Generation and analysis of a mouse intestinal metatranscriptome through Illumina based RNA-sequencing.

PloS One
Xuejian XiongJohn Parkinson

Abstract

With the advent of high through-put sequencing (HTS), the emerging science of metagenomics is transforming our understanding of the relationships of microbial communities with their environments. While metagenomics aims to catalogue the genes present in a sample through assessing which genes are actively expressed, metatranscriptomics can provide a mechanistic understanding of community inter-relationships. To achieve these goals, several challenges need to be addressed from sample preparation to sequence processing, statistical analysis and functional annotation. Here we use an inbred non-obese diabetic (NOD) mouse model in which germ-free animals were colonized with a defined mixture of eight commensal bacteria, to explore methods of RNA extraction and to develop a pipeline for the generation and analysis of metatranscriptomic data. Applying the Illumina HTS platform, we sequenced 12 NOD cecal samples prepared using multiple RNA-extraction protocols. The absence of a complete set of reference genomes necessitated a peptide-based search strategy. Up to 16% of sequence reads could be matched to a known bacterial gene. Phylogenetic analysis of the mapped ORFs revealed a distribution consistent with ribosomal RNA, the majority fr...Continue Reading

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Datasets Mentioned

BETA
SRA051354

Methods Mentioned

BETA
RNA-Seq
Illumina sequencing
antisense oligonucleotides
phosphotransferase
PCR
scraping
electrophoresis

Software Mentioned

megaBLAST
Ensemble
BLASTP
BLAST
InParanoid
BWA
iPath
BLASTX
Velvet

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