SAMFIRE: multi-locus variant calling for time-resolved sequence data.

UNLABELLED: An increasingly common method for studying evolution is the collection of time-resolved short-read sequence data. Such datasets allow for the direct observation of rapid evolutionary processes, as might occur in natural microbial populations and in evolutionary experiments. In many circumstances, evolutionary pressure acting upon single variants can cause genomic changes at multiple nearby loci. SAMFIRE is an open-access software package for processing and analyzing sequence reads from time-resolved data, calling important single- and multi-locus variants over time, identifying alleles potentially affected by selection, calculating linkage disequilibrium statistics, performing haplotype reconstruction and exploiting time-resolved information to estimate the extent of uncertainty in reported genomic data. AVAILABILITY AND IMPLEMENTATION: C ++ code may be found at https://github.com/cjri/samfire/ CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.CI was supported by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (Grant Number 101239/Z/13/Z).This is the author accepted manuscript. The final version is available from Oxford University Press via http://dx.doi.org/10.1093/bioinformatics/btw20

Building a mechanistic mathematical model of hepatitis C virus entry

The mechanism by which hepatitis C virus (HCV) gains entry into cells is a complex one, involving a broad range of host proteins. Entry is a critical phase of the viral lifecycle, and a potential target for therapeutic or vaccine-mediated intervention. However, the mechanics of HCV entry remain poorly understood. Here we describe a novel computational model of viral entry, encompassing the relationship between HCV and the key host receptors CD81 and SR-B1. We conduct experiments to thoroughly quantify the influence of an increase or decrease in receptor availability upon the extent of viral entry. We use these data to build and parameterise a mathematical model, which we then validate by further experiments. Our results are consistent with sequential HCV-receptor interactions, whereby initial interaction between the HCV E2 glycoprotein and SR-B1 facilitates the accumulation CD81 receptors, leading to viral entry. However, we also demonstrate that a small minority of virus can achieve entry in the absence of SR-B1. Our model estimates the impact of the different obstacles that viruses must surmount to achieve entry; among virus particles attaching to the cell surface, around one third of viruses accumulate sufficient CD81 receptors, of which 4-8% then complete the subsequent steps to achieve productive infection. Furthermore, we make estimates of receptor stoichiometry; in excess of 10 receptors are likely to be required to achieve viral entry. Our model provides a tool to investigate the entry characteristics of HCV variants and outlines a framework for future quantitative studies of the multi-receptor dynamics of HCV entry

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.This study was supported by as part of the FP7 EC K aladrug-R project (http://cordis.europa.eu/project/rcn/88823_en.html, grant number: 222895). JAC and MJS are supported by the Wellcome Trust via their core support for the Wellcome Trust Sanger Institute (grant number 098051) . TMF was funded by a BBSRC Research Experience Placement (grant number BB/J014540/1). CJRI was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant number 101239/Z/13/Z). This research was supported in part by the National Science Foundation (grant number: NSF PHY11-25915) and by the Belgian Science Policy Office (TRIT, contract P7/41, to J-C.D.).This is the final version of the article. It was first available from Wiley via http://dx.doi.org/10.1111/mmi.1329

The effective rate of influenza reassortment is limited during human infection

We characterise the evolutionary dynamics of influenza infection described by viral sequence data collected from two challenge studies conducted in human hosts. Viral sequence data were collected at regular intervals from infected hosts. Changes in the sequence data observed across time show that the within-host evolution of the virus was driven by the reversion of variants acquired during previous passaging of the virus. Treatment of some patients with oseltamivir on the first day of infection did not lead to the emergence of drug resistance variants in patients. Using an evolutionary model, we inferred the effective rate of reassortment between viral segments, measuring the extent to which randomly chosen viruses within the host exchange genetic material. We find strong evidence that the rate of effective reassortment is low, such that genetic associations between polymorphic loci in different segments are preserved during the course of an infection in a manner not compatible with epistasis. Combining our evidence with that of previous studies we suggest that spatial heterogeneity in the viral population may reduce the extent to which reassortment is observed. Our results do not contradict previous findings of high rates of viral reassortment in vitro and in small animal studies, but indicate that in human hosts the effective rate of reassortment may be substantially more limited.CJRI is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 101239/Z/13/Z) and received support from the National Science Foundation Research Coordination Network on Infectious Disease Evolution Across Scales. KK, ASL, CWW, and MTM were funded by NIGMS U54-GM111274, the MIDAS Center for Inference and Dynamics of Infectious Disease. ASL acknowledges support from the MSTP training grant number T32 GM007171. GJDS was supported by the Duke-NUS Signature Research Programme funded by the Ministry of Health, Singapore and by contract HHSN272201400006C from the National Institute of Allergy and Infectious Disease, National Institutes of Health, Department of Health and Human Services, USA. DEW, RAH, XL, AR, TBS, SRD and also the influenza whole genome sequencing were supported with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract HHSN272200900007C. GSG was funded by the Defense Advanced Research Projects Agency under grant number DARPA-N66001-07-C-2024. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Evolution Across Scales. KK, ASL, CWW, and MTM were funded by NIGMS U54- GM111274, the MIDAS Center for Inference and Dynamics of Infectious Disease. DEW, RAH, XL, AR, TBS, and SRD were supported with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract HHSN272200900007C. GSG was funded by the Defense Advanced Research Projects Agency under grant number DARPA-N66001-07-C-2024. This work was performed using the Darwin Supercomputer of the University of Cambridge High Performance Computing Service (http://www.hpc.cam.ac.uk/), provided by Dell Inc. using Strategic Research Infrastructure Funding from the Higher Education Funding Council for England and funding from the Science and Technology Facilities Council

On Conduction in a Bacterial Sodium Channel

Voltage-gated Na+-channels are transmembrane proteins that are responsible for the fast depolarizing phase of the action potential in nerve and muscular cells. Selective permeability of Na+ over Ca2+ or K+ ions is essential for the biological function of Na+-channels. After the emergence of the first high-resolution structure of a Na+-channel, an anionic coordination site was proposed to confer Na+ selectivity through partial dehydration of Na+ via its direct interaction with conserved glutamate side chains. By combining molecular dynamics simulations and free-energy calculations, a low-energy permeation pathway for Na+ ion translocation through the selectivity filter of the recently determined crystal structure of a prokaryotic sodium channel from Arcobacter butzleri is characterised. The picture that emerges is that of a pore preferentially occupied by two ions, which can switch between different configurations by crossing low free-energy barriers. In contrast to K+-channels, the movements of the ions appear to be weakly coupled in Na+-channels. When the free-energy maps for Na+ and K+ ions are compared, a selective site is characterised in the narrowest region of the filter, where a hydrated Na+ ion, and not a hydrated K+ ion, is energetically stable

Fitness Landscape of the Fission Yeast Genome

The relationship between DNA sequence, biochemical function and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in non-coding regions, particularly in eukaryote genomes. In part, this is because we lack a complete description of the essential non-coding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66-90% of the genome, including substantial portions of the non-coding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3' and 5' untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary and biochemical data can provide new insights into the relationship between genome function and molecular evolution