Accurate sodium bisulfite sequencing in plants.

DNA cytosine methylation is a conserved epigenetic modification frequently correlating with transcriptional silencing in a wide variety of eukaryotic organisms. Sodium bisulfite treatment of DNA converts unmethylated cytosine to uracil, while 5-methylated cytosine is protected. We describe techniques that ensure reliable sequencing data following sodium bisulfite conversion and to avoid common pitfalls such as amplification of unconverted DNA and inclusion of sibling clones.

Quantitative RNA-seq analysis of the campylobacter jejuni transcriptome

Campylobacter jejuni is the most common bacterial cause of foodborne disease in the developed world. Its general physiology and biochemistry, as well as the mechanisms enabling it to colonize and cause disease in various hosts, are not well understood, and new approaches are required to understand its basic biology. High-throughput sequencing technologies provide unprecedented opportunities for functional genomic research. Recent studies have shown that direct Illumina sequencing of cDNA (RNA-seq) is a useful technique for the quantitative and qualitative examination of transcriptomes. In this study we report RNA-seq analyses of the transcriptomes of C. jejuni (NCTC11168) and its rpoN mutant. This has allowed the identification of hitherto unknown transcriptional units, and further defines the regulon that is dependent on rpoN for expression. The analysis of the NCTC11168 transcriptome was supplemented by additional proteomic analysis using liquid chromatography-MS. The transcriptomic and proteomic datasets represent an important resource for the Campylobacter research community. © 2011 SGM.

A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo.

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.

Identifying cancer subtypes in glioblastoma by combining genomic, transcriptomic and epigenomic data

We present a nonparametric Bayesian method for disease subtype discovery in multi-dimensional cancer data. Our method can simultaneously analyse a wide range of data types, allowing for both agreement and disagreement between their underlying clustering structure. It includes feature selection and infers the most likely number of disease subtypes, given the data. We apply the method to 277 glioblastoma samples from The Cancer Genome Atlas, for which there are gene expression, copy number variation, methylation and microRNA data. We identify 8 distinct consensus subtypes and study their prognostic value for death, new tumour events, progression and recurrence. The consensus subtypes are prognostic of tumour recurrence (log-rank p-value of $3.6 \times 10^{-4}$ after correction for multiple hypothesis tests). This is driven principally by the methylation data (log-rank p-value of $2.0 \times 10^{-3}$) but the effect is strengthened by the other 3 data types, demonstrating the value of integrating multiple data types. Of particular note is a subtype of 47 patients characterised by very low levels of methylation. This subtype has very low rates of tumour recurrence and no new events in 10 years of follow up. We also identify a small gene expression subtype of 6 patients that shows particularly poor survival outcomes. Additionally, we note a consensus subtype that showly a highly distinctive data signature and suggest that it is therefore a biologically distinct subtype of glioblastoma. The code is available from https://sites.google.com/site/multipledatafusion/