myVCF: a desktop application for high-throughput mutations data management

Abstract Summary Next-generation sequencing technologies have become the most powerful tool to discover genetic variants associated with human diseases. Although the dramatic reductions in the costs facilitate the use in the wet-lab and clinics, the huge amount of data generated renders their management by non-expert researchers and physicians extremely difficult. Therefore, there is an urgent need of novel approaches and tools aimed at getting the 'end-users' closer to the sequencing data, facilitating the access by non-bioinformaticians, and to speed-up the functional interpretation of genetic variants. We developed myVCF, a standalone, easy-to-use desktop application, which is based on a browser interface and is suitable for Windows, Mac and UNIX systems. myVCF is an efficient platform that is able to manage multiple sequencing projects created from VCF files within the system; stores genetic variants and samples genotypes from an annotated VCF files into a SQLite database; implements a flexible search engine for data exploration, allowing to query for chromosomal region, gene, single variant or dbSNP ID. Besides, myVCF generates a summary statistics report about mutations distribution across samples and across the genome/exome by aggregating the information within the VCF file. In summary, the myVCF platform allows end-users without strong programming and bioinformatics skills to explore, query, visualize and export mutations data in a simple and straightforward way. Availability and implementation https://apietrelli.github.io/myVCF/ Supplementary information Supplementary data are available at Bioinformatics online

In vitro transcription profiling of the σS subunit of bacterial RNA polymerase: re-definition of the σS regulon and identification of σS-specific promoter sequence elements

Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ70 (the housekeeping σ subunit) and σS (an alternative σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template. We found that Eσ70, in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, EσS efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the −35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the −10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the −10/+1 region could improve promoter recognition by Eσ

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063

The 1.8\u2005\uc5 resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using \u3b2-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography

In vitro transcription profiling of the σS subunit of bacterial RNA polymerase: re-definition of the σS regulon and identification of σS-specific promoter sequence elements

Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ70 (the housekeeping σ subunit) and σS (an alternative σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template. We found that Eσ70, in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, EσS efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the −35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the −10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the −10/+1 region could improve promoter recognition by EσS

Gene expression profile of endothelial cells during perturbation of the gut vascular barrier

It has been widely demonstrated that tolerance against gut microbiota is compartmentalized to mucosal sites where microbes mostly reside. How the commensal bacteria are excluded from the entrance into the blood stream via intestinal capillaries that are located beneath the gut epithelium was not clear. We recently described the existence of a new anatomical structure, the ‘gut vascular barrier’ (GVB), both in murine and human intestines that plays a fundamental role in avoiding indiscriminate trafficking of bacteria from the gut into the blood circulation. The vascular barrier integrity could be altered by Salmonella typhimurium, a pathogen capable of systemic dissemination, through the modulation of the Wnt/β-catenin signaling pathway. Here we have analyzed the differences in gut endothelial gene expression profiles during Salmonella infection and have identified some interesting characteristics of endothelial to mesenchymal transition. These findings add new insights in the gut-liver axis

Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independent of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficient mice (InsR+/-), in the pathogenesis of liver fibrosis in C57BL/6mice.We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation inMCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independent of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization

Journal of Genomics Enly: Improving Draft Genomes through Reads Recycling

© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons Licens

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome

Comprehensive Genomic Characterization of Cutaneous Malignant Melanoma Cell Lines Derived from Metastatic Lesions by Whole-Exome Sequencing and SNP Array Profiling

<div><p>Cutaneous malignant melanoma is the most fatal skin cancer and although improved comprehension of its pathogenic pathways allowed to realize some effective molecular targeted therapies, novel targets and drugs are still needed. Aiming to add genetic information potentially useful for novel targets discovery, we performed an extensive genomic characterization by whole-exome sequencing and SNP array profiling of six cutaneous melanoma cell lines derived from metastatic patients. We obtained a total of 3,325 novel coding single nucleotide variants, including 2,172 non-synonymous variants. We catalogued the coding mutations according to Sanger COSMIC database and to a manually curated list including genes involved in melanoma pathways identified by mining recent literature. Besides confirming the presence of known melanoma driver mutations (<i>BRAF^V600E, NRAS^Q61R</i>), we identified novel mutated genes involved in signalling pathways crucial for melanoma pathogenesis and already addressed by current targeted therapies (such as MAPK and glutamate pathways). We also identified mutations in four genes (<i>MUC19</i>, <i>PAICS</i>, <i>RBMXL1</i>, <i>KIF23</i>) never reported in melanoma, which might deserve further investigations. All data are available to the entire research community in our Melanoma Exome Database (at <a href="https://155.253.6.64/MExDB/" target="_blank">https://155.253.6.64/MExDB/</a>). In summary, these cell lines are valuable biological tools to improve the genetic comprehension of this complex cancer disease and to study functional relevance of individual mutational events, and these findings could provide insights potentially useful for identification of novel therapeutic targets for cutaneous malignant melanoma.</p></div