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Asset Tag: Complex population

July 7, 2019  |  

micropan: an R-package for microbial pan-genomics.

A pan-genome is defined as the set of all unique gene families found in one or more strains of a prokaryotic species. Due to the extensive within-species diversity in the microbial world, the pan-genome is often many times larger than a single genome. Studies of pan-genomes have become popular due to the easy access to whole-genome sequence data for prokaryotes. A pan-genome study reveals species diversity and gene families that may be of special interest, e.g because of their role in bacterial survival or their ability to discriminate strains.We present an R package for the study of prokaryotic pan-genomes. The R computing environment harbors endless possibilities with respect to statistical analyses and graphics. External free software is used for the heavy computations involved, and the R package provides functions for building a computational pipeline.We demonstrate parts of the package on a data set for the gram positive bacterium Enterococcus faecalis. The package is free to download and install from The Comprehensive R Archive Network.

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July 7, 2019  |  

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii.

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.

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July 7, 2019  |  

Mutation assay using single-molecule real-time (SMRT) sequencing technology

Introduction We present here a simple, phenotype-independent mutation assay using a PacBio RSII DNA sequencer employing single-molecule real-time (SMRT) sequencing technology. Salmonella typhimurium YG7108 was treated with the alkylating agent N-ethyl-N-nitrosourea (ENU) and grown though several generations to fix the induced mutations, the DNA was extracted and the mutations were analyzed by using the SMRT DNA sequencer. Results The ENU-induced base-substitution frequency was 15.4 per Megabase pair, which is highly consistent with our previous results based on colony isolation and next-generation sequencing. The induced mutation spectrum (95% G:C???A:T, 5% A:T???G:C) is also consistent with the known ENU signature. The base-substitution frequency of the control was calculated to be less than 0.12 per Megabase pair. A current limitation of the approach is the high frequency of artifactual insertion and deletion mutations it detects. Conclusions Ultra-low frequency base-substitution mutations can be detected directly by using the SMRT DNA sequencer, and this technology provides a phenotype-independent mutation assay.

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July 7, 2019  |  

One Codex: A sensitive and accurate data platform for genomic microbial identification

High-throughput sequencing (HTS) is increasingly being used for broad applications of microbial characterization, such as microbial ecology, clinical diagnosis, and outbreak epidemiology. However, the analytical task of comparing short sequence reads against the known diversity of microbial life has proved to be computationally challenging. The One Codex data platform was created with the dual goals of analyzing microbial data against the largest possible collection of microbial reference genomes, as well as presenting those results in a format that is consumable by applied end-users. One Codex identifies microbial sequences using a “k-mer based” taxonomic classification algorithm through a web-based data platform, using a reference database that currently includes approximately 40,000 bacterial, viral, fungal, and protozoan genomes. In order to evaluate whether this classification method and associated database provided quantitatively different performance for microbial identification, we created a large and diverse evaluation dataset containing 50 million reads from 10,639 genomes, as well as sequences from six organisms novel species not be included in the reference databases of any of the tested classifiers. Quantitative evaluation of several published microbial detection methods shows that One Codex has the highest degree of sensitivity and specificity (AUC = 0.97, compared to 0.82-0.88 for other methods), both when detecting well-characterized species as well as newly sequenced, “taxonomically novel” organisms.

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July 7, 2019  |  

Jitterbug: somatic and germline transposon insertion detection at single-nucleotide resolution.

Transposable elements are major players in genome evolution. Transposon insertion polymorphisms can translate into phenotypic differences in plants and animals and are linked to different diseases including human cancer, making their characterization highly relevant to the study of genome evolution and genetic diseases. Here we present Jitterbug, a novel tool that identifies transposable element insertion sites at single-nucleotide resolution based on the pairedend mapping and clipped-read signatures produced by NGS alignments. Jitterbug can be easily integrated into existing NGS analysis pipelines, using the standard BAM format produced by frequently applied alignment tools (e.g. bwa, bowtie2), with no need to realign reads to a set of consensus transposon sequences. Jitterbug is highly sensitive and able to recall transposon insertions with a very high specificity, as demonstrated by benchmarks in the human and Arabidopsis genomes, and validation using long PacBio reads. In addition, Jitterbug estimates the zygosity of transposon insertions with high accuracy and can also identify somatic insertions. We demonstrate that Jitterbug can identify mosaic somatic transposon movement using sequenced tumor-normal sample pairs and allows for estimating the cancer cell fraction of clones containing a somatic TE insertion. We suggest that the independent methods we use to evaluate performance are a step towards creating a gold standard dataset for benchmarking structural variant prediction tools.

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July 7, 2019  |  

Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis.

Cerebral cavernous malformations (CCMs) are vascular lesions affecting the central nervous system. CCM occurs either sporadically or in an inherited, autosomal dominant manner. Constitutional (germline) mutations in any of three genes, KRIT1, CCM2 and PDCD10, can cause the inherited form. Analysis of CCM lesions from inherited cases revealed biallelic somatic mutations, indicating that CCM follows a Knudsonian two-hit mutation mechanism. It is still unknown, however, if the sporadic cases of CCM also follow this genetic mechanism. We extracted DNA from 11 surgically excised lesions from sporadic CCM patients, and sequenced the three CCM genes in each specimen using a next-generation sequencing approach. Four sporadic CCM lesion samples (36%) were found to contain novel somatic mutations. Three of the lesions contained a single somatic mutation, and one lesion contained two biallelic somatic mutations. Herein, we also describe evidence of somatic mosaicism in a patient presenting with over 130 CCM lesions localized to one hemisphere of the brain. Finally, in a lesion regrowth sample, we found that the regrown CCM lesion contained the same somatic mutation as the original lesion. Together, these data bolster the idea that all forms of CCM have a genetic underpinning of the two-hit mutation mechanism in the known CCM genes. Recent studies have found aberrant Rho kinase activation in inherited CCM pathogenesis, and we present evidence that this pathway is activated in sporadic CCM patients. These results suggest that all CCM patients, including those with the more common sporadic form, are potentially amenable to the same therapy. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019  |  

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera.

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393?Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140?My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.

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July 7, 2019  |  

Life cycles, fitness decoupling and the evolution of multicellularity.

Cooperation is central to the emergence of multicellular life; however, the means by which the earliest collectives (groups of cells) maintained integrity in the face of destructive cheating types is unclear. One idea posits cheats as a primitive germ line in a life cycle that facilitates collective reproduction. Here we describe an experiment in which simple cooperating lineages of bacteria were propagated under a selective regime that rewarded collective-level persistence. Collectives reproduced via life cycles that either embraced, or purged, cheating types. When embraced, the life cycle alternated between phenotypic states. Selection fostered inception of a developmental switch that underpinned the emergence of collectives whose fitness, during the course of evolution, became decoupled from the fitness of constituent cells. Such development and decoupling did not occur when groups reproduced via a cheat-purging regime. Our findings capture key events in the evolution of Darwinian individuality during the transition from single cells to multicellularity.

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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  |  

The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes.

Whole-genome sequences are now available for many microbial species and clades, however, existing whole-genome alignment methods are limited in their ability to perform sequence comparisons of multiple sequences simultaneously. Here we present the Harvest suite of core-genome alignment and visualization tools for the rapid and simultaneous analysis of thousands of intraspecific microbial strains. Harvest includes Parsnp, a fast core-genome multi-aligner, and Gingr, a dynamic visual platform. Together they provide interactive core-genome alignments, variant calls, recombination detection, and phylogenetic trees. Using simulated and real data we demonstrate that our approach exhibits unrivaled speed while maintaining the accuracy of existing methods. The Harvest suite is open-source and freely available from: http://github.com/marbl/harvest.

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July 7, 2019  |  

Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus.

The marine cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism in the oligotrophic oceans, and a model system in marine microbial ecology. Here we report 27 new whole genome sequences (2 complete and closed; 25 of draft quality) of cultured isolates, representing five major phylogenetic clades of Prochlorococcus. The sequenced strains were isolated from diverse regions of the oceans, facilitating studies of the drivers of microbial diversity-both in the lab and in the field. To improve the utility of these genomes for comparative genomics, we also define pre-computed clusters of orthologous groups of proteins (COGs), indicating how genes are distributed among these and other publicly available Prochlorococcus genomes. These data represent a significant expansion of Prochlorococcus reference genomes that are useful for numerous applications in microbial ecology, evolution and oceanography.

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July 7, 2019  |  

Twenty-one novel microsatellite loci for the endangered Florida salt marsh vole (Microtus pennsylvanicus dukecampbelli)

We present 21 microsatellite loci developed for Florida salt marsh voles (Microtus pennsylvanicus dukecampbelli). Microsatellites were identified from single molecule real time sequencing (Pacific Biosciences). We screened 30 loci and identified 21 loci as suitable for genotyping. We screened 17 individuals from Long Cabbage Key, and 3 individuals from an unnamed island. There was no significant departure from Hardy–Weinberg equilibrium or linkage equilibrium. Fifteen of the 21 loci were variable, with overall observed heterozygosity averaging 0.39, and a mean number of alleles of 3.14. Linkage disequilibrium estimate of Ne was 10.7 (95 % CI 6.1–20.1). These markers will be useful for conservation genetics studies of this endangered species.

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July 7, 2019  |  

Feasibility of real time next generation sequencing of cancer genes linked to drug response: results from a clinical trial.

The successes of targeted drugs with companion predictive biomarkers and the technological advances in gene sequencing have generated enthusiasm for evaluating personalized cancer medicine strategies using genomic profiling. We assessed the feasibility of incorporating real-time analysis of somatic mutations within exons of 19 genes into patient management. Blood, tumor biopsy and archived tumor samples were collected from 50 patients recruited from four cancer centers. Samples were analyzed using three technologies: targeted exon sequencing using Pacific Biosciences PacBio RS, multiplex somatic mutation genotyping using Sequenom MassARRAY and Sanger sequencing. An expert panel reviewed results prior to reporting to clinicians. A clinical laboratory verified actionable mutations. Fifty patients were recruited. Nineteen actionable mutations were identified in 16 (32%) patients. Across technologies, results were in agreement in 100% of biopsy specimens and 95% of archival specimens. Profiling results from paired archival/biopsy specimens were concordant in 30/34 (88%) patients. We demonstrated that the use of next generation sequencing for real-time genomic profiling in advanced cancer patients is feasible. Additionally, actionable mutations identified in this study were relatively stable between archival and biopsy samples, implying that cancer mutations that are good predictors of drug response may remain constant across clinical stages. Copyright © 2012 UICC.

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July 7, 2019  |  

Draft genome of Spiribacter salinus M19-40, an abundant gammaproteobacterium in aquatic hypersaline environments.

We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.

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July 7, 2019  |  

Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism.

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

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