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July 7, 2019

Fragmentation of surface adsorbed and aligned DNA molecules using soft lithography for next-generation sequencing

In this study, the enzymatic in situ cutting of linearized DNA molecules at approximately 11 kbp intervals is demonstrated using a soft lithography technique. The ultimate goal is to provide a general ordered cutting method to greatly simplify the assembly process. DNA was stretched onto PMMA (Poly methyl methacrylate) coated silicon by withdrawing the substrate from a DNA solution (a process termed “combing”). The stretched lambda DNA could be linearly cut with a soft lithography stamp used to selectively apply DNase I. After cutting the DNA on the substrate, the DNA fragments are removed from the surface by incubating PMMA in the commercial NEBuffer 3.1 at 75°C. The recovered fragments desorbed into the buffer and were sequenced using the PacBio RS II sequencer without an amplification step. The mean coverage was 2870X for the approximately 11 kbp fragmented sample and 100% of the lambda genome was sequenced. Methods to extend of the technique to ordered fragmentation are discussed.

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July 7, 2019

Two orangutan species have evolved different KIR alleles and haplotypes.

The immune and reproductive functions of human NK cells are regulated by interactions of the C1 and C2 epitopes of HLA-C with C1-specific and C2-specific lineage III killer cell Ig-like receptors (KIR). This rapidly evolving and diverse system of ligands and receptors is restricted to humans and great apes. In this context, the orangutan has particular relevance because it represents an evolutionary intermediate, one having the C1 epitope and corresponding KIR but lacking the C2 epitope. Through a combination of direct sequencing, KIR genotyping, and data mining from the Great Ape Genome Project, we characterized the KIR alleles and haplotypes for panels of 10 Bornean orangutans and 19 Sumatran orangutans. The orangutan KIR haplotypes have between 5 and 10 KIR genes. The seven orangutan lineage III KIR genes all locate to the centromeric region of the KIR locus, whereas their human counterparts also populate the telomeric region. One lineage III KIR gene is Bornean specific, one is Sumatran specific, and five are shared. Of 12 KIR gene-content haplotypes, 5 are Bornean specific, 5 are Sumatran specific, and 2 are shared. The haplotypes have different combinations of genes encoding activating and inhibitory C1 receptors that can be of higher or lower affinity. All haplotypes encode an inhibitory C1 receptor, but only some haplotypes encode an activating C1 receptor. Of 130 KIR alleles, 55 are Bornean specific, 65 are Sumatran specific, and 10 are shared. Copyright © 2017 by The American Association of Immunologists, Inc.

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July 7, 2019

Lactobacillus allii sp. nov. isolated from scallion kimchi.

A novel strain of lactic acid bacteria, WiKim39T, was isolated from a scallion kimchi sample consisting of fermented chili peppers and vegetables. The isolate was a Gram-positive, rod-shaped, non-motile, catalase-negative and facultatively anaerobic lactic acid bacterium. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain WiKim39T belonged to the genus Lactobacillus, and shared 97.1-98.2?%?pair-wise sequence similarities with related type strains, Lactobacillus nodensis, Lactobacillus insicii, Lactobacillus versmoldensis, Lactobacillus tucceti and Lactobacillus furfuricola. The G+C?content of the strain based on its genome sequence was 35.3?mol%. The ANI values between WiKim39T and the closest relatives were lower than 80?%. Based on the phenotypic, biochemical, and phylogenetic analyses, strain WiKim39T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus allii sp. nov. is proposed. The type strain is WiKim39T (=KCTC 21077T=JCM 31938T).

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July 7, 2019

Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus.

Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.

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July 7, 2019

Rapid genetic and developmental morphological change following extreme celerity

Proximate environmental effects on metamorphosis have been explored in many vertebrate systems, but less attention has been devoted to how the environment affects developmental morphological change in mammals. Understanding proximate environmental effects on mammalian morphological change, particularly changes involving skin replacement, may aid in the design of therapeutic strategies to address severe burn or other debilitating injuries. Here, we specifically explore effects of celerity broadly, and we present results showing rapid change in mammalian morphological development following encountering maximum celerity. Morphological changes were pronounced within 96 hours and included at least partial regeneration of skin and organs as well as an elevated somatic mutation rate. Significantly, this high mutation rate did not result in detectable loss of fertility or viability of offspring. Overall, our findings strongly suggest that extreme celerity, an environmental factor rarely considered, can produce strikingly rapid developmental changes in morphology even in mammalian systems and open the door to future studies on the impact of celerity on genetics and morphology.

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July 7, 2019

Systems biology analysis of the key genes of surfactin production in Bacillus subtilis MJ01 (isolated from soil contaminated oil in south of Iran), spizizenii, and 168 isolates

Applying microorganism in oil recovery has attracted attentions recently. Surfactin produced by Bacillus subtilis is widely used industrially in a range of industrial applications in pharmecutical and environmental sectors. Little information about molecular mechanism of suffactin compound is available. In this study, we performed promoter and network analysis of surfactin production genes in Bacillus subtilis subsp. MJ01 (isolated from oil contaminated soil in South of Iran), spizizenii and 168. Our analysis revealed that comQ and comX are the genes with sequence alterations among these three strains of Bacillus subtilis and are involved in surfactin production. Promoter analysis indicated that lrp, argR, rpoD, purr and ihf are overrepresented and have the highest number of transcription factor binding sites (TFBs) on the key surfactin production genes in all 3 strains. Also the pattern of TFBs among these three strains was completely different. Interestingly, there is distinct difference between 168, spizizenii and MJ01 in their frequency of TFs that activate genes involve in surfactin production. Attribute weighting algorithms and decision tree analysis revealed ihf, rpoD and flHCD as the most important TF among surfactin production. Network analysis identified two significant network modules. The first one consists of key genes involved in surfactin production and the second module includes key TFs, involved in regulation of surfactin production. Our findings enhance understanding the molecular mechanism of surfactin production through systems biology analysis.

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July 7, 2019

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes.

Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted “noncoding RNAs” to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.© 2017 Prasad et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Institutional profile: translational pharmacogenomics at the Icahn School of Medicine at Mount Sinai.

For almost 50 years, the Icahn School of Medicine at Mount Sinai has continually invested in genetics and genomics, facilitating a healthy ecosystem that provides widespread support for the ongoing programs in translational pharmacogenomics. These programs can be broadly cataloged into discovery, education, clinical implementation and testing, which are collaboratively accomplished by multiple departments, institutes, laboratories, companies and colleagues. Focus areas have included drug response association studies and allele discovery, multiethnic pharmacogenomics, personalized genotyping and survey-based education programs, pre-emptive clinical testing implementation and novel assay development. This overview summarizes the current state of translational pharmacogenomics at Mount Sinai, including a future outlook on the forthcoming expansions in overall support, research and clinical programs, genomic technology infrastructure and the participating faculty.

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July 7, 2019

Tracing origins of the Salmonella Bareilly strain causing a food-borne outbreak in the United States.

Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India.Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network.Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India.These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general. Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019

Whole-genome sequencing: opportunities and challenges for public health, food-borne outbreak investigations, and the global food supply.

Food-borne disease is burdensome, af- fecting 1 in 6 persons or an estimated 48 million ill, 128 000 hospitalized, and 3000 deaths in the United States annually. In addition, societal costs from lost lives, lost labor, lost wages, and even lost revenue in the food industry are substan- tial. Globally the burden is even higher, and multinational outbreaks due to the global movement of contaminated foods are being described increasingly. The glo- bal food supply links nations and econo- mies, emphasizing the need to view food safety with an integrated farm-to-fork lens. As predicted, advances in molecular techniques and information management have been transformative for food-borne disease investigation.

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July 7, 2019

Characterization of ESBL disseminating plasmids.

Bacteria producing extended-spectrum ß-lactamases (ESBLs) constitute a globally increasing problem that contributes to treatment complications and elevated death rates. The extremely successful dissemination by ESBL-producing Enterobacteriaceae during the latest decades is a result of the combination of mobilization, evolution and horizontal spread of ß-lactamase genes on plasmids. In parallel, spread of these plasmids to particularly well-adapted bacterial clones (outbreak clones) has expanded. In this review we describe ESBL-producing bacteria and the genetic mechanisms for dissemination of ESBL resistance. We describe available methodology for studying plasmids and the importance of including plasmids in epidemiological typing as natural parts of the organisms. Plasmids play a fundamental role in how resistance arises and disseminates.

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July 7, 2019

Microbial bioinformatics for food safety and production.

In the production of fermented foods, microbes play an important role. Optimization of fermentation processes or starter culture production traditionally was a trial-and-error approach inspired by expert knowledge of the fermentation process. Current developments in high-throughput ‘omics’ technologies allow developing more rational approaches to improve fermentation processes both from the food functionality as well as from the food safety perspective. Here, the authors thematically review typical bioinformatics techniques and approaches to improve various aspects of the microbial production of fermented food products and food safety. © The Author 2015. Published by Oxford University Press.

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July 7, 2019

HapCol: accurate and memory-efficient haplotype assembly from long reads.

Haplotype assembly is the computational problem of reconstructing haplotypes in diploid organisms and is of fundamental importance for characterizing the effects of single-nucleotide polymorphisms on the expression of phenotypic traits. Haplotype assembly highly benefits from the advent of ‘future-generation’ sequencing technologies and their capability to produce long reads at increasing coverage. Existing methods are not able to deal with such data in a fully satisfactory way, either because accuracy or performances degrade as read length and sequencing coverage increase or because they are based on restrictive assumptions.By exploiting a feature of future-generation technologies-the uniform distribution of sequencing errors-we designed an exact algorithm, called HapCol, that is exponential in the maximum number of corrections for each single-nucleotide polymorphism position and that minimizes the overall error-correction score. We performed an experimental analysis, comparing HapCol with the current state-of-the-art combinatorial methods both on real and simulated data. On a standard benchmark of real data, we show that HapCol is competitive with state-of-the-art methods, improving the accuracy and the number of phased positions. Furthermore, experiments on realistically simulated datasets revealed that HapCol requires significantly less computing resources, especially memory. Thanks to its computational efficiency, HapCol can overcome the limits of previous approaches, allowing to phase datasets with higher coverage and without the traditional all-heterozygous assumption. Our source code is available under the terms of the GNU General Public License at http://hapcol.algolab.eu/.bonizzoni@disco.unimib.itSupplementary information: Supplementary data are available at Bioinformatics online.© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

High incidence of invasive group A Streptococcus disease caused by strains of uncommon emm types in Thunder Bay, Ontario, Canada.

An outbreak of type emm59 invasive group A Streptococcus (iGAS) disease was declared in 2008 in Thunder Bay District, Northwestern Ontario, two years after a country-wide emm59 epidemic was recognized in Canada. Despite a declining number of emm59 infections since 2010, numerous cases of iGAS disease continue to be reported in the area. We collected clinical information on all iGAS cases recorded in Thunder Bay District from 2008-2013. We also emm typed and sequenced the genomes of all available strains isolated in 2011-2013 from iGAS infections, and from severe cases of soft tissue infections. We used whole-genome data to investigate the population structure of GAS strains of the most frequently isolated emm types. We report increased incidence of iGAS in Thunder Bay compared to the metropolitan area of Toronto/Peel and the province of Ontario. Illicit drug use, alcohol abuse, homelessness and hepatitis C infection were underlying diseases or conditions that might have predisposed patients to iGAS disease. Most cases were caused by clonal strains of “skin” or “generalist” emm types (i.e. emm82, emm87, emm101, emm4, emm83, and emm114), uncommonly seen in other areas of the province. We observed rapid waxing and waning of emm types causing disease and their replacement by other emm types associated with the same tissue tropisms. Thus, iGAS disease in Thunder Bay District predominantly affects a select population of disadvantaged persons and is caused by clonally related strains of a few “skin” and “generalist” emm types less commonly associated with iGAS in other areas of Ontario. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Identification and resolution of microdiversity through metagenomic sequencing of parallel consortia.

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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