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July 7, 2019

Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing.

Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition.An observational prospective cohort study.Burns care ward and critical care ward in the UK.Patients with >7% total burns by surface area were recruited into the study.All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates.WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment.This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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July 7, 2019

The characterization of goat genetic diversity: Towards a genomic approach

The investigation of genetic diversity at molecular level has been proposed as a valuable complement and sometimes proxy to phenotypic diversity of local breeds and is presently considered as one of the FAO priorities for breed characterization. By recommending a set of selected molecular markers for each of the main livestock species, FAO has promoted the meta-analysis of local datasets, to achieve a global view of molecular genetic diversity. Analysis within the EU Globaldiv project of two large goat microsatellite datasets produced by the Econogene Consortium and the IAEA CRP–Asia Consortium, respectively, has generated a picture of goat diversity across continents. This indicates a gradient of decreasing diversity from the domestication centre towards Europe and Asia, a clear phylogeographic structure at the continental and regional levels, and in Asia a limited genetic differentiation among local breeds. The development of SNP panels that assay thousands of markers and the whole genome sequencing of livestock permit an affordable use of genomic technologies in all livestock species, goats included. Preliminary data from the Italian Goat Consortium indicate that the SNP panel developed for this species is highly informative. The existing panel can be improved by integrating additional SNPs identified from the whole genome sequence alignment of goats adapted to extreme climates. Part of this effort is being achieved by international projects (e.g. EU FP7 NextGen and 3SR projects), but a fair representation of the global diversity in goats requires a large panel of samples (i.e. as in the recently launched 1000 cattle genomes initiative). Genomic technologies offer new strategies to investigate complex traits difficult to measure. For example, the comparison of patterns of diversity among the genomes in selected groups of animals (e.g. adapted to different environments) and the integration of genome-wide diversity with new GIScience-based methods are able to identify molecular markers associated with genomic regions of putative importance in adaptation and thus pave the way for the identification of causative genes. Goat breeds adapted to different production systems in extreme and harsh environments will play an important role in this process. The new sequencing technologies also permit the analysis of the entire mitochondrial genome at maximum resolution. The complete mtDNA sequence is now the common standard format for the investigation of human maternal lineages. A preliminary analysis of the complete goat mtDNA genome supports a single Neolithic origin of domestic goats rather than multiple domestication events in different geographic areas.

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July 7, 2019

Diversification of bacterial genome content through distinct mechanisms over different timescales.

Bacterial populations often consist of multiple co-circulating lineages. Determining how such population structures arise requires understanding what drives bacterial diversification. Using 616 systematically sampled genomes, we show that Streptococcus pneumoniae lineages are typically characterized by combinations of infrequently transferred stable genomic islands: those moving primarily through transformation, along with integrative and conjugative elements and phage-related chromosomal islands. The only lineage containing extensive unique sequence corresponds to a set of atypical unencapsulated isolates that may represent a distinct species. However, prophage content is highly variable even within lineages, suggesting frequent horizontal transmission that would necessitate rapidly diversifying anti-phage mechanisms to prevent these viruses sweeping through populations. Correspondingly, two loci encoding Type I restriction-modification systems able to change their specificity over short timescales through intragenomic recombination are ubiquitous across the collection. Hence short-term pneumococcal variation is characterized by movement of phage and intragenomic rearrangements, with the slower transfer of stable loci distinguishing lineages.

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July 7, 2019

Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement.

Advances in modern sequencing technologies allow us to generate sufficient data to analyze hundreds of bacterial genomes from a single machine in a single day. This potential for sequencing massive numbers of genomes calls for fully automated methods to produce high-quality assemblies and variant calls. We introduce Pilon, a fully automated, all-in-one tool for correcting draft assemblies and calling sequence variants of multiple sizes, including very large insertions and deletions. Pilon works with many types of sequence data, but is particularly strong when supplied with paired end data from two Illumina libraries with small e.g., 180 bp and large e.g., 3-5 Kb inserts. Pilon significantly improves draft genome assemblies by correcting bases, fixing mis-assemblies and filling gaps. For both haploid and diploid genomes, Pilon produces more contiguous genomes with fewer errors, enabling identification of more biologically relevant genes. Furthermore, Pilon identifies small variants with high accuracy as compared to state-of-the-art tools and is unique in its ability to accurately identify large sequence variants including duplications and resolve large insertions. Pilon is being used to improve the assemblies of thousands of new genomes and to identify variants from thousands of clinically relevant bacterial strains. Pilon is freely available as open source software.

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July 7, 2019

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.

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July 7, 2019

ProbAlign: a re-alignment method for long sequencing reads

The incorrect alignments are a severe problem in variant calling, and remain as a challenge computational issue in Bioinformatics field. Although there have been some methods utilizing the re-alignment approach to tackle the misalignments, a standalone re-alignment tool for long sequencing reads is lacking. Hence, we present a standalone tool to correct the misalignments, called ProbAlign. It can be integrated into the pipelines of not only variant calling but also other genomic applications. We demonstrate the use of re-alignment in two diverse and important genomics fields: variant calling and viral quasispecies reconstruction. First, variant calling results in the Pacific Biosciences SMRT re-sequencing data of NA12878 show that false positives can be reduced by 43.5%, and true positives can be increased by 24.8% averagely, after re-alignment. Second, results in reconstructing a 5-virus-mix show that the viral population can be completely unraveled, and also the estimation of quasispecies frequencies has been improved, after re-alignment. ProbAlign is freely available in the PyroTools toolkit (https://github.com/homopolymer/PyroTools).

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July 7, 2019

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors.

Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid-a2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.

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July 7, 2019

An in vitro deletion in ribE encoding lumazine synthase contributes to nitrofurantoin resistance in Escherichia coli.

Nitrofurantoin has been used for decades for the treatment of urinary tract infections (UTIs), but clinically significant resistance in Escherichia coli is uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinal E. coli strains (ST540-p and ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 µg/ml) harbored mutations in the known nitrofurantoin resistance determinants nfsA and/or nfsB, which encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC of 32 µg/ml) and exhibited remarkable growth deficits but did not harbor nfsA/nfsB mutations. We identified a 12-nucleotide deletion in ribE, encoding lumazine synthase, an essential enzyme involved in the biosynthesis of flavin mononucleotide (FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-R E. coli isolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations in nfsA and/or nfsB but not ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced nitrofurantoin susceptibility (MICs of 16 to 48 µg/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generated in vitro, presumably because of additional mutations in oxygen-sensitive nitroreductases. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The challenges and importance of structural variation detection in livestock.

Recent studies in humans and other model organisms have demonstrated that structural variants (SVs) comprise a substantial proportion of variation among individuals of each species. Many of these variants have been linked to debilitating diseases in humans, thereby cementing the importance of refining methods for their detection. Despite progress in the field, reliable detection of SVs still remains a problem even for human subjects. Many of the underlying problems that make SVs difficult to detect in humans are amplified in livestock species, whose lower quality genome assemblies and incomplete gene annotation can often give rise to false positive SV discoveries. Regardless of the challenges, SV detection is just as important for livestock researchers as it is for human researchers, given that several productive traits and diseases have been linked to copy number variations (CNVs) in cattle, sheep, and pig. Already, there is evidence that many beneficial SVs have been artificially selected in livestock such as a duplication of the agouti signaling protein gene that causes white coat color in sheep. In this review, we will list current SV and CNV discoveries in livestock and discuss the problems that hinder routine discovery and tracking of these polymorphisms. We will also discuss the impacts of selective breeding on CNV and SV frequencies and mention how SV genotyping could be used in the future to improve genetic selection.

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July 7, 2019

The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line.

The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption–both intentionally and through widespread cross-contamination–and for the past 60?years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500?kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.

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July 7, 2019

Evolution and diversity of copy number variation in the great ape lineage.

Copy number variation (CNV) contributes to disease and has restructured the genomes of great apes. The diversity and rate of this process, however, have not been extensively explored among great ape lineages. We analyzed 97 deeply sequenced great ape and human genomes and estimate 16% (469 Mb) of the hominid genome has been affected by recent CNV. We identify a comprehensive set of fixed gene deletions (n = 340) and duplications (n = 405) as well as >13.5 Mb of sequence that has been specifically lost on the human lineage. We compared the diversity and rates of copy number and single nucleotide variation across the hominid phylogeny. We find that CNV diversity partially correlates with single nucleotide diversity (r(2) = 0.5) and recapitulates the phylogeny of apes with few exceptions. Duplications significantly outpace deletions (2.8-fold). The load of segregating duplications remains significantly higher in bonobos, Western chimpanzees, and Sumatran orangutans-populations that have experienced recent genetic bottlenecks (P = 0.0014, 0.02, and 0.0088, respectively). The rate of fixed deletion has been more clocklike with the exception of the chimpanzee lineage, where we observe a twofold increase in the chimpanzee-bonobo ancestor (P = 4.79 × 10(-9)) and increased deletion load among Western chimpanzees (P = 0.002). The latter includes the first genomic disorder in a chimpanzee with features resembling Smith-Magenis syndrome mediated by a chimpanzee-specific increase in segmental duplication complexity. We hypothesize that demographic effects, such as bottlenecks, have contributed to larger and more gene-rich segments being deleted in the chimpanzee lineage and that this effect, more generally, may account for episodic bursts in CNV during hominid evolution.

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July 7, 2019

In transition: primate genomics at a time of rapid change.

The field of nonhuman primate genomics is undergoing rapid change and making impressive progress. Exploiting new technologies for DNA sequencing, researchers have generated new whole-genome sequence assemblies for multiple primate species over the past 6 years. In addition, investigations of within-species genetic variation, gene expression and RNA sequences, conservation of non-protein-coding regions of the genome, and other aspects of comparative genomics are moving at an accelerating speed. This progress is opening a wide array of new research opportunities in the analysis of comparative primate genome content and evolution. It also creates new possibilities for the use of nonhuman primates as model organisms in biomedical research. This transition, based on both new technology and the new information being generated in regard to human genetics, provides an important justification for reevaluating the research goals, strategies, and study designs used in primate genetics and genomics.

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July 7, 2019

Haplotype assembly in polyploid genomes and identical by descent shared tracts.

Genome-wide haplotype reconstruction from sequence data, or haplotype assembly, is at the center of major challenges in molecular biology and life sciences. For complex eukaryotic organisms like humans, the genome is vast and the population samples are growing so rapidly that algorithms processing high-throughput sequencing data must scale favorably in terms of both accuracy and computational efficiency. Furthermore, current models and methodologies for haplotype assembly (i) do not consider individuals sharing haplotypes jointly, which reduces the size and accuracy of assembled haplotypes, and (ii) are unable to model genomes having more than two sets of homologous chromosomes (polyploidy). Polyploid organisms are increasingly becoming the target of many research groups interested in the genomics of disease, phylogenetics, botany and evolution but there is an absence of theory and methods for polyploid haplotype reconstruction.In this work, we present a number of results, extensions and generalizations of compass graphs and our HapCompass framework. We prove the theoretical complexity of two haplotype assembly optimizations, thereby motivating the use of heuristics. Furthermore, we present graph theory-based algorithms for the problem of haplotype assembly using our previously developed HapCompass framework for (i) novel implementations of haplotype assembly optimizations (minimum error correction), (ii) assembly of a pair of individuals sharing a haplotype tract identical by descent and (iii) assembly of polyploid genomes. We evaluate our methods on 1000 Genomes Project, Pacific Biosciences and simulated sequence data.HapCompass is available for download at http://www.brown.edu/Research/Istrail_Lab/.Supplementary data are available at Bioinformatics online.

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July 7, 2019

Precise breakpoint localization of large genomic deletions using PacBio and Illumina next-generation sequencers.

Herein we present the applicability of single-molecule (PacBio RS) and second-generation sequencing technology (Illumina) to the characterization of large genomic deletions. By testing samples previously characterized using a Sanger approach, our methods determined that both next-generation sequencing platforms were able to identify the position of deletion breakpoints. Our results point out various advantages of next-generation sequencing platforms when characterizing genomic deletions; however, special attention must be dedicated to identical sequences flanking the breakpoints, such as poly(N) motifs.

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