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.

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.

Variation in Genetic Mechanisms for Plumage Polymorphism in Skuas (Stercorarius).

Coloration is evolutionarily labile and so provides an excellent trait for examining the repeatability of evolution. Here, we investigate the repeatability of the evolution of polymorphic variation in ventral plumage coloration in skuas (Stercorarius: Stercorariidae). In 2 species, arctic (S. parasiticus) and pomarine skuas (S. pomarinus), plumage polymorphism was previously shown to be associated with coding changes at the melanocortin-1 receptor (MC1R) locus. Here, we show that polymorphism in a third species, the south polar skua (S. maccormicki), is not associated with coding variation at MC1R or with variation at a Z-linked second candidate locus, tyrosinase-related protein 1 (TYRP1). Hence, convergent evolution of plumage polymorphisms in skuas is only partly repeatable at the level of the genetic locus involved. Interestingly, the pattern of repeatability in skuas is aligned not with phylogeny but with the nature of the phenotypic variation. In particular, south polar skuas show a strong sex bias to coloration that is absent in the other species, and it may be that this has a unique genetic architecture.

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.

The genetic basis and enigmatic origin of melanic polymorphism in pomarine skuas (Stercorarius pomarinus).

A key outstanding issue in adaptive evolution is the relationship between the genetics of intraspecific polymorphism and interspecific evolution. Here, we show that the pale/dark ventral plumage polymorphism that occurs in both the pomarine skua (Stercorarius pomarinus) and Arctic skua (S. parasiticus) is the result of convergent evolution at the same locus (MC1R), involving some of the same amino acid sites. The dark melanic MC1R allele in the pomarine skua is strongly divergent from the pale MC1R alleles. Whereas the dark allele is closely related to MC1R alleles in three species of great skua (S. skua, S. maccormicki, S. lonnbergi), the pale pomarine skua MC1R alleles present a star-like pattern in an intermediate position on the haplotype network, closer to alleles of the long-tailed skua (S. longicaudus). Variation at other nuclear loci confirms a close relationship between the pomarine skua and the great skuas. The plumage polymorphism in pomarine skuas might have arisen in the common ancestor of pomarine and great skuas, only being retained in pomarine skuas. Alternatively, the pale and melanic MC1R alleles may have evolved independently in different lineages and been brought together in pomarine skuas by hybridization. In this case, introgression of a pale MC1R allele into the pomarine skua from another skua lineage is most likely. Our current data do not permit us to distinguish between these hypotheses, and assaying genome-wide variation holds much promise in this regard. Nevertheless, we have uncovered an intriguing example of a functionally important allele within one species that is shared across species.

Molecular population genetics of the melanic plumage polymorphism in Arctic skuas (Stercorarius parasiticus): evidence for divergent selection on plumage colour.

The Arctic skua (Stercorarius parasiticus) is a classic example of an avian plumage polymorphism, with variation in melanin-based ventral plumage coloration defining pale, intermediate and dark morphs in adults of both sexes. However, despite several decades of field research, there is an incomplete understanding of how the polymorphism in ventral plumage colour is maintained and the selective forces involved. Here, we investigate selection on a locus (MC1R) that is strongly associated with plumage colour variation in Arctic skuas using patterns of nucleotide variation and comparison to neutral loci (nuclear introns and mtDNA). We find that three linked nonsynonymous mutations in MC1R, including the single mutation described previously, are associated with plumage colour in the Arctic skua. The position of nonsynonymous mutations on a MC1R haplotype network implies that divergent selection drove the initial evolution of the colour morphs. Comparisons of F(ST)s of MC1R vs. nuclear introns among five skua populations differing in proportion of dark morphs along an approximate north-south cline reveal a signature of divergent selection on MC1R. In contrast, we find limited evidence for balancing selection on MC1R within populations, although the power is low. Our results provide strong evidence for both past and ongoing selection on MC1R, and, by implication, plumage colour in Arctic skuas. The results suggest that a fruitful avenue for future ecological studies will be analysis of selection on morphs in colonies at the extremes along the morph ratio cline.

Organochlorines in antarctic and arctic avian top predators: a comparison between the South Polar Skua and two species of northern hemisphere gulls.

Different organochlorine compounds (OCs) were measured in the blood of breeding south polar skuas (Catharacta maccormicki) at Svarthamaren, Dronning Maud Land (Antarctica) and compared to those in two species of northern hemisphere gulls: the Arctic glaucous gull (Larus hyperboreus) and the subarctic great black-backed gull (Larus marinus). The skuas had 8% and 29% of the SigmaOC levels (45 ng/g, wet weight) of glaucous gulls (591 ng/g) and great black-backed gulls (158 ng/g), respectively. Polychlorinated biphenyls (PCBs) and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) were very low in skuas compared to northern gulls, but the mean hexachlorobenzene (HCB) level was 1.7 times higher than in great black-backed gulls and one-third of the glaucous gull level. Mirex levels in skuas were among the highest reported in birds, the mean level being 3 and 26 times higher than those in glaucous gull and great black-backed gulls, respectively. In skuas, the mean levels of HCB, oxychlordane, p,p'-DDE, and PCBs increased by about 30% during a 2-week period, and mirex increased by nearly 60%. In glacuous gulls, HCB, p,p'-DDE, and PCBs increased by 10-20%. For HCB, mirex, and oxychlordane, only a relatively small proportion of the increase in skuas could be explained by changes in lipid pools and the levels at first sampling, compared to glaucous gulls. Thus, skuas were probably accumulating these compounds when present in Antarctica. p,p'-DDE and PCB levels, in contrast, seemed much more stable in the skuas. Relatively high levels of mirex and HCB in south polar skuas are concerning with regard to potential adverse effects.

Conserved genetic basis of a quantitative plumage trait involved in mate choice.

A key question in evolutionary genetics is whether shared genetic mechanisms underlie the independent evolution of similar phenotypes across phylogenetically divergent lineages. Here we show that in two classic examples of melanic plumage polymorphisms in birds, lesser snow geese (Anser c. caerulescens) and arctic skuas (Stercorarius parasiticus), melanism is perfectly associated with variation in the melanocortin-1 receptor (MC1R) gene. In both species, the degree of melanism correlates with the number of copies of variant MC1R alleles. Phylogenetic reconstructions of variant MC1R alleles in geese and skuas show that melanism is a derived trait that evolved in the Pleistocene.