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

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/. CONTACT: pietrelli@ingm.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High-throughput genetic characterization of a cohort of Brugada syndrome patients.

Brugada syndrome (BrS) is an inherited cardiac arrhythmic disorder that can lead to sudden death, with a prevalence of 1:5000 in Caucasian population and affecting mainly male patients in their third to fourth decade of life. BrS is inherited as an autosomal dominant trait; however, to date genetic bases have been only partially understood. Indeed most mutations are located in the SCN5A gene, encoding the alpha-subunit of the Na(+) cardiac channel, but >70% BrS patients still remain genetically undiagnosed. Although 21 other genes have been associated with BrS susceptibility, their pathogenic role is still unclear. A recent next-generation sequencing study investigated the contribution of 45 arrhythmia susceptibility genes in BrS pathogenesis, observing a significant enrichment only for SCN5A. In our study, we evaluated the distribution of putative functional variants in a wider panel of 158 genes previously associated with arrhythmic and cardiac defects in a cohort of 91 SCN5A-negative BrS patients. In addition, to identify genes significantly enriched in BrS, we performed a mutation burden test by using as control dataset European individuals selected from the 1000Genomes project. We confirmed BrS genetic heterogeneity and identified new potential BrS candidates such as DSG2 and MYH7, suggesting a possible genetic overlap between different cardiac disorders.

The rs2294918 E434K variant modulates patatin-like phospholipase domain-containing 3 expression and liver damage.

UNLABELLED: The patatin-like phosholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is a major determinant of hepatic fat and predisposes to the full spectrum of liver damage in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate whether additional PNPLA3 coding variants contribute to NAFLD susceptibility, first in individuals with contrasting phenotypes (with early-onset NAFLD vs. very low aminotransferases) and then in a large validation cohort. Rare PNPLA3 variants were not detected by sequencing coding regions and intron-exon boundaries either in 142 patients with early-onset NAFLD nor in 100 healthy individuals with alanine aminotransferase <22/20 IU/mL. Besides rs738409 I148M, the rs2294918 G>A polymorphism (E434K sequence variant) was over-represented in NAFLD (adjusted P = 0.01). In 1,447 subjects with and without NAFLD, the 148M-434E (P < 0.0001), but not the 148M-434K, haplotype (P > 0.9), was associated with histological NAFLD and steatohepatitis. Both the I148M (P = 0.0002) and E434K variants (P = 0.044) were associated with serum ALT levels, by interacting with each other, in that the 434K hampered the association with liver damage of the 148M allele (P = 0.006). The E434K variant did not affect PNPLA3 enzymatic activity, but carriers of the rs2294918 A allele (434K) displayed lower hepatic PNPLA3 messenger RNA and protein levels (P < 0.05). CONCLUSIONS: Rare loss-of-function PNPLA3 variants were not detected in early-onset NAFLD. However, PNPLA3 rs2294918 E434K decreased PNPLA3 expression, lessening the effect of the I148M variant on the predisposition to steatosis and liver damage. This suggests that the PNPLA3 I148M variant has a codominant negative effect on triglycerides mobilization from lipid droplets, mediated by inhibition of other lipases.

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 independently 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) haploinsufficiency (InsR+/-) in the pathogenesis of liver fibrosis in C57BL/6 mice. 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 in MCD-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 independently 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.

ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes.

Efflux-mediated macrolide resistance due to mef(E) and mel, carried by the mega element, is common in Streptococcus pneumoniae, for which it was originally characterized, but it is rare in Streptococcus pyogenes In S. pyogenes, mega was previously found to be enclosed in Tn2009, a composite genetic element of the Tn916 family containing tet(M) and conferring erythromycin and tetracycline resistance. In this study, S. pyogenes isolates containing mef(E), apparently not associated with other resistance determinants, were examined to characterize the genetic context of mega. By whole-genome sequencing of one isolate, MB56Spyo009, we identified a novel composite integrative and conjugative element (ICE) carrying mega, designated ICESpy009, belonging to the ICESa2603 family. ICESpy009 was 55 kb long, contained 61 putative open reading frames (ORFs), and was found to be integrated into hylA, a novel integration site for the ICESa2603 family. The modular organization of the ICE was similar to that of members of the ICESa2603 family carried by different streptococcal species. In addition, a novel cluster of accessory resistance genes was found inside a region that encloses mega. PCR mapping targeting ICESpy009 revealed the presence of a similar ICE in five other isolates under study. While in three isolates the integration site was the same as that of ICESpy009, in two isolates the ICE was integrated into rplL, the typical integration site of the ICESa2603 family. ICESpy009 was able to transfer macrolide resistance by conjugation to both S. pyogenes and S. pneumoniae, showing the first evidence of the transferability of mega from S. pyogenes.

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 Å 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 ß-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.

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

The Neanderthal in the karst: First dating, morphometric, and paleogenetic data on the fossil skeleton from Altamura (Italy).

In 1993, a fossil hominin skeleton was discovered in the karst caves of Lamalunga, near Altamura, in southern Italy. Despite the fact that this specimen represents one of the most extraordinary hominin specimens ever found in Europe, for the last two decades our knowledge of it has been based purely on the documented on-site observations. Recently, the retrieval from the cave of a fragment of bone (part of the right scapula) allowed the first dating of the individual, the quantitative analysis of a diagnostic morphological feature, and a preliminary paleogenetic characterization of this hominin skeleton from Altamura. Overall, the results concur in indicating that it belongs to the hypodigm of Homo neanderthalensis, with some phenetic peculiarities that appear consistent with a chronology ranging from 172 ± 15 ka to 130.1 ± 1.9 ka. Thus, the skeleton from Altamura represents the most ancient Neanderthal from which endogenous DNA has ever been extracted.

APPRIS: annotation of principal and alternative splice isoforms.

Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform.

Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering.

Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer.

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, σ(70) (the housekeeping σ subunit) and σ(S) (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 σ(70) (Eσ(70)) or with σ(S) (Eσ(S)) using the whole Escherichia coli genome as DNA template. We found that Eσ(70), in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, Eσ(S) 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(S).

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.

A novel homozygous mutation in the TRDN gene causes a severe form of pediatric malignant ventricular arrhythmia.

BACKGROUND: Triadin is a protein expressed in cardiac and skeletal muscle that has an essential role in the structure and functional regulation of calcium release units and excitation-contraction coupling. Mutations in the triadin gene (TRDN) have been described in different forms of human arrhythmia syndromes with early onset and severe arrhythmogenic phenotype, including triadin knockout syndrome. OBJECTIVE: The purpose of this study was to characterize the pathogenetic mechanism underlying a case of severe pediatric malignant arrhythmia associated with a defect in the TRDN gene. METHODS: We used a trio whole exome sequencing approach to identify the genetic defect in a 2-year-old boy who had been resuscitated from sudden cardiac arrest and had frequent episodes of ventricular fibrillation and a family history positive for sudden death. We then performed in vitro functional analysis to investigate possible pathogenic mechanisms underlying this severe phenotype. RESULTS: We identified a novel homozygous missense variant (p.L56P) in the TRDN gene in the proband that was inherited from the heterozygous unaffected parents. Expression of a green fluorescent protein (GFP)-tagged mutant human cardiac triadin isoform (TRISK32-L56P-GFP) in heterologous systems revealed that the mutation alters protein dynamics. Furthermore, when co-expressed with the type 2 ryanodine receptor, caffeine-induced calcium release from TRISK32-L56P-GFP was relatively lower compared to that observed with the wild-type construct. CONCLUSION: The results of this study allowed us to hypothesize a pathogenic mechanism underlying this rare arrhythmogenic recessive form, suggesting that the mutant protein potentially can trigger arrhythmias by altering calcium homeostasis.

Rare Pathogenic Variants Predispose to Hepatocellular Carcinoma in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a rising cause of hepatocellular carcinoma (HCC). We examined whether inherited pathogenic variants in candidate genes (n = 181) were enriched in patients with NAFLD-HCC. To this end, we resequenced peripheral blood DNA of 142 NAFLD-HCC, 59 NAFLD with advanced fibrosis, and 50 controls, and considered 404 healthy individuals from 1000 G. Pathogenic variants were defined according to ClinVar, likely pathogenic as rare variants predicted to alter protein activity. In NAFLD-HCC patients, we detected an enrichment in pathogenic (p = 0.024), and likely pathogenic variants (p = 1.9*10-6), particularly in APOB (p = 0.047). APOB variants were associated with lower circulating triglycerides and higher HDL cholesterol (p < 0.01). A genetic risk score predicted NAFLD-HCC (OR 4.96, 3.29-7.55; p = 5.1*10-16), outperforming the diagnostic accuracy of common genetic risk variants, and of clinical risk factors (p < 0.05). In conclusion, rare pathogenic variants in genes involved in liver disease and cancer predisposition are associated with NAFLD-HCC development.

A compendium of long non-coding RNAs transcriptional fingerprint in multiple myeloma.

Multiple myeloma (MM) is a clonal proliferation of bone marrow plasma cells characterized by highly heterogeneous genetic background and clinical course, whose pathogenesis remains largely unknown. Long ncRNAs (lncRNAs) are a large class of non-protein-coding RNA, involved in many physiological cellular and genomic processes as well as in carcinogenesis and tumor evolution. Although still in its infancy, the role of lncRNAs in MM is progressively expanding. Besides studies on selected candidates, lncRNAs expression at genome-wide transcriptome level is confined to microarray technologies, thus investigating a limited collection of transcripts. In the present study investigating a cohort of 30 MM patients, a deep RNA-sequencing analysis overwhelmed previous array studies and allowed the most accurate definition of lncRNA transcripts structure and expression, ultimately providing a comprehensive catalogue of lncRNAs specifically associated with the main MM molecular subgroups and genetic alterations. Despite the small number of analyzed samples, the high accuracy of RNA-sequencing approach for complex transcriptome processing led to the identification of 391 deregulated lncRNAs, 67% of which were also detectable and validated by whole-transcript microarrays. In addition, we identified a list of lncRNAs, with potential relevance in MM, co-expressed and in close proximity to genes that might undergo a cis-regulatory relationship.

Telomerase reverse transcriptase germline mutations and hepatocellular carcinoma in patients with nonalcoholic fatty liver disease.

In an increasing proportion of cases, hepatocellular carcinoma (HCC) develops in patients with nonalcoholic fatty liver disease (NAFLD). Mutations in telomerase reverse transcriptase (hTERT) are associated with familial liver diseases. The aim of this study was to examine telomere length and germline hTERT mutations as associated with NAFLD-HCC. In 40 patients with NAFLD-HCC, 45 with NAFLD-cirrhosis and 64 healthy controls, peripheral blood telomere length was evaluated by qRT-PCR and hTERT coding regions and intron-exon boundaries sequenced. We further analyzed 78 patients affected by primary liver cancer (NAFLD-PLC, 76 with HCC). Enrichment of rare coding mutations (allelic frequency <0.001) was evaluated by Burden test. Functional consequences were estimated in silico and by over-expressing protein variants in HEK-293 cells. We found that telomere length was reduced in individuals with NAFLD-HCC versus those with cirrhosis (P = 0.048) and healthy controls (P = 0.0006), independently of age and sex. We detected an enrichment of hTERT mutations in NAFLD-HCC, that was confirmed when we further considered a larger cohort of NAFLD-PLC, and was more marked in female patients (P = 0.03). No mutations were found in cirrhosis and local controls, and only one in 503 healthy Europeans from the 1000 Genomes Project (allelic frequency = 0.025 vs. <0.001; P = 0.0005). Mutations with predicted functional impact, including the frameshift Glu113Argfs*79 and missense Glu668Asp, cosegregated with liver disease in two families. Three patients carried missense mutations (Ala67Val in homozygosity, Pro193Leu and His296Pro in heterozygosity) in the N-terminal template-binding domain (P = 0.037 for specific enrichment). Besides Glu668Asp, the Ala67Val variant resulted in reduced intracellular protein levels. In conclusion, we detected an association between shorter telomeres in peripheral blood and rare germline hTERT mutations and NAFLD-HCC.

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/ß-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.

Glucose availability enhances lipopolysaccharide production and immunogenicity in the opportunistic pathogen Acinetobacter baumannii.

AIM: Acinetobacter baumannii can cause sepsis with high mortality rates. We investigated whether glucose sensing might play a role in A. baumannii pathogenesis. MATERIALS & METHODS: We carried out transcriptome analysis and extracellular polysaccharide determination in an A. baumannii clinical isolate grown on complex medium with or without glucose supplementation, and assessed its ability to induce production of inflammatory cytokines in human macrophages. RESULTS: Growth in glucose-supplemented medium strongly enhanced A. baumannii sugar anabolism, resulting in increasing lipopolysaccharide biosynthesis. In addition, glucose induced active shedding of lipopolysaccharide, in turn triggering a strong induction of inflammatory cytokines in human macrophages. Finally, hemolytic activity was strongly enhanced by growth in glucose-supplemented medium. CONCLUSION: We propose that sensing of exogenous glucose might trigger A. baumannii pathogenesis during sepsis.

Whole-exome sequencing of primary plasma cell leukemia discloses heterogeneous mutational patterns.

Primary plasma cell leukemia (pPCL) is a rare and aggressive form of plasma cell dyscrasia and may represent a valid model for high-risk multiple myeloma (MM). To provide novel information concerning the mutational profile of this disease, we performed the whole-exome sequencing of a prospective series of 12 pPCL cases included in a Phase II multicenter clinical trial and previously characterized at clinical and molecular levels. We identified 1, 928 coding somatic non-silent variants on 1, 643 genes, with a mean of 166 variants per sample, and only few variants and genes recurrent in two or more samples. An excess of C > T transitions and the presence of two main mutational signatures (related to APOBEC over-activity and aging) occurring in different translocation groups were observed. We identified 14 candidate cancer driver genes, mainly involved in cell-matrix adhesion, cell cycle, genome stability, RNA metabolism and protein folding. Furthermore, integration of mutation data with copy number alteration profiles evidenced biallelically disrupted genes with potential tumor suppressor functions. Globally, cadherin/Wnt signaling, extracellular matrix and cell cycle checkpoint resulted the most affected functional pathways. Sequencing results were finally combined with gene expression data to better elucidate the biological relevance of mutated genes. This study represents the first whole-exome sequencing screen of pPCL and evidenced a remarkable genetic heterogeneity of mutational patterns. This may provide a contribution to the comprehension of the pathogenetic mechanisms associated with this aggressive form of PC dyscrasia and potentially with high-risk MM.

Enly: Improving Draft Genomes through Reads Recycling.

The reconstruction of the complete genome sequence of an organism is an important point for comparative, functional and evolutionary genomics. Nevertheless, overcoming the problems encountered while completing the sequence of an entire genome can still be demanding in terms of time and resources. We have developed Enly, a simple tool based on the iterative mapping of sequence reads at contig edges, capable to extend the genomic contigs deriving from high-throughput sequencing, especially those deriving by Newbler-like assemblies. Testing it on a set of de novo draft genomes led to the closure of up to 20% of the gaps originally present. Enly is cross-platform and most of the steps of its pipeline are parallelizable, making easy and fast to improve a draft genome resulting from a de novo assembly.