Identification of horizontally transferred genes in the genus Colletotrichum reveals a steady tempo of bacterial to fungal gene transfer

Background

Horizontal gene transfer (HGT) is the stable transmission of genetic material between organisms by means other than vertical inheritance. HGT has an important role in the evolution of prokaryotes but is relatively rare in eukaryotes. HGT has been shown to contribute to virulence in eukaryotic pathogens. We studied the importance of HGT in plant pathogenic fungi by identifying horizontally transferred genes in the genomes of three members of the genus Colletotrichum.

Results

We identified eleven HGT events from bacteria into members of the genus Colletotrichum or their ancestors. The HGT events include genes involved in amino acid, lipid and sugar metabolism as well as lytic enzymes. Additionally, the putative minimal dates of transference were calculated using a time calibrated phylogenetic tree. This analysis reveals a constant flux of genes from bacteria to fungi throughout the evolution of subphylum Pezizomycotina.

Conclusions

Genes that are typically transferred by HGT are those that are constantly subject to gene duplication and gene loss. The functions of some of these genes suggest roles in niche adaptation and virulence. We found no evidence of a burst of HGT events coinciding with major geological events. In contrast, HGT appears to be a constant, albeit rare phenomenon in the Pezizomycotina, occurring at a steady rate during their evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-16-2) contains supplementary material, which is available to authorized users.     

aTRAM - automated target restricted assembly method: a fast method for assembling loci across divergent taxa from next-generation sequencing data

Background

Assembling genes from next-generation sequencing data is not only time consuming but computationally difficult, particularly for taxa without a closely related reference genome. Assembling even a draft genome using de novo approaches can take days, even on a powerful computer, and these assemblies typically require data from a variety of genomic libraries. Here we describe software that will alleviate these issues by rapidly assembling genes from distantly related taxa using a single library of paired-end reads: aTRAM, automated Target Restricted Assembly Method. The aTRAM pipeline uses a reference sequence, BLAST, and an iterative approach to target and locally assemble the genes of interest.

Results

Our results demonstrate that aTRAM rapidly assembles genes across distantly related taxa. In comparative tests with a closely related taxon, aTRAM assembled the same sequence as reference-based and de novo approaches taking on average < 1 min per gene. As a test case with divergent sequences, we assembled >1,000 genes from six taxa ranging from 25 – 110 million years divergent from the reference taxon. The gene recovery was between 97 – 99% from each taxon.

Conclusions

aTRAM can quickly assemble genes across distantly-related taxa, obviating the need for draft genome assembly of all taxa of interest. Because aTRAM uses a targeted approach, loci can be assembled in minutes depending on the size of the target. Our results suggest that this software will be useful in rapidly assembling genes for phylogenomic projects covering a wide taxonomic range, as well as other applications. The software is freely available http://www.github.com/juliema/aTRAM.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0515-2) contains supplementary material, which is available to authorized users.     

Horizontal gene transfer in an acid mine drainage microbial community

Background

Horizontal gene transfer (HGT) has been widely identified in complete prokaryotic genomes. However, the roles of HGT among members of a microbial community and in evolution remain largely unknown. With the emergence of metagenomics, it is nontrivial to investigate such horizontal flow of genetic materials among members in a microbial community from the natural environment. Because of the lack of suitable methods for metagenomics gene transfer detection, microorganisms from a low-complexity community acid mine drainage (AMD) with near-complete genomes were used to detect possible gene transfer events and suggest the biological significance.

Results

Using the annotation of coding regions by the current tools, a phylogenetic approach, and an approximately unbiased test, we found that HGTs in AMD organisms are not rare, and we predicted 119 putative transferred genes. Among them, 14 HGT events were determined to be transfer events among the AMD members. Further analysis of the 14 transferred genes revealed that the HGT events affected the functional evolution of archaea or bacteria in AMD, and it probably shaped the community structure, such as the dominance of G-plasma in archaea in AMD through HGT.

Conclusions

Our study provides a novel insight into HGT events among microorganisms in natural communities. The interconnectedness between HGT and community evolution is essential to understand microbial community formation and development.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1720-0) contains supplementary material, which is available to authorized users.     

PhyTB: Phylogenetic tree visualisation and sample positioning for M. tuberculosis

Background

Phylogenetic-based classification of M. tuberculosis and other bacterial genomes is a core analysis for studying evolutionary hypotheses, disease outbreaks and transmission events. Whole genome sequencing is providing new insights into the genomic variation underlying intra- and inter-strain diversity, thereby assisting with the classification and molecular barcoding of the bacteria. One roadblock to strain investigation is the lack of user-interactive solutions to interrogate and visualise variation within a phylogenetic tree setting.

Results

We have developed a web-based tool called PhyTB (http://pathogenseq.lshtm.ac.uk/phytblive/index.php) to assist phylogenetic tree visualisation and identification of M. tuberculosis clade-informative polymorphism. Variant Call Format files can be uploaded to determine a sample position within the tree. A map view summarises the geographical distribution of alleles and strain-types. The utility of the PhyTB is demonstrated on sequence data from 1,601 M. tuberculosis isolates.

Conclusion

PhyTB contextualises M. tuberculosis genomic variation within epidemiological, geographical and phylogenic settings. Further tool utility is possible by incorporating large variants and phenotypic data (e.g. drug-resistance profiles), and an assessment of genotype-phenotype associations. Source code is available to develop similar websites for other organisms (http://sourceforge.net/projects/phylotrack).     

Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

Background

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.

Results

Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.

Conclusions

We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1135) contains supplementary material, which is available to authorized users.     

Detection of internal exon deletion with exon Del

Background

Exome sequencing allows researchers to study the human genome in unprecedented detail. Among the many types of variants detectable through exome sequencing, one of the most over looked types of mutation is internal deletion of exons. Internal exon deletions are the absence of consecutive exons in a gene. Such deletions have potentially significant biological meaning, and they are often too short to be considered copy number variation. Therefore, to the need for efficient detection of such deletions using exome sequencing data exists.

Results

We present ExonDel, a tool specially designed to detect homozygous exon deletions efficiently. We tested ExonDel on exome sequencing data generated from 16 breast cancer cell lines and identified both novel and known IEDs. Subsequently, we verified our findings using RNAseq and PCR technologies. Further comparisons with multiple sequencing-based CNV tools showed that ExonDel is capable of detecting unique IEDs not found by other CNV tools.

Conclusions

ExonDel is an efficient way to screen for novel and known IEDs using exome sequencing data. ExonDel and its source code can be downloaded freely at https://github.com/slzhao/ExonDel.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-332) contains supplementary material, which is available to authorized users.     

Linear-time computation of minimal absent words using suffix array

Background

An absent word of a word y of length n is a word that does not occur in y. It is a minimal absent word if all its proper factors occur in y. Minimal absent words have been computed in genomes of organisms from all domains of life; their computation also provides a fast alternative for measuring approximation in sequence comparison. There exists an ??(n)-time and ??(n)-space algorithm for computing all minimal absent words on a fixed-sized alphabet based on the construction of suffix automata (Crochemore et al., 1998). No implementation of this algorithm is publicly available. There also exists an ??(n²)-time and ??(n)-space algorithm for the same problem based on the construction of suffix arrays (Pinho et al., 2009). An implementation of this algorithm was also provided by the authors and is currently the fastest available.

Results

Our contribution in this article is twofold: first, we bridge this unpleasant gap by presenting an ??(n)-time and ??(n)-space algorithm for computing all minimal absent words based on the construction of suffix arrays; and second, we provide the respective implementation of this algorithm. Experimental results, using real and synthetic data, show that this implementation outperforms the one by Pinho et al. The open-source code of our implementation is freely available at http://github.com/solonas13/maw.

Conclusions

Classical notions for sequence comparison are increasingly being replaced by other similarity measures that refer to the composition of sequences in terms of their constituent patterns. One such measure is the minimal absent words. In this article, we present a new linear-time and linear-space algorithm for the computation of minimal absent words based on the suffix array.     

MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems

Motivation

Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose.

Results

Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. We show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles.

Availability and Implementation

MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfinder. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information.     

The Duplicated Genes Database: Identification and Functional Annotation of Co-Localised Duplicated Genes across Genomes

Background

There has been a surge in studies linking genome structure and gene expression, with special focus on duplicated genes. Although initially duplicated from the same sequence, duplicated genes can diverge strongly over evolution and take on different functions or regulated expression. However, information on the function and expression of duplicated genes remains sparse. Identifying groups of duplicated genes in different genomes and characterizing their expression and function would therefore be of great interest to the research community. The ‘Duplicated Genes Database’ (DGD) was developed for this purpose.

Methodology

Nine species were included in the DGD. For each species, BLAST analyses were conducted on peptide sequences corresponding to the genes mapped on a same chromosome. Groups of duplicated genes were defined based on these pairwise BLAST comparisons and the genomic location of the genes. For each group, Pearson correlations between gene expression data and semantic similarities between functional GO annotations were also computed when the relevant information was available.

Conclusions

The Duplicated Gene Database provides a list of co-localised and duplicated genes for several species with the available gene co-expression level and semantic similarity value of functional annotation. Adding these data to the groups of duplicated genes provides biological information that can prove useful to gene expression analyses. The Duplicated Gene Database can be freely accessed through the DGD website at http://dgd.genouest.org.     

YOC,A new strategy for pairwise alignment of collinear genomes

Background

Comparing and aligning genomes is a key step in analyzing closely related genomes. Despite the development of many genome aligners in the last 15 years, the problem is not yet fully resolved, even when aligning closely related bacterial genomes of the same species. In addition, no procedures are available to assess the quality of genome alignments or to compare genome aligners.

Results

We designed an original method for pairwise genome alignment, named YOC, which employs a highly sensitive similarity detection method together with a recent collinear chaining strategy that allows overlaps. YOC improves the reliability of collinear genome alignments, while preserving or even improving sensitivity. We also propose an original qualitative evaluation criterion for measuring the relevance of genome alignments. We used this criterion to compare and benchmark YOC with five recent genome aligners on large bacterial genome datasets, and showed it is suitable for identifying the specificities and the potential flaws of their underlying strategies.

Conclusions

The YOC prototype is available at https://github.com/ruricaru/YOC. It has several advantages over existing genome aligners: (1) it is based on a simplified two phase alignment strategy, (2) it is easy to parameterize, (3) it produces reliable genome alignments, which are easier to analyze and to use.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0530-3) contains supplementary material, which is available to authorized users.     

tcR: an R package for T cell receptor repertoire advanced data analysis

Background

The Immunoglobulins (IG) and the T cell receptors (TR) play the key role in antigen recognition during the adaptive immune response. Recent progress in next-generation sequencing technologies has provided an opportunity for the deep T cell receptor repertoire profiling. However, a specialised software is required for the rational analysis of massive data generated by next-generation sequencing.

Results

Here we introduce tcR, a new R package, representing a platform for the advanced analysis of T cell receptor repertoires, which includes diversity measures, shared T cell receptor sequences identification, gene usage statistics computation and other widely used methods. The tool has proven its utility in recent research studies.

Conclusions

tcR is an R package for the advanced analysis of T cell receptor repertoires after primary TR sequences extraction from raw sequencing reads. The stable version can be directly installed from The Comprehensive R Archive Network (http://cran.r-project.org/mirrors.html). The source code and development version are available at tcR GitHub (http://imminfo.github.io/tcr/) along with the full documentation and typical usage examples.     

Identifying Cis-Regulatory Sequences by Word Profile Similarity

Background

Recognizing regulatory sequences in genomes is a continuing challenge, despite a wealth of available genomic data and a growing number of experimentally validated examples.

Methodology/Principal Findings

We discuss here a simple approach to search for regulatory sequences based on the compositional similarity of genomic regions and known cis-regulatory sequences. This method, which is not limited to searching for predefined motifs, recovers sequences known to be under similar regulatory control. The words shared by the recovered sequences often correspond to known binding sites. Furthermore, we show that although local word profile clustering is predictive for the regulatory sequences involved in blastoderm segmentation, local dissimilarity is a more universal feature of known regulatory sequences in Drosophila.

Conclusions/Significance

Our method leverages sequence motifs within a known regulatory sequence to identify co-regulated sequences without explicitly defining binding sites. We also show that regulatory sequences can be distinguished from surrounding sequences by local sequence dissimilarity, a novel feature in identifying regulatory sequences across a genome. Source code for WPH-finder is available for download at http://rana.lbl.gov/downloads/wph.tar.gz.     

An assembly and alignment-free method of phylogeny reconstruction from next-generation sequencing data

Background

Next-generation sequencing technologies are rapidly generating whole-genome datasets for an increasing number of organisms. However, phylogenetic reconstruction of genomic data remains difficult because de novo assembly for non-model genomes and multi-genome alignment are challenging.

Results

To greatly simplify the analysis, we present an Assembly and Alignment-Free (AAF) method (https://sourceforge.net/projects/aaf-phylogeny) that constructs phylogenies directly from unassembled genome sequence data, bypassing both genome assembly and alignment. Using mathematical calculations, models of sequence evolution, and simulated sequencing of published genomes, we address both evolutionary and sampling issues caused by direct reconstruction, including homoplasy, sequencing errors, and incomplete sequencing coverage. From these results, we calculate the statistical properties of the pairwise distances between genomes, allowing us to optimize parameter selection and perform bootstrapping. As a test case with real data, we successfully reconstructed the phylogeny of 12 mammals using raw sequencing reads. We also applied AAF to 21 tropical tree genome datasets with low coverage to demonstrate its effectiveness on non-model organisms.

Conclusion

Our AAF method opens up phylogenomics for species without an appropriate reference genome or high sequence coverage, and rapidly creates a phylogenetic framework for further analysis of genome structure and diversity among non-model organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1647-5) contains supplementary material, which is available to authorized users.     

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background

Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.

Results

We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.

Conclusions

This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.     

Oral Doxycycline Reduces Pterygium Lesions; Results from a Double Blind,Randomized, Placebo Controlled Clinical Trial

Purpose

To determine whether oral doxycycline treatment reduces pterygium lesions.

Design

Double blind, randomized, placebo controlled clinical trial.

Participants

98 adult patients with primary pterygium.

Methods

Patients were randomly assigned to receive 100 mg oral doxycycline twice a day (49 subjects), or placebo (49 subjects), for 30 days. Photographs of the lesion were taken at the time of recruitment and at the end of the treatment. Follow-up sessions were performed 6 and 12 months post-treatment. Statistical analyses for both continuous and categorical variables were applied. p values of less than 0.05 were considered to indicate statistical significance.

Main Outcome Measures

The primary endpoint was the change in lesion size after 30 days of treatment.

Results

The primary endpoint was not met for the whole population but subgroup analysis showed that doxycycline was effective in patients of Caucasian origin while other ethnicities, mostly Hispanic, did not respond to the treatment. Moreover, there was a correlation between age and better response (p = 0.003). Adverse events were uncommon, mild, and in agreement with previous reports on short doxycycline treatments.

Conclusions

Oral doxycycline was superior to placebo for the treatment of primary pterygia in older Caucasian patients. These findings support the use of doxycycline for pterygium treatment in particular populations.

Trial Registration

European Union Clinical Trials Register EudraCT 2008-007178-39     

Thymidylate Synthase Genotype-Directed Chemotherapy for Patients with Gastric and Gastroesophageal Junction Cancers

Background

Retrospective studies indicate associations between TSER (thymidylate synthase enhancer region) genotypes and clinical outcomes in patients receiving 5-FU based chemotherapy, but well-controlled prospective validation has been lacking.

Methods

In this phase II study ({"type":"clinical-trial","attrs":{"text":"NCT00515216","term_id":"NCT00515216"}}NCT00515216 registered through ClinicalTrials.gov, http://clinicaltrials.gov/show/{"type":"clinical-trial","attrs":{"text":"NCT00515216","term_id":"NCT00515216"}}NCT00515216), patients with “good risk” TSER genotypes (at least one TSER*2 allele) were treated with FOLFOX chemotherapy to determine whether prospective patient selection can improve overall response rates (ORR) in patients with gastric and gastroesophageal junction (GEJ) cancers, compared with historical outcomes in unselected patients (estimated 43%).

Results

The ORR in genotype-selected patients was 39.1% (9 partial responses out of 23 evaluable patients, 95% CI, 22.2 to 59.2), not achieving the primary objective of improving ORR. An encouraging disease control rate (DCR, consisting of partial responses and stable diseases) of 95.7% was noted and patients with homozygous TSER*2 genotype showed better tumor response.

Conclusions

In this first prospective, multi-institutional study in patients with gastric or GEJ cancers, selecting patients with at least one TSER*2 allele did not improve the ORR but led to an encouraging DCR. Further studies are needed to investigate the utility of selecting patients homozygous for the TSER*2 allele and additional genomic markers in improving clinical outcomes for patients with gastric and GEJ cancers.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00515216","term_id":"NCT00515216"}}NCT00515216