Clustering and Alignment of Polymorphic Sequences for HLA-DRB1 Genotyping

Located on Chromosome 6p21, classical human leukocyte antigen genes are highly polymorphic. HLA alleles associate with a variety of phenotypes, such as narcolepsy, autoimmunity, as well as immunologic response to infectious disease. Moreover, high resolution genotyping of these loci is critical to achieving long-term survival of allogeneic transplants. Development of methods to obtain high resolution analysis of HLA genotypes will lead to improved understanding of how select alleles contribute to human health and disease risk. Genomic DNAs were obtained from a cohort of n = 383 subjects recruited as part of an Ulcerative Colitis study and analyzed for HLA-DRB1. HLA genotypes were determined using sequence specific oligonucleotide probes and by next-generation sequencing using the Roche/454 GSFLX instrument. The Clustering and Alignment of Polymorphic Sequences (CAPSeq) software application was developed to analyze next-generation sequencing data. The application generates HLA sequence specific 6-digit genotype information from next-generation sequencing data using MUMmer to align sequences and the R package diffusionMap to classify sequences into their respective allelic groups. The incorporation of Bootstrap Aggregating, Bagging to aid in sorting of sequences into allele classes resulted in improved genotyping accuracy. Using Bagging iterations equal to 60, the genotyping results obtained using CAPSeq when compared with sequence specific oligonucleotide probe characterized 4-digit genotypes exhibited high rates of concordance, matching at 759 out of 766 (99.1%) alleles. © 2013 Ringquist et al

Sequence and functional analyses of Haemophilus spp. genomic islands

Comparative analysis of genomic islands of Haemophilus species shows that they are co-evolving as semi-autonomous genomes within the host genome

MultiMetEval: comparative and multi-objective analysis of genome-scale metabolic models

Comparative metabolic modelling is emerging as a novel field, supported by the development of reliable and standardized approaches for constructing genome-scale metabolic models in high throughput. New software solutions are needed to allow efficient comparative analysis of multiple models in the context of multiple cellular objectives. Here, we present the user-friendly software framework Multi-Metabolic Evaluator (MultiMetEval), built upon SurreyFBA, which allows the user to compose collections of metabolic models that together can be subjected to flux balance analysis. Additionally, MultiMetEval implements functionalities for multi-objective analysis by calculating the Pareto front between two cellular objectives. Using a previously generated dataset of 38 actinobacterial genome-scale metabolic models, we show how these approaches can lead to exciting novel insights. Firstly, after incorporating several pathways for the biosynthesis of natural products into each of these models, comparative flux balance analysis predicted that species like Streptomyces that harbour the highest diversity of secondary metabolite biosynthetic gene clusters in their genomes do not necessarily have the metabolic network topology most suitable for compound overproduction. Secondly, multi-objective analysis of biomass production and natural product biosynthesis in these actinobacteria shows that the well-studied occurrence of discrete metabolic switches during the change of cellular objectives is inherent to their metabolic network architecture. Comparative and multi-objective modelling can lead to insights that could not be obtained by normal flux balance analyses. MultiMetEval provides a powerful platform that makes these analyses straightforward for biologists. Sources and binaries of MultiMetEval are freely available from https://github.com/PiotrZakrzewski/MetEv​al/downloads

Functional assays to determine the significance of two common XPC 3'UTR variants found in bladder cancer patients

<p>Abstract</p> <p>Background</p> <p><it>XPC </it>is involved in the nucleotide excision repair of DNA damaged by carcinogens known to cause bladder cancer. Individuals homozygous for the variant allele of <it>XPC </it>c.1496C > T (p.Ala499Val) were shown in a large pooled analysis to have an increased bladder cancer risk, and we found two 3'UTR variants, *611T > A and c.*618A > G, to be in strong linkage disequilibrium with c.1496T. Here we determined if these two 3'UTR variants can affect mRNA stability and assessed the impact of all three variants on mRNA and protein expression.</p> <p>Methods</p> <p><it>In vitro </it>mRNA stability assays were performed and mRNA and protein expression measured both in plasmid-based assays and in lymphocytes and lymphoblastoid cell lines from bladder and breast cancer patients.</p> <p>Results</p> <p>The two 3'UTR variants were associated with reduced protein and mRNA expression in plasmid-based assays, suggesting an effect on mRNA stability and/or transcription/translation. A near-significant reduction in XPC protein expression (p = 0.058) was detected in lymphoblastoid cell lines homozygous for these alleles but no differences in mRNA stability in these lines was found or in mRNA or protein levels in lymphocytes heterozygous for these alleles.</p> <p>Conclusion</p> <p>The two 3'UTR variants may be the variants underlying the association of c.1496C > T and bladder cancer risk acting via a mechanism modulating protein expression.</p

Improving Ethical Review of Research Involving Incentives for Health Promotion

Alex London and colleagues propose new ethical frameworks for evaluating the risks associated with research in which financial or other incentives are used to promote healthy behavior

Differences in genotype and virulence among four multidrug-resistant <i>Streptococcus pneumoniae</i> isolates belonging to the PMEN1 clone

We report on the comparative genomics and characterization of the virulence phenotypes of four &lt;i&gt;S. pneumoniae&lt;/i&gt; strains that belong to the multidrug resistant clone PMEN1 (Spain&lt;sup&gt;23F&lt;/sup&gt; ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinant

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

The evolution of the urinary bladder as a storage organ: scent trails and selective pressure of the first land animals in a computational simulation

The function of waste control in all living organisms is one of the vital importance. Almost universally, terrestrial tetrapods have a urinary bladder with a storage function. It is well documented that many marine and aerial species do not have an organ of such a function, or have one with very depressed storage functionality. Bladder morphology indicates it has evolved from a thin-walled structure used for osmoregulatory purposes, as it is currently used in many marine animals. It is hypothesised that the storage function of the urinary bladder allows for an evolutionary selective advantage in reducing the likelihood of successful predation. Random walks simulating predator and prey movements with simplified scent trails were utilised to represent various stages of the hunt: Detection and pursuit. A final evolutionary model is proposed in order to display the advantages over inter-generational time scales and illustrates how a bladder may evolve from an osmoregulatory organ to one of the storage. Data sets were generated for each case and analysed indicating the viability of such advantages. From the highly consistent results, three distinct characteristics of having a storage function in the urinary bladder are suggested: reduced scent trail detection rate; increased prey–predator separation (upon scent trail detection); and a reduced probability of successful capture upon scent detection by the predator. Furthered by the evolutionary model indicating such characteristics are conserved and augmented over many generations, it is concluded that prey–predator interactions provide a large selective pressure in the evolution of the urinary bladder and its storage function

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated