Divergent evolution of male-determining loci on proto-Y chromosomes of the housefly.

Houseflies provide a good experimental model to study the initial evolutionary stages of a primary sex-determining locus because they possess different recently evolved proto-Y chromosomes that contain male-determining loci (M) with the same male-determining gene, Mdmd. We investigate M-loci genomically and cytogenetically revealing distinct molecular architectures among M-loci. M on chromosome V (MV) has two intact Mdmd copies in a palindrome. M on chromosome III (MIII) has tandem duplications containing 88 Mdmd copies (only one intact) and various repeats, including repeats that are XY-prevalent. M on chromosome II (MII) and the Y (MY) share MIII-like architecture, but with fewer repeats. MY additionally shares MV-specific sequence arrangements. Based on these data and karyograms using two probes, one derives from MIII and one Mdmd-specific, we infer evolutionary histories of polymorphic M-loci, which have arisen from unique translocations of Mdmd, embedded in larger DNA fragments, and diverged independently into regions of varying complexity.

Application of long-read sequencing to elucidate complex pharmacogenomic regions: a proof of principle.

The use of pharmacogenomics in clinical practice is becoming standard of care. However, due to the complex genetic makeup of pharmacogenes, not all genetic variation is currently accounted for. Here, we show the utility of long-read sequencing to resolve complex pharmacogenes by analyzing a well-characterised sample. This data consists of long reads that were processed to resolve phased haploblocks. 73% of pharmacogenes were fully covered in one phased haploblock, including 9/15 genes that are 100% complex. Variant calling accuracy in the pharmacogenes was high, with 99.8% recall and 100% precision for SNVs and 98.7% precision and 98.0% recall for Indels. For the majority of gene-drug interactions in the DPWG and CPIC guidelines, the associated genes could be fully resolved (62% and 63% respectively). Together, these findings suggest that long-read sequencing data offers promising opportunities in elucidating complex pharmacogenes and haplotype phasing while maintaining accurate variant calling.

Toward predicting CYP2D6-mediated variable drug response from CYP2D6 gene sequencing data.

Pharmacogenomics is a key component of personalized medicine that promises safer and more effective drug treatment by individualizing drug choice and dose based on genetic profiles. In clinical practice, genetic biomarkers are used to categorize patients into *-alleles to predict CYP450 enzyme activity and adjust drug dosages accordingly. However, this approach leaves a large part of variability in drug response unexplained. Here, we present a proof-of-concept approach that uses continuous-scale (instead of categorical) assignments to predict enzyme activity. We used full CYP2D6 gene sequences obtained with long-read amplicon-based sequencing and cytochrome P450 (CYP) 2D6-mediated tamoxifen metabolism data from a prospective study of 561 patients with breast cancer to train a neural network. The model explained 79% of interindividual variability in CYP2D6 activity compared to 54% with the conventional *-allele approach, assigned enzyme activities to known alleles with previously reported effects, and predicted the activity of previously uncharacterized combinations of variants. The results were replicated in an independent cohort of tamoxifen-treated patients (model R 2 adjusted = 0.66 versus *-allele R 2 adjusted = 0.35) and a cohort of patients treated with the CYP2D6 substrate venlafaxine (model R 2 adjusted = 0.64 versus *-allele R 2 adjusted = 0.55). Human embryonic kidney cells were used to confirm the effect of five genetic variants on metabolism of the CYP2D6 substrate bufuralol in vitro. These results demonstrate the advantage of a continuous scale and a completely phased genotype for prediction of CYP2D6 enzyme activity and could potentially enable more accurate prediction of individual drug response.

A single QTL with large effect is associated with female functional virginity in an asexual parasitoid wasp.

During the transition from sexual to asexual reproduction, a suite of reproduction-related sexual traits become superfluous, and may be selected against if costly. Female functional virginity refers to asexual females resisting to mate or not fertilizing eggs after mating. These traits appear to be among the first that evolve during transitions from sexual to asexual reproduction. The genetic basis of female functional virginity remains elusive. Previously, we reported that female functional virginity segregates as expected for a single recessive locus in the asexual parasitoid wasp Asobara japonica. Here, we investigate the genetic basis of this trait by quantitative trait loci (QTL) mapping and candidate gene analyses. Consistent with the segregation of phenotypes, we found a single QTL of large effect, spanning over 4.23 Mb and comprising at least 131 protein-coding genes, of which 15 featured sex-biased expression in the related sexual species Asobara tabida. Two of the 15 sex-biased genes were previously identified to differ between related sexual and asexual population/species: CD151 antigen and nuclear pore complex protein Nup50. A third gene, hormone receptor 4, is involved in steroid hormone mediated mating behaviour. Overall, our results are consistent with a single locus, or a cluster of closely linked loci, underlying rapid evolution of female functional virginity in the transition to asexuality. Once this variant, causing rejection to mate, has swept through a population, the flanking region does not get smaller owing to lack of recombination in asexuals.

Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes.

What Is in Umbilicaria pustulata? A Metagenomic Approach to Reconstruct the Holo-Genome of a Lichen.

Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis-the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)-were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

Resolving the complete genome of Kuenenia stuttgartiensis from a membrane bioreactor enrichment using Single-Molecule Real-Time sequencing.

Anaerobic ammonium-oxidizing (anammox) bacteria are a group of strictly anaerobic chemolithoautotrophic microorganisms. They are capable of oxidizing ammonium to nitrogen gas using nitrite as a terminal electron acceptor, thereby facilitating the release of fixed nitrogen into the atmosphere. The anammox process is thought to exert a profound impact on the global nitrogen cycle and has been harnessed as an environment-friendly method for nitrogen removal from wastewater. In this study, we present the first closed genome sequence of an anammox bacterium, Kuenenia stuttgartiensis MBR1. It was obtained through Single-Molecule Real-Time (SMRT) sequencing of an enrichment culture constituting a mixture of at least two highly similar Kuenenia strains. The genome of the novel MBR1 strain is different from the previously reported Kuenenia KUST reference genome as it contains numerous structural variations and unique genomic regions. We find new proteins, such as a type 3b (sulf)hydrogenase and an additional copy of the hydrazine synthase gene cluster. Moreover, multiple copies of ammonium transporters and proteins regulating nitrogen uptake were identified, suggesting functional differences in metabolism. This assembly, including the genome-wide methylation profile, provides a new foundation for comparative and functional studies aiming to elucidate the biochemical and metabolic processes of these organisms.

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

BACKGROUND: The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. RESULTS: In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. CONCLUSIONS: Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

Coping with living in the soil: the genome of the parthenogenetic springtail Folsomia candida.

BACKGROUND: Folsomia candida is a model in soil biology, belonging to the family of Isotomidae, subclass Collembola. It reproduces parthenogenetically in the presence of Wolbachia, and exhibits remarkable physiological adaptations to stress. To better understand these features and adaptations to life in the soil, we studied its genome in the context of its parthenogenetic lifestyle. RESULTS: We applied Pacific Bioscience sequencing and assembly to generate a reference genome for F. candida of 221.7 Mbp, comprising only 162 scaffolds. The complete genome of its endosymbiont Wolbachia, was also assembled and turned out to be the largest strain identified so far. Substantial gene family expansions and lineage-specific gene clusters were linked to stress response. A large number of genes (809) were acquired by horizontal gene transfer. A substantial fraction of these genes are involved in lignocellulose degradation. Also, the presence of genes involved in antibiotic biosynthesis was confirmed. Intra-genomic rearrangements of collinear gene clusters were observed, of which 11 were organized as palindromes. The Hox gene cluster of F. candida showed major rearrangements compared to arthropod consensus cluster, resulting in a disorganized cluster. CONCLUSIONS: The expansion of stress response gene families suggests that stress defense was important to facilitate colonization of soils. The large number of HGT genes related to lignocellulose degradation could be beneficial to unlock carbohydrate sources in soil, especially those contained in decaying plant and fungal organic matter. Intra- as well as inter-scaffold duplications of gene clusters may be a consequence of its parthenogenetic lifestyle. This high quality genome will be instrumental for evolutionary biologists investigating deep phylogenetic lineages among arthropods and will provide the basis for a more mechanistic understanding in soil ecology and ecotoxicology.

Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing.

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.

Variations in the Genome: The Mutation Detection 2015 Meeting on Detection, Genome Sequencing, and Interpretation.

The content of the 13th Mutation Detection meeting (Leiden, April 2015) is summarized in this report. Topics discussed at the meeting included current challenges of clinical NGS, advances in bioinformatics, data quality control, single cell analysis and RNA sequencing, among others. Social, ethical and regulatory challenges of genomic data handling and data sharing were the focus of an expert panel debate. The 14th International Symposium on Variants in the Genome will take place in Santiago de Compostela, June 5-8, 2017. http://isv.variome.org.

Gene Family Evolution Reflects Adaptation to Soil Environmental Stressors in the Genome of the Collembolan Orchesella cincta.

Collembola (springtails) are detritivorous hexapods that inhabit the soil and its litter layer. The ecology of the springtail Orchesella cincta is extensively studied in the context of adaptation to anthropogenically disturbed areas. Here, we present a draft genome of an O. cincta reference strain with an estimated size of 286.8 Mbp, containing 20,249 genes. In total, 446 gene families are expanded and 1,169 gene families evolved specific to this lineage. Besides these gene families involved in general biological processes, we observe gene clusters participating in xenobiotic biotransformation. Furthermore, we identified 253 cases of horizontal gene transfer (HGT). Although the largest percentage of them originated from bacteria (37.5%), we observe an unusually high percentage (30.4%) of such genes of fungal origin. The majority of foreign genes are involved in carbohydrate metabolism and cellulose degradation. Moreover, some foreign genes (e.g., bacillopeptidases) expanded after HGT. We hypothesize that horizontally transferred genes could be advantageous for food processing in a soil environment that is full of decaying organic material. Finally, we identified several lineage-specific genes, expanded gene families, and horizontally transferred genes, associated with altered gene expression as a consequence of genetic adaptation to metal stress. This suggests that these genome features may be preadaptations allowing natural selection to act on. In conclusion, this genome study provides a solid foundation for further analysis of evolutionary mechanisms of adaptation to environmental stressors.

Decay of Sexual Trait Genes in an Asexual Parasitoid Wasp.

Trait loss is a widespread phenomenon with pervasive consequences for a species' evolutionary potential. The genetic changes underlying trait loss have only been clarified in a small number of cases. None of these studies can identify whether the loss of the trait under study was a result of neutral mutation accumulation or negative selection. This distinction is relatively clear-cut in the loss of sexual traits in asexual organisms. Male-specific sexual traits are not expressed and can only decay through neutral mutations, whereas female-specific traits are expressed and subject to negative selection. We present the genome of an asexual parasitoid wasp and compare it to that of a sexual lineage of the same species. We identify a short-list of 16 genes for which the asexual lineage carries deleterious SNP or indel variants, whereas the sexual lineage does not. Using tissue-specific expression data from other insects, we show that fifteen of these are expressed in male-specific reproductive tissues. Only one deleterious variant was found that is expressed in the female-specific spermathecae, a trait that is heavily degraded and thought to be under negative selection in L. clavipes. Although the phenotypic decay of male-specific sexual traits in asexuals is generally slow compared with the decay of female-specific sexual traits, we show that male-specific traits do indeed accumulate deleterious mutations as expected by theory. Our results provide an excellent starting point for detailed study of the genomics of neutral and selected trait decay.

Complete genome sequence of the Clostridium difficile laboratory strain 630Δerm reveals differences from strain 630, including translocation of the mobile element CTn5.

BACKGROUND: Clostridium difficile strain 630Δerm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns. RESULTS: In addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain. CONCLUSIONS: Together, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile.

Complete genome sequence of BS49 and draft genome sequence of BS34A, Bacillus subtilis strains carrying Tn916.

Bacillus subtilis strains BS49 and BS34A, both derived from a common ancestor, carry one or more copies of Tn916, an extremely common mobile genetic element capable of transfer to and from a broad range of microorganisms. Here, we report the complete genome sequence of BS49 and the draft genome sequence of BS34A, which have repeatedly been used as donors to transfer Tn916, Tn916 derivatives or oriTTn916-containing plasmids to clinically important pathogens.

The Complete Genome Sequence of the Murine Pathobiont Helicobacter typhlonius.

BACKGROUND: Immuno-compromised mice infected with Helicobacter typhlonius are used to model microbially inducted inflammatory bowel disease (IBD). The specific mechanism through which H. typhlonius induces and promotes IBD is not fully understood. Access to the genome sequence is essential to examine emergent properties of this organism, such as its pathogenicity. To this end, we present the complete genome sequence of H. typhlonius MIT 97-6810, obtained through single-molecule real-time sequencing. RESULTS: The genome was assembled into a single circularized contig measuring 1.92 Mbp with an average GC content of 38.8%. In total 2,117 protein-encoding genes and 43 RNA genes were identified. Numerous pathogenic features were found, including a putative pathogenicity island (PAIs) containing components of type IV secretion system, virulence-associated proteins and cag PAI protein. We compared the genome of H. typhlonius to those of the murine pathobiont H. hepaticus and human pathobiont H. pylori. H. typhlonius resembles H. hepaticus most with 1,594 (75.3%) of its genes being orthologous to genes in H. hepaticus. Determination of the global methylation state revealed eight distinct recognition motifs for adenine and cytosine methylation. H. typhlonius shares four of its recognition motifs with H. pylori. CONCLUSION: The complete genome sequence of H. typhlonius MIT 97-6810 enabled us to identify many pathogenic features suggesting that H. typhlonius can act as a pathogen. Follow-up studies are necessary to evaluate the true nature of its pathogenic capabilities. We found many methylated sites and a plethora of restriction-modification systems. The genome, together with the methylome, will provide an essential resource for future studies investigating gene regulation, host interaction and pathogenicity of H. typhlonius. In turn, this work can contribute to unraveling the role of Helicobacter in enteric disease.

Determining the quality and complexity of next-generation sequencing data without a reference genome.

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL webcite.

The genomic landscape of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV.

BACKGROUND: Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture. RESULTS: In this study, we present the complete genome sequence of Methylacidiphilum fumariolicum SolV, obtained using Pacific Biosciences single-molecule real-time (SMRT) sequencing technology. The genome assembles to a single 2.5 Mbp chromosome with an average GC content of 41.5%. The genome contains 2,741 annotated genes and 314 functional subsystems including all key metabolic pathways that are associated with Methylacidiphilum strains, including the CBB pathway for CO2 fixation. However, it does not encode the serine cycle and ribulose monophosphate pathways for carbon fixation. Phylogenetic analysis of the particulate methane mono-oxygenase operon separates the Methylacidiphilum strains from other verrucomicrobial methanotrophs. RNA-Seq analysis of cell cultures growing in three different conditions revealed the deregulation of two out of three pmoCAB operons. In addition, genes involved in nitrogen fixation were upregulated in cell cultures growing in nitrogen fixing conditions, indicating the presence of active nitrogenase. Characterization of the global methylation state of M. fumariolicum SolV revealed methylation of adenines and cytosines mainly in the coding regions of the genome. Methylation of adenines was predominantly associated with 5'-m6ACN4GT-3' and 5'-CCm6AN5CTC-3' methyltransferase recognition motifs whereas methylated cytosines were not associated with any specific motif. CONCLUSIONS: Our findings provide novel insights into the global methylation state of verrucomicrobial methanotroph M. fumariolicum SolV. However, partial conservation of methyltransferases between M. fumariolicum SolV and M. infernorum V4 indicates potential differences in the global methylation state of Methylacidiphilum strains. Unravelling the M. fumariolicum SolV genome and its epigenetic regulation allow for robust characterization of biological processes that are involved in oxidizing methane. In turn, they offer a better understanding of the evolution, the underlying physiological and ecological properties of SolV and other Methylacidiphilum strains.

TSSV: a tool for characterization of complex allelic variants in pure and mixed genomes.

MOTIVATION: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive or uncharacterized regions. RESULTS: We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by transcription activator-like effector nucleases (TALENs) and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics. AVAILABILITY AND IMPLEMENTATION: We have implemented TSSV as a Python package that can be installed through the command-line using pip install TSSV command. Its source code and documentation are available at https://pypi.python.org/pypi/tssv and http://www.lgtc.nl/tssv.