Suite of tools for statistical N-gram language modeling for pattern mining in whole genome sequences

Genome sequences contain a number of patterns that have biomedical significance. Repetitive sequences of various kinds are a primary component of most of the genomic sequence patterns. We extended the suffix-array based Biological Language Modeling Toolkit to compute n-gram frequencies as well as n-gram language-model based perplexity in windows over the whole genome sequence to find biologically relevant patterns. We present the suite of tools and their application for analysis on whole human genome sequence.

Comparison against 186 canid whole genome sequences reveals survival strategies of an ancient clonally transmissible canine tumor.

Canine transmissible venereal tumor (CTVT) is a parasitic cancer clone that has propagated for thousands of years via sexual transfer of malignant cells. Little is understood about the mechanisms that converted an ancient tumor into the world's oldest known continuously propagating somatic cell lineage. We created the largest existing catalog of canine genome-wide variation and compared it against two CTVT genome sequences, thereby separating alleles derived from the founder's genome from somatic drivers of clonal transmissibility. We show that CTVT has undergone continuous adaptation to its transmissible allograft niche, with overlapping mutations at every step of immunosurveillance, particularly self-antigen presentation and apoptosis. We also identified chronologically early somatic mutations in oncogenesis- and immune-related genes that may represent key initiators of clonal transmissibility. Thus, we provide the first insights into the specific genomic aberrations that underlie CTVT's dogged perseverance in canids around the world.

Complete Genome Sequences of Three Propionibacterium acnes Isolates from the Type IA(2) Cluster

Propionibacterium acnes is an anaerobic Gram-positive bacterium that has been linked to a wide range of opportunistic human infections and conditions, most notably acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821-832, 2009). We now present the whole-genome sequences of three P. acnes strains from the type IA(2) cluster which were recovered from ophthalmic infections (A. McDowell et al., Microbiology 157:1990-2003, 2011).

PrimerSNP: a web tool for whole-genome selection of allele-specific and common primers of phylogenetically-related bacterial genomic sequences

Background: The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the identification of the few loci in the genome that can serve as unique markers for strain differentiation. PrimerSNP attempts to identify reliable strain-specific markers, on which specific primers are designed for pathogen detection purpose.Results: PrimerSNP is an online tool to design primers based on strain specific SNPs for multiple strains/species of microorganisms at the whole genome level. The allele-specific primers could distinguish query sequences of one strain from other homologous sequences by standard PCR reaction. Additionally, PrimerSNP provides a feature for designing common primers that can amplify all the homologous sequences of multiple strains/species of microorganisms. PrimerSNP is freely available at http://cropdisease.ars.usda.gov/similar to primer.Conclusion: PrimerSNP is a high-throughput specific primer generation tool for the differentiation of phylogenetically-related strains/species. Experimental validation showed that this software had a successful prediction rate of 80.4 - 100% for strain specific primer design.

Whole genome analysis reveals a high incidence of non-optimal codons in secretory signal sequences of Escherichia coli

Translational pausing may occur due to a number of mechanisms, including the presence of non-optimal codons, and it is thought to play a role in the folding of specific polypeptide domains during translation and in the facilitation of signal peptide recognition during see-dependent protein targeting. In this whole genome analysis of Escherichia coli we have found that non-optimal codons in the signal peptide-encoding sequences of secretory genes are overrepresented relative to the mature portions of these genes; this is in addition to their overrepresentation in the 5'-regions of genes encoding non-secretory proteins. We also find increased non-optimal codon usage at the 3' ends of most E. coli genes, in both non-secretory and secretory sequences. Whereas presumptive translational pausing at the 5' and 3' ends of E. coli messenger RNAs may clearly have a general role in translation, we suggest that it also has a specific role in sec-dependent protein export, possibly in facilitating signal peptide recognition. This finding may have important implications for our understanding of how the majority of non-cytoplasmic proteins are targeted, a process that is essential to all biological cells. (C) 2004 Elsevier Inc. All rights reserved.

Whole Genome Sequencing Reveals Local Transmission Patterns of Mycobacterium bovis in Sympatric Cattle and Badger Populations

Whole genome sequencing (WGS) technology holds great promise as a tool for the forensic epidemiology of bacterial pathogens. It is likely to be particularly useful for studying the transmission dynamics of an observed epidemic involving a largely unsampled 'reservoir' host, as for bovine tuberculosis (bTB) in British and Irish cattle and badgers. BTB is caused by Mycobacterium bovis, a member of the M. tuberculosis complex that also includes the aetiological agent for human TB. In this study, we identified a spatio-temporally linked group of 26 cattle and 4 badgers infected with the same Variable Number Tandem Repeat (VNTR) type of M. bovis. Single-nucleotide polymorphisms (SNPs) between sequences identified differences that were consistent with bacterial lineages being persistent on or near farms for several years, despite multiple clear whole herd tests in the interim. Comparing WGS data to mathematical models showed good correlations between genetic divergence and spatial distance, but poor correspondence to the network of cattle movements or within-herd contacts. Badger isolates showed between zero and four SNP differences from the nearest cattle isolate, providing evidence for recent transmissions between the two hosts. This is the first direct genetic evidence of M. bovis persistence on farms over multiple outbreaks with a continued, ongoing interaction with local badgers. However, despite unprecedented resolution, directionality of transmission cannot be inferred at this stage. Despite the often notoriously long timescales between time of infection and time of sampling for TB, our results suggest that WGS data alone can provide insights into TB epidemiology even where detailed contact data are not available, and that more extensive sampling and analysis will allow for quantification of the extent and direction of transmission between cattle and badgers. © 2012 Biek et al.

Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing

Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism’s phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Tracking a tuberculosis outbreak over 21 years: strain-specific single nucleotide polymorphism-typing combined with targeted whole genome sequencing.

BACKGROUND  Whole genome sequencing (WGS) is increasingly used in molecular-epidemiological investigations of bacterial pathogens, despite cost- and time-intensive analyses. We combined strain-specific single nucleotide polymorphism (SNP)-typing and targeted WGS to investigate a tuberculosis cluster spanning 21 years in Bern, Switzerland. METHODS  Based on genome sequences of three historical outbreak Mycobacterium tuberculosis isolates, we developed a strain-specific SNP-typing assay to identify further cases. We screened 1,642 patient isolates, and performed WGS on all identified cluster isolates. We extracted SNPs to construct genomic networks. Clinical and social data were retrospectively collected. RESULTS  We identified 68 patients associated with the outbreak strain. Most were diagnosed in 1991-1995, but cases were observed until 2011. Two thirds belonged to the homeless and substance abuser milieu. Targeted WGS revealed 133 variable SNP positions among outbreak isolates. Genomic network analyses suggested a single origin of the outbreak, with subsequent division into three sub-clusters. Isolates from patients with confirmed epidemiological links differed by 0-11 SNPs. CONCLUSIONS  Strain-specific SNP-genotyping allowed rapid and inexpensive identification of M. tuberculosis outbreak isolates in a population-based strain collection. Subsequent targeted WGS provided detailed insights into transmission dynamics. This combined approach could be applied to track bacterial pathogens in real-time and at high resolution.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Comparing genotyping algorithms for Illumina's Infinium whole-genome SNP BeadChips

Background Illumina's Infinium SNP BeadChips are extensively used in both small and large-scale genetic studies. A fundamental step in any analysis is the processing of raw allele A and allele B intensities from each SNP into genotype calls (AA, AB, BB). Various algorithms which make use of different statistical models are available for this task. We compare four methods (GenCall, Illuminus, GenoSNP and CRLMM) on data where the true genotypes are known in advance and data from a recently published genome-wide association study. Results In general, differences in accuracy are relatively small between the methods evaluated, although CRLMM and GenoSNP were found to consistently outperform GenCall. The performance of Illuminus is heavily dependent on sample size, with lower no call rates and improved accuracy as the number of samples available increases. For X chromosome SNPs, methods with sex-dependent models (Illuminus, CRLMM) perform better than methods which ignore gender information (GenCall, GenoSNP). We observe that CRLMM and GenoSNP are more accurate at calling SNPs with low minor allele frequency than GenCall or Illuminus. The sample quality metrics from each of the four methods were found to have a high level of agreement at flagging samples with unusual signal characteristics. Conclusions CRLMM, GenoSNP and GenCall can be applied with confidence in studies of any size, as their performance was shown to be invariant to the number of samples available. Illuminus on the other hand requires a larger number of samples to achieve comparable levels of accuracy and its use in smaller studies (50 or fewer individuals) is not recommended.

Prospects for whole genome linkage disequilibrium mapping in domestic dog breeds

Linkage disequilibrium (LD) mapping is commonly used as a fine mapping tool in human genome mapping and has been used with some success for initial disease gene isolation in certain isolated in-bred human populations. An understanding of the population history of domestic dog breeds suggests that LD mapping could be routinely utilized in this species for initial genome-wide scans. Such an approach offers significant advantages over traditional linkage analysis. Here, we demonstrate, using canine copper toxicosis in the Bedlington terrier as the model, that LD mapping could be reasonably expected to be a useful strategy in low-resolution, genome-wide scans in pure-bred dogs. Significant LD was demonstrated over distances up to 33.3 cM. It is very unlikely, for a number of reasons discussed, that this result could be extrapolated to the rest of the genome. It is, however, consistent with the expectation given the population structure of canine breeds and, in this breed at least, with the hypothesis that it may be possible to utilize LD in a genome-wide scan. In this study, LD mapping confirmed the location of the copper toxicosis in Bedlington terrier gene (CT-BT) and was able to do so in a population that was refractory to traditional linkage analysis.

Genetic susceptibility to complex traits : moving towards informed analysis of whole-genome screens

Susceptibility to complex traits, by definition, involves aetiological polymorphisms at multiple genetic loci combined with variable contributions by environmental factors. However, the approaches taken to identifying genetic loci implicated in susceptibility to complex traits frequently overlooks the compounding contribution of multiple loci in favour of highlighting a single gene solely responsible for predisposition. It is only in a small minority of cases that this has resulted in clear disease heritability associated with polymorphisms in a single gene. More often, this approach has led to an accumulation of single-gene associations with minor contributions to disease susceptibility. As the genomic era advances and genome-wide screens become higher in resolution and throughput, the need for simultaneous consideration of multiple loci is becoming more important. With special reference to non-Hodgkin’s lymphoma (NHL), this chapter will overview the current progress made in elucidating genetic polymorphisms associated with disease susceptibility. We also present novel data from a high-resolution single nucleotide polymorphism (SNP) microarray screen for susceptibility loci that are involved in NHL. Using an ‘informed approach’, the findings are highlighted within the context of cellular pathways, and provide insight and new ideas for methods of analysis for genome-wide screens for susceptibility.