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.

Mitochondrial Transport from Mesenchymal Stromal Cells to Chondrocytes Increases DNA Content and Proteoglycan Deposition In Vitro in 3D Cultures.

OBJECTIVE: Allogeneic mesenchymal stromal cells (MSCs) are used in the 1-stage treatment of articular cartilage defects. The aim of this study is to investigate whether transport of mitochondria exists between chondrocytes and MSCs and to investigate whether the transfer of mitochondria to chondrocytes contributes to the mechanism of action of MSCs. DESIGN: Chondrocytes and MSCs were stained with MitoTracker, and CellTrace was used to distinguish between cell types. The uptake of fluorescent mitochondria was measured in cocultures using flow cytometry. Transport was visualized using fluorescence microscopy. Microvesicles were isolated and the presence of mitochondria was assessed. Mitochondria were isolated from MSCs and transferred to chondrocytes using MitoCeption. Pellets of chondrocytes, chondrocytes with transferred MSC mitochondria, and cocultures were cultured for 28 days. DNA content and proteoglycan content were measured. Mitochondrial DNA of cultured pellets and of repair cartilage tissue was quantified. RESULTS: Mitochondrial transfer occurred bidirectionally within the first 4 hours until 16 hours of coculture. Transport took place via tunneling nanotubes, direct cell-cell contact, and extracellular vesicles. After 28 days of pellet culture, DNA content and proteoglycan deposition were higher in chondrocyte pellets to which MSC mitochondria were transferred than the control groups. No donor mitochondrial DNA was traceable in the biopsies, whereas an increase in MSC mitochondrial DNA was seen in the pellets. CONCLUSIONS: These results suggest that mitochondrial transport plays a role in the chondroinductive effect of MSCs on chondrocytes in vitro. However, in vivo no transferred mitochondria could be traced back after 1 year.

DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets: Validation with camera-recorded diets.

Ecological research is often hampered by the inability to quantify animal diets. Diet composition can be tracked through DNA metabarcoding of fecal samples, but whether (complex) diets can be quantitatively determined with metabarcoding is still debated and needs validation using free-living animals. This study validates that DNA metabarcoding of feces can retrieve actual ingested taxa, and most importantly, that read numbers retrieved from sequencing can also be used to quantify the relative biomass of dietary taxa. Validation was done with the hole-nesting insectivorous Pied Flycatcher whose diet was quantified using camera footage. Size-adjusted counts of food items delivered to nestlings were used as a proxy for provided biomass of prey orders and families, and subsequently, nestling feces were assessed through DNA metabarcoding. To explore potential effects of digestion, gizzard and lower intestine samples of freshly collected birds were subjected to DNA metabarcoding. For metabarcoding with Cytochrome Oxidase subunit I (COI), we modified published invertebrate COI primers LCO1490 and HCO1777, which reduced host reads to 0.03%, and amplified Arachnida DNA without significant changing the recovery of other arthropod taxa. DNA metabarcoding retrieved all commonly camera-recorded taxa. Overall, and in each replicate year (N = 3), the relative scaled biomass of prey taxa and COI read numbers correlated at R = .85 (95CI:0.68-0.94) at order level and at R = .75 (CI:0.67-0.82) at family level. Similarity in arthropod community composition between gizzard and intestines suggested limited digestive bias. This DNA metabarcoding validation demonstrates that quantitative analyses of arthropod diet is possible. We discuss the ecological applications for insectivorous birds.

On the Forensic Use of Y-Chromosome Polymorphisms.

Nowadays, the use of Y-chromosome polymorphisms forms an essential part of many forensic DNA investigations. However, this was not always the case. Only since 1992 have we seen that some forensic scientists started to have an interest in this chromosome. In this review, I will sketch a brief history focusing on the forensic use of Y-chromosome polymorphisms. Before describing the various applications of short-tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) on the Y-chromosome, I will discuss a few often ignored aspects influencing proper use and interpretation of Y-chromosome information: (i) genotyping Y-SNPs and Y-STRs, (ii) Y-STR haplotypes shared identical by state (IBS) or identical by descent (IBD), and (iii) Y-haplotype database frequencies.

Application of a probabilistic genotyping software to MPS mixture STR data is supported by similar trends in LRs compared with CE data.

The interpretation of short tandem repeat (STR) profiles can be challenging when, for example, alleles are masked due to allele sharing among contributors and/or when they are subject to drop-out, for instance from sample degradation. Mixture interpretation can be improved by increasing the number of STRs and/or loci with a higher discriminatory power. Both capillary electrophoresis (CE, 6-dye) and massively parallel sequencing (MPS) provide a platform for analysing relatively large numbers of autosomal STRs. In addition, MPS enables distinguishing between sequence variants, resulting in enlarged discriminatory power. Also, MPS allows for small amplicon sizes for all loci as spacing is not an issue, which is beneficial with degraded DNA. Altogether, MPS has the potential to increase the weights of evidence for true contributors to (complex) DNA profiles. In this study, likelihood ratio (LR) calculations were performed using STR profiles obtained with two different MPS systems and analysed using different settings: 1) MPS PowerSeq™ Auto System profiles analysed using FDSTools equipped with optimized settings such as noise correction, 2) ForenSeq™ DNA Signature Prep Kit profiles analysed using the default settings in the Universal Analysis Software (UAS), and 3) ForenSeq™ DNA Signature Prep Kit profiles analysed using FDSTools empirically adapted to cope with one-directional reads and provisional, basic settings. The LR calculations used genotyping data for two- to four-person mixtures varying for mixture proportion, level of drop-out and allele sharing and were generated with the continuous model EuroForMix. The LR results for the over 2000 sets of propositions were affected by the variation for the number of markers and analysis settings used in the three approaches. Nevertheless, trends for true and non-contributors, effects of replicates, assigned number of contributors, and model validation results were comparable for the three MPS approaches and alike the trends known for CE data. Based on this analogy, we regard the probabilistic interpretation of MPS STR data fit for forensic DNA casework. In addition, guidelines were derived on when to apply LR calculations to MPS autosomal STR data and report the corresponding results.

Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing.

Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.

Subdividing Y-chromosome haplogroup R1a1 reveals Norse Viking dispersal lineages in Britain.

The influence of Viking-Age migrants to the British Isles is obvious in archaeological and place-names evidence, but their demographic impact has been unclear. Autosomal genetic analyses support Norse Viking contributions to parts of Britain, but show no signal corresponding to the Danelaw, the region under Scandinavian administrative control from the ninth to eleventh centuries. Y-chromosome haplogroup R1a1 has been considered as a possible marker for Viking migrations because of its high frequency in peninsular Scandinavia (Norway and Sweden). Here we select ten Y-SNPs to discriminate informatively among hg R1a1 sub-haplogroups in Europe, analyse these in 619 hg R1a1 Y chromosomes including 163 from the British Isles, and also type 23 short-tandem repeats (Y-STRs) to assess internal diversity. We find three specifically Western-European sub-haplogroups, two of which predominate in Norway and Sweden, and are also found in Britain; star-like features in the STR networks of these lineages indicate histories of expansion. We ask whether geographical distributions of hg R1a1 overall, and of the two sub-lineages in particular, correlate with regions of Scandinavian influence within Britain. Neither shows any frequency difference between regions that have higher (≥10%) or lower autosomal contributions from Norway and Sweden, but both are significantly overrepresented in the region corresponding to the Danelaw. These differences between autosomal and Y-chromosomal histories suggest either male-specific contribution, or the influence of patrilocality. Comparison of modern DNA with recently available ancient DNA data supports the interpretation that two sub-lineages of hg R1a1 spread with the Vikings from peninsular Scandinavia.

DNA commission of the International Society of Forensic Genetics (ISFG): Recommendations on the interpretation of Y-STR results in forensic analysis.

Forensic genetic laboratories perform a large amount of STR analyses of the Y chromosome, in particular to analyze the male part of complex DNA mixtures. However, the statistical interpretation of evidence retrieved from Y-STR haplotypes is challenging. Due to the uni-parental inheritance mode, Y-STR loci are connected to each other and thus haplotypes show patterns of relationship on the familial and population level. This precludes the treatment of Y-STR loci as independently inherited variables and the application of the product rule. Instead, the dependency structure of Y-STRs needs to be included in the haplotype frequency estimation process affecting also the current paradigm of a random match probability that is in the autosomal case approximated by the population frequency assuming unrelatedness of sampled individuals. Information on the degree of paternal relatedness in the suspect population as well as on the familial network is however needed to interpret Y-chromosomal results in the best possible way. The previous recommendations of the DNA commission of the ISFG on the use of Y-STRs in forensic analysis published more than a decade ago [1] cover the interpretation issue only marginally. The current recommendations address a number of topics (frequency estimators, databases, metapopulations, LR formulation, triage, rapidly mutating Y-STRs) with relevance for the Y-STR statistics and recommend a decision-based procedure, which takes into account legal requirements as well as availability of population data and statistical methods.

Generation and genetic repair of 2 iPSC clones from a patient bearing a heterozygous c.1120del18 mutation in the ACVRL1 gene leading to Hereditary Hemorrhagic Telangiectasia (HHT) type 2.

Fibroblasts from a patient carrying a heterozygous 18bp deletion in exon 8 of the ACVRL1 gene (c.1120del18) were reprogrammed using episomal vectors. The in-frame deletion in ACVRL1 causes the loss of 6 amino acids of the protein, which is associated with Hereditary Hemorrhagic Telangiectasia (HHT) type 2 (Letteboer et al., 2005). CRISPR-Cas9 editing was used to genetically correct the mutation in the induced pluripotent stem cells (iPSCs). The top5-predicted off-target sites were not altered. Patient and isogenic iPSCs showed high pluripotent marker expression, in vitro differentiation capacity into all three germ layers and displayed a normal karyotype. The obtained isogenic pairs will enable proper in vitro disease modelling of HHT (Roman and Hinck, 2017).

Taxonomic classification and abundance estimation using 16S and WGS-A comparison using controlled reference samples.

The assessment of microbiome biodiversity is the most common application of metagenomics. While 16S sequencing remains standard procedure for taxonomic profiling of metagenomic data, a growing number of studies have clearly demonstrated biases associated with this method. By using Whole Genome Shotgun sequencing (WGS) metagenomics, most of the known restrictions associated with 16S data are alleviated. However, due to the computationally intensive data analyses and higher sequencing costs, WGS based metagenomics remains a less popular option. Selecting the experiment type that provides a comprehensive, yet manageable amount of information is a challenge encountered in many metagenomics studies. In this work, we created a series of artificial bacterial mixes, each with a different distribution of skin-associated microbial species. These mixes were used to estimate the resolution of two different metagenomic experiments - 16S and WGS - and to evaluate several different bioinformatics approaches for taxonomic read classification. In all test cases, WGS approaches provide much more accurate results, in terms of taxa prediction and abundance estimation, in comparison to those of 16S. Furthermore, we demonstrate that a 16S dataset, analysed using different state of the art techniques and reference databases, can produce widely different results. In light of the fact that most forensic metagenomic analysis are still performed using 16S data, our results are especially important.

The Dutch Y-chromosomal landscape.

Previous studies indicated existing, albeit limited, genetic-geographic population substructure in the Dutch population based on genome-wide data and a lack of this for mitochondrial SNP based data. Despite the aforementioned studies, Y-chromosomal SNP data from the Netherlands remain scarce and do not cover the territory of the Netherlands well enough to allow a reliable investigation of genetic-geographic population substructure. Here we provide the first substantial dataset of detailed spatial Y-chromosomal haplogroup information in 2085 males collected across the Netherlands and supplemented with previously published data from northern Belgium. We found Y-chromosomal evidence for genetic-geographic population substructure, and several Y-haplogroups demonstrating significant clinal frequency distributions in different directions. By means of prediction surface maps we could visualize (complex) distribution patterns of individual Y-haplogroups in detail. These results highlight the value of a micro-geographic approach and are of great use for forensic and epidemiological investigations and our understanding of the Dutch population history. Moreover, the previously noted absence of genetic-geographic population substructure in the Netherlands based on mitochondrial DNA in contrast to our Y-chromosome results, hints at different population histories for women and men in the Netherlands.

Correction to: The Dutch Y-chromosomal landscape.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

From next generation sequencing to now generation sequencing in forensics.

In contrast to genetic diagnostic disciplines such as Oncogenetics and Cinical Genetics, where worldwide, since 2010, tens of thousands of DNA samples are routinely screened annually using either targeted genome sequencing or whole genome sequencing using massively parallel sequencing (MPS), the forensic use of MPS is still far from being a routine diagnostic tool. This perspectives focusses on issues that are essential in order to fully understand (i) why MPS of short tandem repeats (STRs) is very different from the capillary electrophoresis (CE) based genotyping of STRs, (ii) what we, DNA experts, should know before explaining MPS-based evidence in court, and (iii) what information should be present in a forensic investigation report that is MPS-based. Here one has to keep in mind that the forensic use of CE was first introduced in 1992-1993 and that it took some time to fully appreciate all intricacies. Obviously, I might be biased in my opinion, having worked on this topic since 2008, but I sincerely hope that MPS will soon be widely accepted and used because, especially in case of mixed-source DNA samples, MPS is much better in the deconvolution of the individual contributors and invariably reveals genetic information that cannot be inferred otherwise.

Diagnostics of short tandem repeat expansion variants using massively parallel sequencing and componential tools.

Short tandem repeats (STRs) are scattered throughout the human genome. Some STRs, like trinucleotide repeat expansion (TRE) variants, cause hereditable disorders. Unambiguous molecular diagnostics of TRE disorders is hampered by current technical limitations imposed by traditional PCR and DNA sequencing methods. Here we report a novel pipeline for TRE variant diagnosis employing the massively parallel sequencing (MPS) combined with an opensource software package (FDSTools), which together are designed to distinguish true STR sequences from STR sequencing artifacts. We show that this approach can improve TRE diagnosis, such as Oculopharyngeal muscular dystrophy (OPMD). OPMD is caused by a trinucleotide expansion in the PABPN1 gene. A short GCN expansion, (GCN[10]), coding for a 10 alanine repeat is not pathogenic, but an alanine expansion is pathogenic. Applying this novel procedure in  a Dutch OPMD patient cohort, we found expansion variants from GCN[11] to GCN[16], with the GCN[16] as the most abundant variant. The repeat expansion length did not correlate with clinical features. However, symptom severity was found to correlate with age and with the initial affected muscles, suggesting that aging and muscle-specific factors can play a role in modulating OPMD.

Short hypervariable microhaplotypes: A novel set of very short high discriminating power loci without stutter artefacts.

Since two decades, short tandem repeats (STRs) are the preferred markers for human identification, routinely analysed by fragment length analysis. Here we present a novel set of short hypervariable autosomal microhaplotypes (MH) that have four or more SNPs in a span of less than 70 nucleotides (nt). These MHs display a discriminating power approaching that of STRs and provide a powerful alternative for the analysis;1;is of forensic samples that are problematic when the STR fragment size range exceeds the integrity range of severely degraded DNA or when multiple donors contribute to an evidentiary stain and STR stutter artefacts complicate profile interpretation. MH typing was developed using the power of massively parallel sequencing (MPS) enabling new powerful, fast and efficient SNP-based approaches. MH candidates were obtained from queries in data of the 1000 Genomes, and Genome of the Netherlands (GoNL) projects. Wet-lab analysis of 276 globally dispersed samples and 97 samples of nine large CEPH families assisted locus selection and corroboration of informative value. We infer that MHs represent an alternative marker type with good discriminating power per locus (allowing the use of a limited number of loci), small amplicon sizes and absence of stutter artefacts that can be especially helpful when unbalanced mixed samples are submitted for human identification.

Demographic History and Genetic Adaptation in the Himalayan Region Inferred from Genome-Wide SNP Genotypes of 49 Populations.

We genotyped 738 individuals belonging to 49 populations from Nepal, Bhutan, North India, or Tibet at over 500,000 SNPs, and analyzed the genotypes in the context of available worldwide population data in order to investigate the demographic history of the region and the genetic adaptations to the harsh environment. The Himalayan populations resembled other South and East Asians, but in addition displayed their own specific ancestral component and showed strong population structure and genetic drift. We also found evidence for multiple admixture events involving Himalayan populations and South/East Asians between 200 and 2,000 years ago. In comparisons with available ancient genomes, the Himalayans, like other East and South Asian populations, showed similar genetic affinity to Eurasian hunter-gatherers (a 24,000-year-old Upper Palaeolithic Siberian), and the related Bronze Age Yamnaya. The high-altitude Himalayan populations all shared a specific ancestral component, suggesting that genetic adaptation to life at high altitude originated only once in this region and subsequently spread. Combining four approaches to identifying specific positively selected loci, we confirmed that the strongest signals of high-altitude adaptation were located near the Endothelial PAS domain-containing protein 1 and Egl-9 Family Hypoxia Inducible Factor 1 loci, and discovered eight additional robust signals of high-altitude adaptation, five of which have strong biological functional links to such adaptation. In conclusion, the demographic history of Himalayan populations is complex, with strong local differentiation, reflecting both genetic and cultural factors; these populations also display evidence of multiple genetic adaptations to high-altitude environments.

Erratum: The Beaker phenomenon and the genomic transformation of northwest Europe.

This corrects the article DOI: 10.1038/nature25738.