The frequency of amyotrophic lateral sclerosis (ALS) mutations has been extensively investigated in several populations; however, a systematic analysis in Turkish cases has not been reported so far. In this study, we screened 477 ALS patients for mutations, including 116 familial ALS patients from 82 families and 361 sporadic ALS (sALS) cases. Patients were genotyped for C9orf72 (18.3%), SOD1 (12.2%), FUS (5%), TARDBP (3.7%), and UBQLN2 (2.4%) gene mutations, which together account for approximately 40% of familial ALS in Turkey. No SOD1 mutations were detected in sALS patients; however, C9orf72 (3.1%) and UBQLN2 (0.6%) explained 3.7% of sALS in the population. Exome sequencing revealed mutations in OPTN, SPG11, DJ1, PLEKHG5, SYNE1, TRPM7, and SQSTM1 genes, many of them novel. The spectrum of mutations reflect both the distinct genetic background and the heterogeneous nature of the Turkish ALS population.

BackgroundTurkey is a crossroads of major population movements throughout history and has been a hotspot of cultural interactions. Several studies have investigated the complex population history of Turkey through a limited set of genetic markers. However, to date, there have been no studies to assess the genetic variation at the whole genome level using whole genome sequencing. Here, we present whole genome sequences of 16 Turkish individuals resequenced at high coverage (32 × -48×).ResultsWe show that the genetic variation of the contemporary Turkish population clusters with South European populations, as expected, but also shows signatures of relatively recent contribution from ancestral East Asian populations. In addition, we document a significant enrichment of non-synonymous private alleles, consistent with recent observations in European populations. A number of variants associated with skin color and total cholesterol levels show frequency differentiation between the Turkish populations and European populations. Furthermore, we have analyzed the 17q21.31 inversion polymorphism region (MAPT locus) and found increased allele frequency of 31.25% for H1/H2 inversion polymorphism when compared to European populations that show about 25% of allele frequency.ConclusionThis study provides the first map of common genetic variation from 16 western Asian individuals and thus helps fill an important geographical gap in analyzing natural human variation and human migration. Our data will help develop population-specific experimental designs for studies investigating disease associations and demographic history in Turkey.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-963) contains supplementary material, which is available to authorized users.

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MotivationDespite recent advances in algorithms design to characterize structural variation using high-throughput short read sequencing (HTS) data, characterization of novel sequence insertions longer than the average read length remains a challenging task. This is mainly due to both computational difficulties and the complexities imposed by genomic repeats in generating reliable assemblies to accurately detect both the sequence content and the exact location of such insertions. Additionally, de novo genome assembly algorithms typically require a very high depth of coverage, which may be a limiting factor for most genome studies. Therefore, characterization of novel sequence insertions is not a routine part of most sequencing projects.There are only a handful of algorithms that are specifically developed for novel sequence insertion discovery that can bypass the need for the whole genome de novo assembly. Still, most such algorithms rely on high depth of coverage, and to our knowledge there is only one method (PopIns) that can use multi-sample data to “collectively” obtain a very high coverage dataset to accurately find insertions common in a given population.ResultHere, we present Pamir, a new algorithm to efficiently and accurately discover and genotype novel sequence insertions using either single or multiple genome sequencing datasets. Pamir is able to detect breakpoint locations of the insertions and calculate their zygosity (i.e. heterozygous versus homozygous) by analyzing multiple sequence signatures, matching one-end-anchored sequences to small-scale de novo assemblies of unmapped reads, and conducting strand-aware local assembly. We test the efficacy of Pamir on both simulated and real data, and demonstrate its potential use in accurate and routine identification of novel sequence insertions in genome projects.Availability and implementationPamir is available at https://github.com/vpc-ccg/pamir.Supplementary information Supplementary data are available at Bioinformatics online.

Fasciola hepatica is a trematode helminth causing a damaging disease, fasciolosis, in ruminants and humans. Comprehensive proteomic studies broaden our knowledge of the parasite's protein profile, and provide new insights into the development of more effective strategies to deal with fasciolosis. The objective of this study was to generate a comprehensive profile of F. hepatica proteins expressed during the chronic stage of infection in cattle by building on previous efforts in this area. The approach included an improved sample preparation procedure for surface and internal layers of the parasite, the application of nano-UPLC-ESI-qTOF-MS (nano-ultra-performance LC and ESI quadrupole TOF MS) integrated with different acquisition methods and in silico database search against various protein databases and a transcript database including a new assembly of publically available EST. Of a total of 776 identified proteins, 206 and 332 were specific to the surface and internal layers of the parasite, respectively. Furthermore, 238 proteins were common to both layers, with comparative differences of 172 proteins detected. Specific proteins not previously identified in F. hepatica, but shown to be immunomodulatory or potential drug targets for other parasites, are discussed.

BackgroundFasciola hepatica causes chronic liver disease, fasciolosis, leading to significant losses in the livestock economy and concerns for human health in many countries. The identification of F. hepatica genes involved in the parasite’s virulence through modulation of host immune system is utmost important to comprehend evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, to identify the parasite’s putative virulence genes which are associated with host immunomodulation, we explored whole transcriptome of an adult F. hepatica using current transcriptome profiling approaches integrated with detailed in silico analyses. In brief, the comparison of the parasite transcripts with the specialised public databases containing sequence data of non-parasitic organisms (Dugesiidae species and Caenorhabditis elegans) or of numerous pathogens and investigation of the sequences in terms of nucleotide evolution (directional selection) and cytokine signaling relation were conducted.ResultsNGS of the whole transcriptome resulted in 19,534,766 sequence reads, yielding a total of 40,260 transcripts (N50 = 522 bp). A number of the parasite transcripts (n = 1,671) were predicted to be virulence-related on the basis of the exclusive homology with the pathogen-associated data, positive selection or relationship with cytokine signaling. Of these, a group of the virulence-related genes (n = 62), not previously described, were found likely to be associated with immunomodulation based on in silico functional categorisation, showing significant sequence similarities with various immune receptors (i.e. MHC I class, TGF-β receptor, toll/interleukin-1 receptor, T-cell receptor, TNF receptor, and IL-18 receptor accessory protein), cytokines (i.e. TGF-β, interleukin-4/interleukin-13 and TNF-α), cluster of differentiations (e.g. CD48 and CD147) or molecules associated with other immunomodulatory mechanisms (such as regulation of macrophage activation). Some of the genes (n = 5) appeared to be under positive selection (Ka/Ks > 1), imitating proteins associated with cytokine signaling (through sequence homologies with thrombospondin type 1, toll/interleukin-1 receptor, TGF-β receptor and CD147).ConclusionsWith a comparative transcriptome profiling approach, we have identified a number of potential immunomodulator genes of F. hepatica (n = 62), which are firstly described here, could be employed for the development of better strategies (including RNAi) in the battle against both zoonotically and economically important disease, fasciolosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1539-8) contains supplementary material, which is available to authorized users.

The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are significant improvements in accuracy and reproducibility of HTS based analyses, the usability of these types of data for diagnostic and prognostic applications necessitates a near perfect data generation. To assess the usability of a widely used HTS platform for accurate and reproducible clinical applications in terms of robustness, we generated whole genome shotgun (WGS) sequence data from the genomes of two human individuals in two different genome sequencing centers. After analyzing the data to characterize SNPs and indels using the same tools (BWA, SAMtools, and GATK), we observed significant number of discrepancies in the call sets. As expected, the most of the disagreements between the call sets were found within genomic regions containing common repeats and segmental duplications, albeit only a small fraction of the discordant variants were within the exons and other functionally relevant regions such as promoters. We conclude that although HTS platforms are sufficiently powerful for providing data for first-pass clinical tests, the variant predictions still need to be confirmed using orthogonal methods before using in clinical applications.

Background Natural killer cell lymphomas (NKCLs) are rare diseases with poor prognosis. There are few studies that reported oncogenic mutations in this disease. Identifying mutations critical to the neoplastic transformation of NK cells is crucial for the development of targeted therapies. Methods We performed RNA sequencing (RNA-Seq) on NKCL cases (n=17), malignant NK cell lines (n=3) and resting or activated normal NK cells (n=3) to analyze the genome-wide mutation profile in NKCLs. All SNVs detected by RNA-Seq were validated by Sanger sequencing using the corresponding genomic DNA (gDNA). We expanded our analysis to include specific sequencing of the SH2 domains of STAT3 and STAT5B using 37 additional NKCL cases and 6 NK cell lines. Retroviral or lentiviral transduction was performed to express SOCS6 or STAT3 shRNA in NK cell lines, respectively. After transduction, cells were tracked by quantifying the % of GFP+ cells. Apoptosis was assessed by quantifying AnnexinV-PE stained cells. Western blot was performed on 6 NK cell lines using p-STAT3 (Tyr705) antibody. q-MSP and q-RT-PCR were used to detect SOCS6 promoter methylation and mRNA expression in NK cell lines, respectively. Results RNA-Seq showed frequent oncogenic mutations in JAK/STAT pathway members STAT3 (18%), STAT5B (6%), BRAF (6%) and MAP2K1 (6%). Targeted Sanger sequencing of 37 additional NKCL cases showed one patient with activating STAT3 and two patients with activating STAT5B mutations leading to 7.4% (4 of 54) and 6% (3 of 50) total mutation frequency, respectively. STAT3 and STAT5B mutations were located in the SH2 domain and three of four STAT3 mutations and all STAT5B mutations were previously observed in NK- or T-LGL leukemia cases. Oncogenic activities of two other JAK-STAT pathway genes, BRAF (G469A), MAP2K1 (K57N) have been reported in solid tumors and leukemias. The JAK/STAT pathway mutations were present in a total of 53% of the NKCL cases with RNA-Seq data available (n=17). Intriguingly, targeted sequencing revealed oncogenic STAT3 mutations in 50% of malignant NK cell lines (n=6), which were associated with p-STAT3 expression detected by western blot.STAT3 knock-down resulted in reduced growth in a NK cell line with STAT3 mutation. In agreement with Kimura et. al. Leuk Lymphoma 2013, we were not able to detect JAK3 mutations as reported previously. In an accompanying DNA methylation analysis, we observed epigenetic silencing of SOCS6, a negative regulator of JAK-STAT3 signaling, in NKCL samples. Reintroduction of SOCS6 showed negative selection pressure associated with increased apoptosis in limiting concentrations of IL2 in two SOCS6-null NK cell lines with activating STAT3 mutations suggesting a possible cooperation of oncogenic JAK-STAT pathway mutations and the epigenetic silencing of a negative regulator of this pathway. Conclusions We have identified a high incidence of activating mutations of STAT3, STAT5B and other oncogenic JAK-STAT pathway genes in NKCLs. SOCS6 was frequently methylated in NKCLs with corresponding low gene expression. There was evidence suggestive of cooperation of genetic and epigenetic mechanisms in the activation of the JAK-STAT pathway in NKCLs. This study suggests that JAK-STAT pathway inhibition may be a therapeutic option in NKCLs. Disclosures: No relevant conflicts of interest to declare.