VariantClassifier: A hierarchical variant classifier for annotated genomes

<p>Abstract</p> <p>Background</p> <p>High-throughput DNA sequencing has produced a large number of closed and well annotated genomes. As the focus from whole genome sequencing and assembly moves towards resequencing, variant data is becoming more accessible and large quantities of polymorphisms are being detected. An easy-to-use tool for quickly assessing the potential importance of these discovered variants becomes ever important.</p> <p>Findings</p> <p>Written in Perl, the VariantClassifier receives a list of polymorphisms and genome annotation, and generates a hierarchically-structured classification for each variant. Depending on the available annotation, the VariantClassifier may assign each polymorphism to a large variety of feature types, such as intergenic or genic; upstream promoter region, intronic region, exonic region or downstream transcript region; 5' splice site or 3' splice site; 5' untranslated region (UTR), 3' UTR or coding sequence (CDS); impacted protein domain; substitution, insertion or deletion; synonymous or non-synonymous; conserved or unconserved; and frameshift or amino acid insertion or deletion (indel). If applicable, the truncated or altered protein sequence is also predicted. For organisms with annotation maintained at Ensembl, a software application for downloading the necessary annotation is also provided, although the classifier will function with properly formatted annotation provided through alternative means.</p> <p>Conclusions</p> <p>We have utilized the VariantClassifier for several projects since its implementation to quickly assess hundreds of thousands of variations on several genomes and have received requests to make the tool publically available. The project website can be found at: <url>http://www.jcvi.org/cms/research/projects/variantclassifier</url>.</p

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

BACKGROUND: Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. RESULTS: Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts) in the new templates. CONCLUSION: Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications

A Review of Existing Studies Reporting the Negative Effects of Alcohol Access and Positive Effects of Alcohol Control Policies on Interpersonal Violence

Alcohol consumption often leads to elevated rates of violence yet alcohol access policies continue to relax across the globe. Our review establishes the extent alcohol policy can moderate violent crime through alcohol availability restrictions. Results were informed from comprehensive selection of peer-reviewed journals from 1950 to October 2015. Our search identified 88 relevant studies on alcohol access and violence conducted across 12 countries. Seventeen studies included quasi-control design, and 23 conducted intervention analysis. Seventy-two (82%) reported a significant relationship between alcohol access and violent offences. Alcohol outlet studies reported the greatest percentage of significant results (93%), with trading hours (63%), and alcohol price following (58%). Results from baseline studies indicated the effectiveness of increasing the price of commonly consumed alcohol, restricting the hours of alcohol trading, and limiting the number of alcohol outlets per region to prevent violent offences. Unclear are the effects of tax reductions, restriction of on-premises re-entry, and different outlet types on violent crime. Further, the generalization of statistics over broad areas and the low number of control/intervention studies poses some concern for confounding or correlated effects on study results, and amount of information for local level prevention of interpersonal violence. Future studies should focus on gathering longitudinal data, validating models, limiting crime data to peak drinking days and times, and wherever possible collecting the joint distribution between violent crime, intoxication, and place. A greater up take of local level analysis will benefit studies comparing the influence of multiple alcohol establishment types by relating the location of a crime to establishment proximity. Despite, some uncertainties particular studies showed that even modest policy changes such as 1% increases in alcohol price, one hour changes to closing times, and limiting establishment densities to less than 25 outlets per postal code substantively reduce violent crime

Genetic Variation in an Individual Human Exome

There is much interest in characterizing the variation in a human individual, because this may elucidate what contributes significantly to a person's phenotype, thereby enabling personalized genomics. We focus here on the variants in a person's ‘exome,’ which is the set of exons in a genome, because the exome is believed to harbor much of the functional variation. We provide an analysis of the ∼12,500 variants that affect the protein coding portion of an individual's genome. We identified ∼10,400 nonsynonymous single nucleotide polymorphisms (nsSNPs) in this individual, of which ∼15–20% are rare in the human population. We predict ∼1,500 nsSNPs affect protein function and these tend be heterozygous, rare, or novel. Of the ∼700 coding indels, approximately half tend to have lengths that are a multiple of three, which causes insertions/deletions of amino acids in the corresponding protein, rather than introducing frameshifts. Coding indels also occur frequently at the termini of genes, so even if an indel causes a frameshift, an alternative start or stop site in the gene can still be used to make a functional protein. In summary, we reduced the set of ∼12,500 nonsilent coding variants by ∼8-fold to a set of variants that are most likely to have major effects on their proteins' functions. This is our first glimpse of an individual's exome and a snapshot of the current state of personalized genomics. The majority of coding variants in this individual are common and appear to be functionally neutral. Our results also indicate that some variants can be used to improve the current NCBI human reference genome. As more genomes are sequenced, many rare variants and non-SNP variants will be discovered. We present an approach to analyze the coding variation in humans by proposing multiple bioinformatic methods to hone in on possible functional variation

Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells

Evaluation of next generation sequencing platforms for population targeted sequencing studies

Human sequence generated from three next-generation sequencing platforms reveals systematic variability in sequence coverage due to local sequence characteristics

Face Masks and Cough Etiquette Reduce the Cough Aerosol Concentration of Pseudomonas aeruginosa in People with Cystic Fibrosis

People with cystic fibrosis (CF) generate Pseudomonas aeruginosa in droplet nuclei during coughing. The use of surgical masks has been recommended in healthcare settings to minimize pathogen transmission between patients with CF.To determine if face masks and cough etiquette reduce viable P. aeruginosa aerosolized during coughing.Twenty-five adults with CF and chronic P. aeruginosa infection were recruited. Participants performed six talking and coughing maneuvers, with or without face masks (surgical and N95) and hand covering the mouth when coughing (cough etiquette) in an aerosol-sampling device. An Andersen Cascade Impactor was used to sample the aerosol at 2 meters from each participant. Quantitative sputum and aerosol bacterial cultures were performed, and participants rated the mask comfort levels during the cough maneuvers.During uncovered coughing (reference maneuver), 19 of 25 (76%) participants produced aerosols containing P. aeruginosa, with a positive correlation found between sputum P. aeruginosa concentration (measured as cfu/ml) and aerosol P. aeruginosa colony-forming units. There was a reduction in aerosol P. aeruginosa load during coughing with a surgical mask, coughing with an N95 mask, and cough etiquette compared with uncovered coughing (P

Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARSCoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn’t attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses

Ecosystem Interactions Underlie the Spread of Avian Influenza A Viruses with Pandemic Potential

Despite evidence for avian influenza A virus (AIV) transmission between wild and domestic ecosystems, the roles of bird migration and poultry trade in the spread of viruses remain enigmatic. In this study, we integrate ecosystem interactions into a phylogeographic model to assess the contribution of wild and domestic hosts to AIV distribution and persistence. Analysis of globally sampled AIV datasets shows frequent two-way transmission between wild and domestic ecosystems. In general, viral flow from domestic to wild bird populations was restricted to within a geographic region. In contrast, spillover from wild to domestic populations occurred both within and between regions. Wild birds mediated long-distance dispersal at intercontinental scales whereas viral spread among poultry populations was a major driver of regional spread. Viral spread between poultry flocks frequently originated from persistent lineages circulating in regions of intensive poultry production. Our analysis of long-term surveillance data demonstrates that meaningful insights can be inferred from integrating ecosystem into phylogeographic reconstructions that may be consequential for pandemic preparedness and livestock protection.National Institutes of Health (U.S.) (NIH Centers for Excellence in Influenza Research and Surveillance (CEIRS, contract # HHSN266200700010C))National Institutes of Health (U.S.) (NIH Centers for Excellence in Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C))National Institutes of Health (U.S.) (NIH Centers for Excellence in Influenza Research and Surveillance (CEIRS, contract # HHSN272201400006C)