Biogeographical Ancestry Analyses Using the ForenSeqTM DNA Signature Prep Kit and Multiple Prediction Tools.

The inference of biogeographical ancestry (BGA) can assist in police investigations of serious crime cases and help to identify missing people and victims of mass disasters. In this study, we evaluated the typing performance of 56 ancestry-informative SNPs in 177 samples using the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx system. Furthermore, we compared the prediction accuracy of the tools Universal Analysis Software v1.2 (UAS), the FROG-kb, and GenoGeographer when inferring the ancestry of 503 Europeans, 22 non-Europeans, and 5 individuals with co-ancestry. The kit was highly sensitive with complete aiSNP profiles in samples with as low as 250pg input DNA. However, in line with others, we observed low read depth and occasional drop-out in some SNPs. Therefore, we suggest not using less than the recommended 1ng of input DNA. FROG-kb and GenoGeographer accurately predicted both Europeans (99.6% and 91.8% correct, respectively) and non-Europeans (95.4% and 90.9% correct, respectively). The UAS was highly accurate when predicting Europeans (96.0% correct) but performed poorer when predicting non-Europeans (40.9% correct). None of the tools were able to correctly predict individuals with co-ancestry. Our study demonstrates that the use of multiple prediction tools will increase the prediction accuracy of BGA inference in forensic casework.

Association between Variants in the OCA2-HERC2 Region and Blue Eye Colour in HERC2 rs12913832 AA and AG Individuals.

The OCA2-HERC2 region is strongly associated with human pigmentation, especially eye colour. The HERC2 SNP rs12913832 is currently the best-known predictor for blue and brown eye colour. However, in a previous study we found that 43 of 166 Norwegians with the brown eye colour genotype rs12913832:AA or AG, did not have the expected brown eye colour. In this study, we carried out massively parallel sequencing of a ~500 kbp HERC2-OCA2 region in 94 rs12913832:AA and AG Norwegians (43 blue-eyed and 51 brown-eyed) to search for novel blue eye colour variants. The new candidate variants were subsequently typed in a Norwegian biobank population (total n = 519) for population specific association analysis. We identified five new variants, rs74409036:A, rs78544415:T, rs72714116:T, rs191109490:C and rs551217952:C, to be the most promising candidates for explaining blue eye colour in individuals with the rs12913832:AA and AG genotype. Additionally, we confirmed the association of the missense variants rs74653330:T and rs121918166:T with blue eye colour, and observed lighter skin colour in rs74653330:T individuals. In total, 37 (86%) of the 43 blue-eyed rs12913832:AA and AG Norwegians could potentially be explained by these seven variants, and we suggest including them in future prediction models.

Predicting eye and hair colour in a Norwegian population using Verogen's ForenSeq™ DNA signature prep kit.

Prediction of eye and hair colour from DNA can be an important investigative tool in forensic cases if conventional DNA profiling fails to match DNA from any known suspects or cannot obtain a hit in a DNA database. The HIrisPlex model for simultaneous eye and hair colour predictions was developed for forensic usage. To genotype a DNA sample, massively parallel sequencing (MPS) has brought new possibilities to the analysis of forensic DNA samples. As part of an in-house validation, this study presents the genotyping and predictive performance of the HIrisPlex SNPs in a Norwegian study population, using Verogen's ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx system and the HIrisPlex webtool. DNA-profiles were successfully typed with DNA input down to 125 pg. In samples with DNA input < 125 pg, false homozygotes were observed with as many as 92 reads. Prediction accuracies in terms of AUC were high for red (0.97) and black (0.93) hair colours, as well as blue (0.85) and brown (0.94) eye colours. The AUCs for blond (0.72) and brown (0.70) hair colour were considerably lower. None of the individuals was predicted to have intermediate eye colour. Therefore, the error rates of the overall eye colour predictions were 37% with no predictive probability threshold (pmax) and 26% with a probability threshold of 0.7. We also observed that more than half of the incorrect predictions were for individuals carrying the rs12913832 GG genotype. For hair colour, 65% of the individuals were correctly predicted when using the highest probability category approach. The main error was observed for individuals with brown hair colour that were predicted to have blond hair. Utilising the prediction guide approach increased the correct predictions to 75%. Assessment of phenotype-genotype associations of eye colours using a quantitative eye colour score (PIE-score), revealed that rs12913832 AA individuals of Norwegian descent had statistically significantly higher PIE-score (less brown eye colour) than individuals of non-northern European descent. To our knowledge, this has not been reported in other studies. Our study suggests that careful assessment of the target population prior to the implementation of forensic DNA phenotyping to case work is beneficial.

Prediction of Eye Colour in Scandinavians Using the EyeColour 11 (EC11) SNP Set.

Description of a perpetrator's eye colour can be an important investigative lead in a forensic case with no apparent suspects. Herein, we present 11 SNPs (Eye Colour 11-EC11) that are important for eye colour prediction and eye colour prediction models for a two-category reporting system (blue and brown) and a three-category system (blue, intermediate, and brown). The EC11 SNPs were carefully selected from 44 pigmentary variants in seven genes previously found to be associated with eye colours in 757 Europeans (Danes, Swedes, and Italians). Mathematical models using three different reporting systems: a quantitative system (PIE-score), a two-category system (blue and brown), and a three-category system (blue, intermediate, brown) were used to rank the variants. SNPs with a sufficient mean variable importance (above 0.3%) were selected for EC11. Eye colour prediction models using the EC11 SNPs were developed using leave-one-out cross-validation (LOOCV) in an independent data set of 523 Norwegian individuals. Performance of the EC11 models for the two- and three-category system was compared with models based on the IrisPlex SNPs and the most important eye colour locus, rs12913832. We also compared model performances with the IrisPlex online tool (IrisPlex Web). The EC11 eye colour prediction models performed slightly better than the IrisPlex and rs12913832 models in all reporting systems and better than the IrisPlex Web in the three-category system. Three important points to consider prior to the implementation of eye colour prediction in a forensic genetic setting are discussed: (1) the reference population, (2) the SNP set, and (3) the reporting strategy.

Genetic relationships of European, Mediterranean, and SW Asian populations using a panel of 55 AISNPs.

The set of 55 ancestry informative SNPs (AISNPs) originally developed by the Kidd Lab has been studied on a large number of populations and continues to be applied to new population samples. The existing reference database of population samples allows the relationships of new population samples to be inferred on a global level. Analyses show that these autosomal markers constitute one of the better panels of AISNPs. Continuing to build this reference database enhances its value. Because more than half of the 25 ethnic groups recently studied with these AISNPs are from Southwest Asia and the Mediterranean region, we present here various analyses focused on populations from these regions along with selected reference populations from nearby regions where genotype data are available. Many of these ethnic groups have not been previously studied for forensic markers. Data on populations from other world regions have also been added to the database but are not included in these focused analyses. The new population samples added to ALFRED and FROG-kb increase the total to 164 population samples that have been studied for all 55 AISNPs.

Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation.

High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.

Functional analysis of polyomavirus BK non-coding control region quasispecies from kidney transplant recipients.

Replication of the human polyomavirus BK (BKV) in renal tubular epithelial cells causes viruria and BKV-nephropathy in kidney transplant recipients. Following prolonged high-level BKV replication, rearrangement of the archetype non-coding control region (NCCR) leads to a mixture of BKV variants. The aim of this study was to compare potential functional differences of 12 rearranged (rr)-NCCR variants with the archetype (ww)-NCCR (WWT) found in allograft biopsies or urine from three kidney transplant recipients including two with BKV-nephropathy. Twelve different rr-NCCRs and one archetype ww-NCCR were inserted between the early and late protein coding region of BKV(Dunlop) to make recombinant BKV genomes for transfection into Vero cells. Immunoblotting, immunofluorescence staining, and quantitative PCR demonstrated that viral protein expression and extracellular BKV loads of 10 rr-NCCR variants were similar or higher than observed for the ww-NCCR BKV. Two rr-NCCR variants (RH-2 and RH-19) were non-functional. The functional rr-NCCRs produced infectious progeny successfully infecting primary renal proximal tubular epithelial cells. The number of infected cells and extracellular BKV loads corresponded to the activity seen in Vero cells. Three rr-NCCR variants (RH-1, RH-10, RH-13) only gave rise to a few infected cells similar to ww-NCCR, whereas seven variants had intermediate activity (RH-5, RH-6, RH-8, RH-9, RH-11) or high replication activity (RH-7 and RH-18) with several hundred infected cells per well. The results indicate that both functional and non-functional BKV rr-NCCR variants arise during BKV replication in kidney transplant recipients and that most functional rr-NCCR variants confer a higher replication capacity than archetype ww-NCCR.

Genetic variability in BK Virus regulatory regions in urine and kidney biopsies from renal-transplant patients.

The human Polyomavirus BK (BKV) contains a hypervariable non-coding control region (NCCR), which regulates DNA replication and RNA transcription. The aim of this study was to characterize BKV NCCR-variants in kidney biopsies and urine samples from renal-transplant patients and to see whether there is any association between NCCR variability and BKV-nephropathy. Kidney biopsies and urine samples were examined from 11 patients with elevated serum creatinine and >5,000 genomic BKV copies per ml of urine. BKV-nephropathy was diagnosed in seven patients. Using PCR, BKV NCCR was amplified from urine from all BKV-nephropathy patients. The dominant NCCR corresponded to the archetype (WWT). In addition, a total of 14 non-archetype NCCR-variants were detected. Thirteen of these NCCR-variants were found in urine from one single BKV-nephropathy patient also suffering from hepatitis C. The NCCR of BKV was amplified from kidney biopsies of six BKV-nephropathy patients. Three patients demonstrated WWT NCCR, while three other patients harbored rearranged NCCR variants. The WWT NCCR was also detected in urine from control patients, except for one patient who harbored two non-archetypal NCCR variants. However, these variants were not resulting from complex rearrangements but instead had a linear NCCR anatomy with deletion(s) in the P-block. No BKV DNA was detected in biopsies from control patients. The results indicate that rearranged BKV NCCR is associated with BKV-nephropathy.