Large-scale pedigree analysis highlights rapidly mutating Y-chromosomal short tandem repeats for differentiating patrilineal relatives and predicting their degrees of consanguinity.

Rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) were suggested for differentiating patrilineally related men as relevant in forensic genetics, anthropological genetics, and genetic genealogy. Empirical data are available for closely related males, while differentiation rates for more distant relatives are scarce. Available RM Y-STR mutation rate estimates are typically based on father-son pair data, while pedigree-based studies for efficient analysis requiring less samples are rare. Here, we present a large-scale pedigree analysis in 9379 pairs of men separated by 1-34 meioses on 30 Y-STRs with increased mutation rates including all known RM Y-STRs (RMplex). For comparison, part of the samples were genotyped at 25 standard Y-STRs mostly with moderate mutation rates (Yfiler Plus). For 43 of the 49 Y-STRs analyzed, pedigree-based mutation rates were similar to previous father-son based estimates, while for six markers significant differences were observed. Male relative differentiation rates from the 30 RMplex Y-STRs were 43%, 84%, 96%, 99%, and 100% for relatives separated by one, four, six, nine, and twelve meioses, respectively, which largely exceeded rates obtained by 25 standard Y-STRs. Machine learning based models for predicting the degree of patrilineal consanguinity yielded accurate and reasonably precise predictions when using RM Y-STRs. Fully matching haplotypes resulted in a 95% confidence interval of 1-6 meioses with RMplex compared to 1-25 with Yfiler Plus. Our comprehensive pedigree study demonstrates the value of RM Y-STRs for differentiating male relatives of various types, in many cases achieving individual identification, thereby overcoming the largest limitation of forensic Y-chromosome analysis.

Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers.

Short tandem repeat polymorphisms on the male-specific part of the human Y-chromosome (Y-STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (~10-3 mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y-STR markers with untypically high mutation rates (>10-2 ), termed rapidly mutating (RM) Y-STRs, and showed that they improved male relative differentiation over standard Y-STRs. By applying a newly developed in silico search approach to the Y-chromosome reference sequence, we identified 27 novel RM Y-STR candidates. Genotyping them in 1,616 DNA-confirmed father-son pairs for mutation rate estimation empirically highlighted 12 novel RM Y-STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y-STRs, it was 44%, 69%, and 83%. Of the 647 Y-STR mutations observed in total, almost all were single repeat changes, repeat gains, and losses were well balanced; allele length and fathers' age were positively correlated with mutation rate. We expect these new RM Y-STRs, together with the previously known ones, to significantly improving male relative differentiation in future human genetic applications.

Yleaf: Software for Human Y-Chromosomal Haplogroup Inference from Next-Generation Sequencing Data.

Next-generation sequencing (NGS) technologies offer immense possibilities given the large genomic data they simultaneously deliver. The human Y-chromosome serves as good example how NGS benefits various applications in evolution, anthropology, genealogy, and forensics. Prior to NGS, the Y-chromosome phylogenetic tree consisted of a few hundred branches, based on NGS data, it now contains many thousands. The complexity of both, Y tree and NGS data provide challenges for haplogroup assignment. For effective analysis and interpretation of Y-chromosome NGS data, we present Yleaf, a publically available, automated, user-friendly software for high-resolution Y-chromosome haplogroup inference independently of library and sequencing methods.

Population data of 17 Y-STRs (Yfiler) from Punjabis and Kashmiris of Pakistan.

Pakistan harbors 16 major ethnic groups including Punjabis (56% of total population) and Kashmiri (6% of total population). Here, we report data of 17 Y-chromosomal short tandem repeats (Y-STRs) genotyped with the AmpFlSTR Y-filer™ PCR Amplification kit in 94 Punjabis and 101 Kashmiris. The estimated haplotype diversity was higher in Punjabis (0.996) than that in Kashmiris (0.983). Furthermore, we performed population genetic analyses by including data from six other Pakistani groups. The presented haplotype data were recently included in the Y-Chromosome Haplotype Reference Database (YHRD) for future forensic and other usage.

Simultaneous analysis of hundreds of Y-chromosomal SNPs for high-resolution paternal lineage classification using targeted semiconductor sequencing.

SNPs from the non-recombining part of the human Y chromosome (Y-SNPs) are informative to classify paternal lineages in forensic, genealogical, anthropological, and evolutionary studies. Although thousands of Y-SNPs were identified thus far, previous Y-SNP multiplex tools target only dozens of markers simultaneously, thereby restricting the provided Y-haplogroup resolution and limiting their applications. Here, we overcome this shortcoming by introducing a high-resolution multiplex tool for parallel genotyping-by-sequencing of 530 Y-SNPs using the Ion Torrent PGM platform, which allows classification of 432 worldwide Y haplogroups. Contrary to previous Y-SNP multiplex tools, our approach covers branches of the entire Y tree, thereby maximizing the paternal lineage classification obtainable. We used a default DNA input amount of 10 ng per reaction but preliminary sensitivity testing revealed positive results from as little as 100 pg input DNA. Furthermore, we demonstrate that sample pooling using barcodes is feasible, allowing increased throughput for lower per-sample costs. In addition to the wetlab protocol, we provide a software tool for automated data quality control and haplogroup classification. The unique combination of ultra-high marker density and high sensitivity achievable from low amounts of potentially degraded DNA makes this new multiplex tool suitable for a wide range of Y-chromosome applications.

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Common DNA variants predict tall stature in Europeans.

Genomic prediction of the extreme forms of adult body height or stature is of practical relevance in several areas such as pediatric endocrinology and forensic investigations. Here, we examine 770 extremely tall cases and 9,591 normal height controls in a population-based Dutch European sample to evaluate the capability of known height-associated DNA variants in predicting tall stature. Among the 180 normal height-associated single nucleotide polymorphisms (SNPs) previously reported by the Genetic Investigation of ANthropocentric Traits (GIANT) genome-wide association study on normal stature, in our data 166 (92.2 %) showed directionally consistent effects and 75 (41.7 %) showed nominally significant association with tall stature, indicating that the 180 GIANT SNPs are informative for tall stature in our Dutch sample. A prediction analysis based on the weighted allele sums method demonstrated a substantially improved potential for predicting tall stature (AUC = 0.75; 95 % CI 0.72-0.79) compared to a previous attempt using 54 height-associated SNPs (AUC = 0.65). The achieved accuracy is approaching practical relevance such as in pediatrics and forensics. Furthermore, a reanalysis of all SNPs at the 180 GIANT loci in our data identified novel secondary association signals for extreme tall stature at TGFB2 (P = 1.8 × 10(-13)) and PCSK5 (P = 7.8 × 10(-11)) suggesting the existence of allelic heterogeneity and underlining the importance of fine analysis of already discovered loci. Extrapolating from our results suggests that the genomic prediction of at least the extreme forms of common complex traits in humans including common diseases are likely to be informative if large numbers of trait-associated common DNA variants are available.

Male Pedigree Toolbox: A Versatile Software for Y-STR Data Analyses.

Y-chromosomal short tandem repeats (Y-STRs) are widely used in forensic, genealogical, and population genetics. With the recent increase in the number of rapidly mutating (RM) Y-STRs, an unprecedented level of male differentiation can be achieved, widening and improving the applications of Y-STRs in various fields, including forensics. The growing complexity of Y-STR data increases the need for automated data analyses, but dedicated software tools are scarce. To address this, we present the Male Pedigree Toolbox (MPT), a software tool for the automated analysis of Y-STR data in the context of patrilineal genealogical relationships. The MPT can estimate mutation rates and male relative differentiation rates from input Y-STR pedigree data. It can aid in determining ancestral haplotypes within a pedigree and visualize the genetic variation within pedigrees in all branches of family trees. Additionally, it can provide probabilistic classifications using machine learning, helping to establish or prove the structure of the pedigree and the level of relatedness between males, even for closely related individuals with highly similar haplotypes. The tool is flexible and easy to use and can be adjusted to any set of Y-STR markers by modifying the intuitive input file formats. We introduce the MPT software tool v1.0 and make it publicly available with the goal of encouraging and supporting forensic, genealogical, and other geneticists in utilizing the full potential of Y-STRs for both research purposes and practical applications, including criminal casework.

Multiplex genotyping assays for fine-resolution subtyping of the major human Y-chromosome haplogroups E, G, I, J, and R in anthropological, genealogical, and forensic investigations.

Inherited DNA polymorphisms located within the nonrecombing portion of the human Y chromosome provide a powerful means of tracking the patrilineal ancestry of male individuals. Recently, we introduced an efficient genotyping method for the detection of the basal Y-chromosome haplogroups A to T, as well as an additional method for the dissection of haplogroup O into its sublineages. To further extend the use of the Y chromosome as an evolutionary marker, we here introduce a set of genotyping assays for fine-resolution subtyping of haplogroups E, G, I, J, and R, which make up the bulk of Western Eurasian and African Y chromosomes. The marker selection includes a total of 107 carefully selected bi-allelic polymorphisms that were divided into eight hierarchically organized multiplex assays (two for haplogroup E, one for I, one for J, one for G, and three for R) based on the single-base primer extension (SNaPshot) technology. Not only does our method allow for enhanced Y-chromosome lineage discrimination, the more restricted geographic distribution of the subhaplogroups covered also enables more fine-scaled estimations of patrilineal bio-geographic origin. Supplementing our previous method for basal Y-haplogroup detection, the currently introduced assays are thus expected to be of major relevance for future DNA studies targeting male-specific ancestry for forensic, anthropological, and genealogical purposes.

SMapper: visualizing spatial prevalence data of all types, including sparse and incomplete datasets.

Motivation: We introduce SMapper, a novel web and software tool for visualizing spatial prevalence data of all types including those suffering from incomplete geographic coverage and insufficient sample sizes. We demonstrate the benefits of our tool in overcoming interpretational issues with existing tools caused by such data limitations. We exemplify the use of SMapper by applications to human genotype and phenotype data relevant in an epidemiological, anthropological and forensic context. Availability and implementation: A web implementation is available at https://rhodos.ccg.uni-koeln.de/smapper/. A stand-alone version, released under the GNU General Public License version 3 as published by the Free Software Foundation, is available from https://rhodos.ccg.uni-koeln.de/smapper/software-download.php as a Singularity container (https://docs.sylabs.io/guides/latest/user-guide/index.html) and a native Linux Python installation.

Improving the differentiation of closely related males by RMplex analysis of 30 Y-STRs with high mutation rates.

The discovery of rapidly mutating (RM) Y-STRs started to move the field of forensic Y-STR analysis from male lineage identification towards male individual identification. Previously, the forensic value of RM Y-STRs for differentiating male relatives was limited due to the modest number of 13 identified RM Y-STRs. Recently, new RM Y-STRs were discovered, with strong expectations for significantly improving male relative differentiation; however, empirical evidence is missing yet. More recently, the genotyping method RMplex for efficiently analyzing 30 Y-STRs with high mutation rates, including all 26 currently known RM Y-STRs, was introduced. Here, we applied RMplex as well as the current state-of-the-art commercial Y-STR kit: Yfiler™ Plus PCR Amplification kit, to several hundreds of DNA-confirmed father-son pairs. Newly established estimates confirmed the high mutation rates of novel and previous RM Y-STRs. By combining current with previous data, we provide updated consensus estimates of mutation rates for all 49 Y-STRs targeted with both methods. Based on RMplex, 42% of 499 father-son pairs were differentiated, while 14% of 530 pairs based on Yfiler™ Plus, and 48% of 499 pairs based on both methods combined. Regarding brothers, RMplex also clearly outperformed Yfiler™ Plus, with differentiation rates of 62% and 33%, respectively. By combining both methods 72.9% of the brothers showed at least one mutation. For unrelated males, both methods achieved a discrimination capacity of 99.8% and a haplotype diversity of 0.999991, since all males had different haplotypes, except for two, perhaps indicating a hidden paternal relationship. Overall, this study underlines the value of RM Y-STRs in general and RMplex in particular for differentiating male relatives highly relevant in forensic genetics. It provides the first empirical evidence on the high value of RMplex for differentiating close male relatives, which for father-son pairs was almost 60% higher than with the initial set of 13 RM Y-STRs and three times higher than with Yfiler™ Plus. Based on our results from closely related males, we expect RMplex to also improve the differentiation of more distantly related males significantly, which needs empirical demonstration in future studies. We encourage the forensic community to apply RMplex in all forensic cases where a match with a commercial Y-STR kit was obtained between the male suspect and the evidence material, or to solely use RMplex in such cases, aiming to find out if the male suspect or any of his male paternal relatives left the evidence material at the crime scene.

RMplex reveals population differences in RM Y-STR mutation rates and provides improved father-son differentiation in Japanese.

Rapidly mutating Y chromosomal short tandem repeat markers (RM Y-STRs) -characterized by at least one mutation per 100 generations- are suitable for differentiating both related and unrelated males. The recently introduced multiplex method RMplex allows for the efficient analysis of 30 Y-STRs with increased mutation rates, including all 26 currently known RM Y-STRs. While currently available RM Y-STR mutation rates were established mostly from European individuals, here we applied RMplex to DNA samples of 178 genetically confirmed father-son pairs from East Asia. For several Y-STRs, we found significantly higher mutation rates in Japanese compared to previous estimates. The consequent father-son differentiation rate based on RMplex was significantly higher (52%) in Japanese than previously reported for Europeans (42%), and much higher than with Yfiler Plus in both sample sets (14% and 13%, respectively). Further analysis suggests that the higher mutation and relative differentiation rates in Japanese can in part be explained by on average longer Y-STR alleles relative to Europeans. Moreover, we show that the most striking difference, which was found in DYS712, could be linked to a Y-SNP haplogroup (O1b2-P49) that is common in Japanese and rare in other populations. We encourage the forensic Y-STR community to generate more RMplex data from more population samples of sufficiently large sample size in combination with Y-SNP data to further investigate population effects on mutation and relative differentiation rates. Until more RMplex data from more populations become available, caution shall be placed when applying RM Y-STR mutation rate estimates established in one population, such as Europeans, to forensic casework involving male suspects of paternal origin from other populations, such as non-Europeans.

An efficient multiplex genotyping approach for detecting the major worldwide human Y-chromosome haplogroups.

The Y chromosome is paternally inherited and therefore serves as an evolutionary marker of patrilineal descent. Worldwide DNA variation within the non-recombining portion of the Y chromosome can be represented as a monophyletic phylogenetic tree in which the branches (haplogroups) are defined by at least one SNP. Previous human population genetics research has produced a wealth of knowledge about the worldwide distribution of Y-SNP haplogroups. Here, we apply previous and very recent knowledge on the Y-SNP phylogeny and Y-haplogroup distribution by introducing two multiplex genotyping assays that allow for the hierarchical detection of 28 Y-SNPs defining the major worldwide Y haplogroups. PCR amplicons were kept small to make the method sensitive and thereby applicable to DNA of limited amount and/or quality such as in forensic settings. These Y-SNP assays thus form a valuable tool for researchers in the fields of forensic genetics and genetic anthropology to infer a man's patrilineal bio-geographic ancestry from DNA.

Investigative DNA analysis of two-person mixed crime scene trace in a murder case.

It has been advocated before that appearance prediction of unknown suspects from crime scene DNA, in the context of Forensic DNA Phenotyping (FDP), is mostly suitable for single source DNA samples, whereas FDP from DNA mixtures to which more than one person contributed, is viewed challenging. With this report on a murder case, we practically demonstrate the feasibility of appearance DNA prediction of an unknown suspect from a mixed crime scene trace, to which the unknown suspect and the known victim had contributed. From this two-person DNA mixture, we successfully predicted eye, hair and skin color of the unknown suspect with the HIrisPlex-S system by applying targeted massively parallel sequencing (MPS). We argue that at least three factors benefit appearance DNA prediction of unknown suspects from mixed crime scene traces, which were met in this murder case: i) SNP genotype knowledge from reference DNA analysis for one of the two persons in the mixture (here the known victim), ii) about equal DNA contributions by both donors to the mixed crime scene stain, and iii) the use of MPS allowing quantitative SNP analysis. Moreover, we show that additionally analyzing animal DNA in this mixed crime scene trace provides further investigative information. We envision that the investigative DNA strategy that we applied here for analyzing a two-person mixed crime scene trace in a murder case, will be applied in the future to more criminal cases with two-person DNA mixtures, for instance sexual assault cases.

RMplex: An efficient method for analyzing 30 Y-STRs with high mutation rates.

Y-chromosomal short tandem repeats (Y-STRs) with high mutation rates are recognized as valuable genetic markers for differentiating paternally related men, who typically cannot be separated with standard Y-STRs, and were shown to provide paternal lineage differentiation on a higher resolution level than standard Y-STRs. Both features make Y-STRs with high mutation rates relevant in criminal casework, particularly in sexual assault cases involving highly unbalanced male-female DNA mixtures that often fail autosomal forensic STR profiling for the male donor. Previously, the number of known Y-STRs with mutation rates higher than 10-2 per locus per generation termed rapidly mutating Y-STRs (RM Y-STRs) was limited to 13, which has recently been overcome by the discovery and characterization of 12 additional RM Y-STRs. Here, we present the development and validation of RMplex, an efficient genotyping system for analyzing 30 Y-STRs with high mutation rates, including all currently known RM Y-STRs, using multiplex PCR with capillary electrophoresis (CE) or massively parallel sequencing (MPS), overall targeting a total of 44 male-specific loci. If previously unavailable, repeat number assignations were provided based on newly generated MPS data. Validation tests based on the CE method demonstrated that the results were both repeatable and reproducible, full profiles were achieved with minimal input DNA of 250 pg for RMplex 1 and 100 pg for RMplex 2, and in the presence of inhibitors, or with a surplus of female DNA, the assays performed reasonably well. Application of RMplex to differentiate between paternally related men was exemplified in 32 males belonging to five different paternal pedigrees. Given further successful forensic validation testing, we envision the future application of RMplex in criminal cases where it is suspected, or cannot be excluded, that the crime scene trace originated from a male relatives of the suspect who is highlighted with standard Y-STR matching. Other applications of RMplex are in criminal cases without known suspects to differentiate between male relatives highlighted in familial searching based on standard Y-STR matching.

Forensic Y-SNP analysis beyond SNaPshot: High-resolution Y-chromosomal haplogrouping from low quality and quantity DNA using Ion AmpliSeq and targeted massively parallel sequencing.

Y-chromosomal haplogroups assigned from male-specific Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal bio-geographic ancestry inference, both being relevant in forensic genetics. However, most previously developed forensic Y-SNP tools did not provide Y haplogroup resolution on the high level needed in forensic applications, because the limited multiplex capacity of the DNA technologies used only allowed the inclusion of a relatively small number of Y-SNPs. In a proof-of-principle study, we recently demonstrated that high-resolution Y haplogrouping is feasible via two AmpliSeq PCR analyses and simultaneous massively parallel sequencing (MPS) of 530 Y-SNPs allowing the inference of 432 Y-haplogroups. With the current study, we present a largely improved Y-SNP MPS lab tool that we specifically designed for the analysis of low quality and quantity DNA often confronted with in forensic DNA analysis. Improvements include i) Y-SNP marker selection based on the "minimal reference phylogeny for the human Y chromosome" (PhyloTree Y), ii) strong increase of the number of targeted Y-SNPs allowing many more Y haplogroups to be inferred, iii) focus on short amplicon length enabling successful analysis of degraded DNA, and iv) combination of all amplicons in a single AmpliSeq PCR and simultaneous sequencing allowing single DNA aliquot use. This new MPS tool simultaneously analyses 859 Y-SNPs and allows inferring 640 Y haplogroups. Preliminary forensic developmental validation testing revealed that this tool performs highly accurate, is sensitive and robust. We also provide a revised software tool for analysing the sequencing data produced by the new MPS lab tool including final Y haplogroup assignment. We envision the tools introduced here for high-resolution Y-chromosomal haplogrouping to determine a man's paternal lineage and/or paternal bio-geographic ancestry to become widely used in forensic Y-chromosome DNA analysis and other applications were Y haplogroup information from low quality / quantity DNA samples is required.

Novel taxonomy-independent deep learning microbiome approach allows for accurate classification of different forensically relevant human epithelial materials.

Correct identification of different human epithelial materials such as from skin, saliva and vaginal origin is relevant in forensic casework as it provides crucial information for crime reconstruction. However, the overlap in human cell type composition between these three epithelial materials provides challenges for their differentiation and identification when using previously proposed human cell biomarkers, while their microbiota composition largely differs. By using validated 16S rRNA gene massively parallel sequencing data from the Human Microbiome Project of 1636 skin, oral and vaginal samples, 50 taxonomy-independent deep learning networks were trained to classify these three tissues. Validation testing was performed in de-novo generated high-throughput 16S rRNA gene sequencing data using the Ion Torrent™ Personal Genome Machine from 110 test samples: 56 hand skin, 31 saliva and 23 vaginal secretion specimens. Body-site classification accuracy of these test samples was very high as indicated by AUC values of 0.99 for skin, 0.99 for oral, and 1 for vaginal secretion. Misclassifications were limited to 3 (5%) skin samples. Additional forensic validation testing was performed in mock casework samples by de-novo high-throughput sequencing of 19 freshly-prepared samples and 22 samples aged for 1 up to 7.6 years. All of the 19 fresh and 20 (91%) of the 22 aged mock casework samples were correctly tissue-type classified. Moreover, comparing the microbiome results with outcomes from previous human mRNA-based tissue identification testing in the same 16 aged mock casework samples reveals that our microbiome approach performs better in 12 (75%), similarly in 2 (12.5%), and less good in 2 (12.5%) of the samples. Our results demonstrate that this new microbiome approach allows for accurate tissue-type classification of three human epithelial materials of skin, oral and vaginal origin, which is highly relevant for future forensic investigations.

HIrisPlex-S system for eye, hair, and skin color prediction from DNA: Massively parallel sequencing solutions for two common forensically used platforms.

Forensic DNA Phenotyping (FDP) provides the ability to predict externally visible characteristics from minute amounts of crime scene DNA, which can help find unknown perpetrators who are typically unidentifiable via conventional forensic DNA profiling. Fundamental human genetics research has led to a better understanding of the specific DNA variants responsible for physical appearance characteristics, particularly eye, hair, and skin color. Recently, we introduced the HIrisPlex-S system for the simultaneous prediction of eye, hair, and skin color based on 41 DNA variants generated from two forensically validated SNaPshot multiplex assays using capillary electrophoresis (CE). Here we introduce massively parallel sequencing (MPS) solutions for the HIrisPlex-S (HPS) system on two MPS platforms commonly used in forensics, Ion Torrent and MiSeq, that cover all 41 DNA variants in a single assay, respectively. Additionally, we present the forensic developmental validation of the two HPS-MPS assays. The Ion Torrent MPS assay, based on Ion AmpliSeq technology, illustrated the successful generation of full HIrisPlex-S genotypic profiles from 100 pg of input control DNA, while the MiSeq MPS assay based on an in-house design yielded complete profiles from 250 pg of input DNA. Assessing simulated forensic casework samples such as saliva, hair (bulb), blood, semen, and low quantity touch DNA, as well as artificially damaged DNA samples, concordance testing, and samples from numerous species, all illustrated the ability of both versions of the HIrisPlex-S MPS assay to produce results that motivate forensic applications. By also providing an integrated bioinformatics analysis pipeline, MPS data can now be analyzed and a file generated for upload to the publically accessible HIrisPlex online webtool (https://hirisplex.erasmusmc.nl). In addition, we updated the website to accept VCF input data for those with genome sequence data. We thus provide a user-friendly and semi-automated MPS workflow from DNA sample to individual eye, hair, and skin color prediction probabilities. Furthermore, we present a 2-person mixture separation tool that not only assesses genotype reliability with regards genotyping confidence but also provides the most fitting mixture scenario for both minor and major contributors, including profile separation. We envision this MPS implementation of the HIrisPlex-S system for eye, hair, and skin color prediction from DNA as a starting point for further expanding MPS-based forensic DNA phenotyping. This may include the future addition of SNPs predictive for more externally visible characteristics, as well as SNPs for bio-geographic ancestry inference, provided the statistical framework for DNA prediction of these traits is in place.