SLMSuite: a suite of algorithms for segmenting genomic profiles.

BACKGROUND: The identification of copy number variants (CNVs) is essential to study human genetic variation and to understand the genetic basis of mendelian disorders and cancers. At present, genome-wide detection of CNVs can be achieved using microarray or second generation sequencing (SGS) data. Although these technologies are very different, the genomic profiles that they generate are mathematically very similar and consist of noisy signals in which a decrease or increase of consecutive data represent deletions or duplication of DNA. In this framework, the most important step of the analysis consists of segmenting genomic profiles for the identification of the boundaries of genomic regions with increased or decreased signal. RESULTS: Here we introduce SLMSuite, a collection of algorithms, based on shifting level models (SLM), to segment genomic profiles from array and SGS experiments. The SLM algorithms take as input the log-transformed genomic profiles from SGS or microarray experiments and output segmentation results. We apply our method to the analysis of synthetic genomic profiles and real whole genome sequencing data and we demonstrate that it outperforms the state of the art circular binary segmentation algorithm in terms of sensitivity, specificity and computational speed. CONCLUSION: The SLMSuite contains an R library with the segmentation methods and three wrappers that allow to use them in Python, Ruby and C++. SLMSuite is freely available at https://sourceforge.net/projects/slmsuite .

The pangenome of (Antarctic) Pseudoalteromonas bacteria: evolutionary and functional insights.

BACKGROUND: Pseudoalteromonas is a genus of ubiquitous marine bacteria used as model organisms to study the biological mechanisms involved in the adaptation to cold conditions. A remarkable feature shared by these bacteria is their ability to produce secondary metabolites with a strong antimicrobial and antitumor activity. Despite their biotechnological relevance, representatives of this genus are still lacking (with few exceptions) an extensive genomic characterization, including features involved in the evolution of secondary metabolites production. Indeed, biotechnological applications would greatly benefit from such analysis. RESULTS: Here, we analyzed the genomes of 38 strains belonging to different Pseudoalteromonas species and isolated from diverse ecological niches, including extreme ones (i.e. Antarctica). These sequences were used to reconstruct the largest Pseudoalteromonas pangenome computed so far, including also the two main groups of Pseudoalteromonas strains (pigmented and not pigmented strains). The downstream analyses were conducted to describe the genomic diversity, both at genus and group levels. This allowed highlighting a remarkable genomic heterogeneity, even for closely related strains. We drafted all the main evolutionary steps that led to the current structure and gene content of Pseudoalteromonas representatives. These, most likely, included an extensive genome reduction and a strong contribution of Horizontal Gene Transfer (HGT), which affected biotechnologically relevant gene sets and occurred in a strain-specific fashion. Furthermore, this study also identified the genomic determinants related to some of the most interesting features of the Pseudoalteromonas representatives, such as the production of secondary metabolites, the adaptation to cold temperatures and the resistance to abiotic compounds. CONCLUSIONS: This study poses the bases for a comprehensive understanding of the evolutionary trajectories followed in time by this peculiar bacterial genus and for a focused exploitation of their biotechnological potential.

The genetic and clinical spectrum of a large cohort of patients with distal renal tubular acidosis.

Primary distal renal tubular acidosis is a rare genetic disease. Mutations in SLC4A1, ATP6V0A4, and ATP6V1B1 genes have been described as the cause of the disease, transmitted as either an autosomal dominant or recessive trait. Particular clinical features, such as sensorineural hearing loss, have been mainly described in association with mutations in one gene instead of the others. Nevertheless, the diagnosis of distal renal tubular acidosis is essentially based on clinical and laboratory findings, and the series of patients described so far are usually represented by small cohorts. Therefore, a strict genotype-phenotype correlation is still lacking, and questions about whether clinical and laboratory data should direct the genetic analysis remain open. Here, we applied next-generation sequencing in 89 patients with a clinical diagnosis of distal renal tubular acidosis, analyzing the prevalence of genetic defects in SLC4A1, ATP6V0A4, and ATP6V1B1 genes and the clinical phenotype. A genetic cause was determined in 71.9% of cases. In our group of sporadic cases, clinical features, including sensorineural hearing loss, are not specific indicators of the causal underlying gene. Mutations in the ATP6V0A4 gene are quite as frequent as mutations in ATP6V1B1 in patients with recessive disease. Chronic kidney disease was frequent in patients with a long history of the disease. Thus, our results suggest that when distal renal tubular acidosis is suspected, complete genetic testing could be considered, irrespective of the clinical phenotype of the patient.

Origin, duplication and reshuffling of plasmid genes: Insights from Burkholderia vietnamiensis G4 genome.

Using a computational pipeline based on similarity networks reconstruction we analysed the 1133 genes of the Burkholderia vietnamiensis (Bv) G4 five plasmids, showing that gene and operon duplication played an important role in shaping the plasmid architecture. Several single/multiple duplications occurring at intra- and/or interplasmids level involving 253 paralogous genes (stand-alone, clustered or operons) were detected. An extensive gene/operon exchange between plasmids and chromosomes was also disclosed. The larger the plasmid, the higher the number and size of paralogous fragments. Many paralogs encoded mobile genetic elements and duplicated very recently, suggesting that the rearrangement of the Bv plastic genome is ongoing. Concerning the "molecular habitat" and the "taxonomical status" (the Preferential Organismal Sharing) of Bv plasmid genes, most of them have been exchanged with other plasmids of bacteria belonging (or phylogenetically very close) to Burkholderia, suggesting that taxonomical proximity of bacterial strains is a crucial issue in plasmid-mediated gene exchange.

Phenotypic and genomic characterization of the Antarctic bacterium Gillisia sp. CAL575, a producer of antimicrobial compounds.

Microorganisms from Antarctica have evolved particular strategies to cope with cold. Moreover, they have been recently reported as producers of antimicrobial compounds, which inhibit the growth of other bacteria. In this work we characterized from different viewpoints the Gillisia sp. CAL575 strain, a psychrotrophic bacterium that produces microbial volatile organic compounds involved in the growth inhibition of Burkholderia cepacia complex members. Sequencing and analysis of the whole genome of Gillisia sp. CAL575 revealed that it includes genes that are involved in secondary metabolite production, adaptation to cold conditions, and different metabolic pathways for the production of energy. All these features make Gillisia sp. CAL575 a possible tool for biotechnology.

Draft genome sequence of the hydrocarbon-degrading and emulsan-producing strain Acinetobacter venetianus RAG-1T.

We report the draft genome sequence of Acinetobacter venetianus strain RAG-1(T), which is able to degrade hydrocarbons and to synthesize a powerful biosurfactant (emulsan) that can be employed for oil removal and as an adjuvant for vaccine delivery. The genome sequence of A. venetianus RAG-1(T) might be useful for bioremediation and/or clinical purposes.

Draft genome sequence of the volatile organic compound-producing Antarctic bacterium Arthrobacter sp. strain TB23, able to inhibit cystic fibrosis pathogens belonging to the Burkholderia cepacia complex.

Arthrobacter sp. strain TB23 was isolated from the Antarctic sponge Lissodendoryx nobilis. This bacterium is able to produce antimicrobial compounds and volatile organic compounds (VOCs) that inhibit the growth of other Antarctic bacteria and of cystic fibrosis opportunistic pathogens, respectively. Here we report the draft genome sequence of Arthrobacter sp. TB23.

Genome of the Komodo dragon reveals adaptations in the cardiovascular and chemosensory systems of monitor lizards.

Monitor lizards are unique among ectothermic reptiles in that they have high aerobic capacity and distinctive cardiovascular physiology resembling that of endothermic mammals. Here, we sequence the genome of the Komodo dragon Varanus komodoensis, the largest extant monitor lizard, and generate a high-resolution de novo chromosome-assigned genome assembly for V. komodoensis using a hybrid approach of long-range sequencing and single-molecule optical mapping. Comparing the genome of V. komodoensis with those of related species, we find evidence of positive selection in pathways related to energy metabolism, cardiovascular homoeostasis, and haemostasis. We also show species-specific expansions of a chemoreceptor gene family related to pheromone and kairomone sensing in V. komodoensis and other lizard lineages. Together, these evolutionary signatures of adaptation reveal the genetic underpinnings of the unique Komodo dragon sensory and cardiovascular systems, and suggest that selective pressure altered haemostasis genes to help Komodo dragons evade the anticoagulant effects of their own saliva. The Komodo dragon genome is an important resource for understanding the biology of monitor lizards and reptiles worldwide.

Xome-Blender: A novel cancer genome simulator.

The adoption of next generation sequencing based methods in cancer research allowed for the investigation of the complex genetic structure of tumor samples. In the last few years, considerable importance was given to the research of somatic variants and several computational approaches were developed for this purpose. Despite continuous improvements to these programs, the validation of their results it's a hard challenge due to multiple sources of error. To overcome this drawback different simulation approaches are used to generate synthetic samples but they are often based on the addition of artificial mutations that mimic the complexity of genomic variations. For these reasons, we developed a novel software, Xome-Blender, that generates synthetic cancer genomes with user defined features such as the number of subclones, the number of somatic variants and the presence of copy number alterations (CNAs), without the addition of any synthetic element. The singularity of our method is the "morphological approach" used to generate mutation events. To demonstrate the power of our tool we used it to address the hard challenge of evaluating the performance of nine state-of-the-art somatic variant calling methods for small and large variants (VarScan2, MuTect, Shimmer, BCFtools, Strelka, EXCAVATOR2, Control-FREEC and CopywriteR). Through these analyses we observed that by using Xome-Blender data it is possible to appraise small differences between their performance and we have designated VarScan2 and EXCAVATOR2 as best tool for this kind of applications. Xome-Blender is unix-based, licensed under the GPLv3 and freely available at https://github.com/rsemeraro/XomeBlender.

Genomic and phenotypic characterization of the species Acinetobacter venetianus.

Crude oil is a complex mixture of hydrocarbons and other organic compounds that can produce serious environmental problems and whose removal is highly demanding in terms of human and technological resources. The potential use of microbes as bioremediation agents is one of the most promising fields in this area. Members of the species Acinetobacter venetianus have been previously characterized for their capability to degrade n-alkanes and thus may represent interesting model systems to implement this process. Although a preliminary experimental characterization of the overall hydrocarbon degradation capability has been performed for five of them, to date, the genetic/genomic features underlying such molecular processes have not been identified. Here we have integrated genomic and phenotypic information for six A. venetianus strains, i.e. VE-C3, RAG-1(T), LUH 13518, LUH 7437, LUH 5627 and LUH 8758. Besides providing a thorough description of the A. venetianus species, these data were exploited to infer the genetic features (presence/absence patterns of genes) and the short-term evolutionary events possibly responsible for the variability in n-alkane degradation efficiency of these strains, including the mechanisms of interaction with the fuel droplet and the subsequent catabolism of this pollutant.

Methods for assembling reads and producing contigs.

Determining the genome sequence of an organism is often the first step towards its molecular characterization. Once a difficult and expensive task, nowadays it is an almost routine practice in many molecular biology labs. In this chapter we discuss in depth the various methods to assemble the short sequences (called reads) obtained from a massive sequencing system, using different software and strategies, and how to perform some fundamental quality controls on the data obtained.

Network Analysis of Plasmidomes: The Azospirillum brasilense Sp245 Case.

Azospirillum brasilense is a nitrogen-fixing bacterium living in association with plant roots. The genome of the strain Sp245, isolated in Brazil from wheat roots, consists of one chromosome and six plasmids. In this work, the A. brasilense Sp245 plasmids were analyzed in order to shed some light on the evolutionary pathways they followed over time. To this purpose, a similarity network approach was applied in order to identify the evolutionary relationships among all the A. brasilense plasmids encoded proteins; in this context a computational pipeline specifically devoted to the analysis and the visualization of the network-like evolutionary relationships among different plasmids molecules was developed. This information was supplemented with a detailed (in silico) functional characterization of both the connected (i.e., sharing homology with other sequences in the dataset) and the unconnected (i.e., not sharing homology) components of the network. Furthermore, the most likely source organism for each of the genes encoded by A. brasilense plasmids was checked, allowing the identification of possible trends of gene loss/gain in this microorganism. Data obtained provided a detailed description of the evolutionary landscape of the plasmids of A. brasilense Sp245, suggesting some of the molecular mechanisms responsible for the present-day structure of these molecules.

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.

Genomic analysis of three sponge-associated Arthrobacter Antarctic strains, inhibiting the growth of Burkholderia cepacia complex bacteria by synthesizing volatile organic compounds.

In this work we analyzed the ability of three Arthrobacter strains (namely TB23, TB26 and CAL618), which were isolated from the Antarctic sponges Haliclonissa verrucosa and Lyssodendrix nobilis, to specifically inhibit the growth of a panel of 40 Burkholderia cepacia complex strains, representing a major cause of infections in patients that are affected by Cystic Fibrosis. The inhibitory activity was due to the synthesis of antimicrobial compounds, very likely volatile organic compounds (VOCs), and was partially dependent on the growth media that were used for Antarctic strains growth. The phylogenetic analysis revealed that two of them (i.e. CAL 618 and TB23) were very close and very likely belonged to the same Arthrobacter species, whereas the strain TB26 was placed in a distant branch. The genome of the strains TB26 and CAL618 was also sequenced and compared with that of the strain TB23. The analysis revealed that TB23 and CAL618 shared more genomic properties (GC content, genome size, number of genes) than with TB26. Since the three strains exhibited very similar inhibition pattern vs Bcc strains, it is quite possible that genes involved in the biosynthesis of antimicrobial compounds very likely belong to the core genome.

Draft genomes of three Antarctic Psychrobacter strains producing antimicrobial compounds against Burkholderia cepacia complex, opportunistic human pathogens.

Herein we present the draft genomes of three Psychrobacter strains isolated from Antarctic sponges and able to inhibit the growth of bacteria belonging to the Burkholderia cepacia complex, responsible for infections of the respiratory system in patients affected by Cystic Fibrosis. The comparative analysis of the annotated genomes of these Psychrobacter strains highlighted their differences in terms of overall genomic content (e.g. shared gene sets) and allowed the identification of gene clusters hypothetically involved in the biosynthesis of antimicrobial compounds.

Draft Genome Sequence of the Fast-Growing Bacterium Vibrio natriegens Strain DSMZ 759.

Vibrio natriegens is a Gram-negative bacterium known for its extremely short doubling time. Here we present the annotated draft genome sequence of Vibrio natriegens strain DSMZ 759, with the aim of providing insights about its high growth rate.

The genome sequence of the hydrocarbon-degrading Acinetobacter venetianus VE-C3.

Here we report the genome sequence of Acinetobacter venetianus VE-C3, a strain isolated from the Venice Lagoon and known to be able to degrade n-alkanes. Post sequencing analyses revealed that this strain is relatively distantly related to the other Acinetobacter strains completely sequenced so far as shown by phylogenetic analysis and pangenome analysis (1285 genes shared with all the other Acinetobacter genomes sequenced so far). A. venetianus VE-C3 possesses a wide range of determinants whose molecular functions are probably related to the survival in a strongly impacted ecological niche. Among them, genes probably involved in the metabolism of long-chain n-alkanes and in the resistance to toxic metals (e.g. arsenic, cadmium, cobalt and zinc) were found. Genes belonging to these processes were found both on the chromosome and on plasmids. Also, our analysis documented one of the possible genetic bases underlying the strategy adopted by A. venetianus VE-C3 for the adhesion to oil fuel droplets, which could account for the differences existing in this process with other A. venetianus strains. Finally, the presence of a number of DNA mobilization-related genes (i.e. transposases, integrases, resolvases) strongly suggests an important role played by horizontal gene transfer in shaping the genome of A. venetianus VE-C3 and in its adaptation to its special ecological niche.

Bioactive volatile organic compounds from Antarctic (sponges) bacteria.

Antarctic bacteria represent a reservoir of unexplored biodiversity, which, in turn, might be correlated to the synthesis of still undescribed bioactive molecules, such as antibiotics. In this work we have further characterized a panel of four marine Antarctic bacteria able to inhibit the growth of human opportunistic multiresistant pathogenic bacteria belonging to the Burkholderia cepacia complex (responsible for the 'cepacia' syndrome in Cystic Fibrosis patients) through the production of a set of microbial Volatile Organic Compounds (mVOCs). A list of 30 different mVOCs synthesized under aerobic conditions by Antarctic bacteria was identified by GC-SPME analysis. Cross-streaking experiments suggested that Antarctic bacteria might also synthesize non-volatile molecules able to enhance the anti-Burkholderia activity. The biosynthesis of such a mixture of mVOCs was very probably influenced by both the presence/absence of oxygen and the composition of media used to grow the Antarctic strains. The antimicrobial activity exhibited by Antarctic strains also appeared to be more related to their taxonomical position rather than to the sampling site. Different Bcc bacteria were differently sensitive to the 'Antarctic' mVOCs and this was apparently related neither to the taxonomical position of the different strains nor to their source. The genome sequence of three new Antarctic strains was determined revealing that only P. atlantica TB41 possesses some genes belonging to the nrps-pks cluster. The comparative genomic analysis performed on the genome of the four strains also revealed the presence of a few genes belonging to the core genome and involved in the secondary metabolites biosynthesis. Data obtained suggest that the antimicrobial activity exhibited by Antarctic bacteria might rely on a (complex) mixture of mVOCs whose relative concentration may vary depending on the growth conditions. Besides, it is also possible that the biosynthesis of these compounds might occur through still unknown metabolic pathways.