, EVCs and BGA of the DNA donors to assess concordance. The kit was able to produce high quality SNP profiles from 1.0 ng down to 0.1 ng of DNA, with high repeatability and reproducibility, and minimal background noise. The prediction accuracy for biological sex (95%), hair colour (58%), eye colour (74%) and BGA inferences (consistent: 74%; partially consistent: 10%; inconclusive: 16%) was determined based on self-declared data. Additionally, SNP profiles from a volunteer family group of ten related individuals were uploaded to GEDmatch PRO™ to assess kinship accuracy. The kit was capable of detecting (97%) and accurately classifying (90%) genetic relationships spanning from first to fifth degree. The Kintelligence Kit provides the AFP Program with a robust and reliable genetic intelligence tool for unidentified and missing persons investigations, which has been designed to sequence multiple challenging samples in a single multiplexed assay using existing laboratory instrumentation.