Functional Profiling of Unfamiliar Microbial Communities Using a Validated De Novo Assembly Metatranscriptome Pipeline

Abstract

BackgroundMetatranscriptomic landscapes can provide insights in functional relationships within natural microbial communities. Analysis of complex metatranscriptome datasets of these communities poses a considerable bioinformatic challenge since they are non-restricted with a varying number of participating strains and species. For RNA-Seq data a standard approach is to align the generated reads to a set of closely related reference genomes. This only works well for microbial communities for which a near complete catalogue of reference genomes is available at a small evolutionary distance. In this study, we focus on the design of a validated de novo metatranscriptome assembly pipeline for single-end Illumina RNA-Seq data to obtain functional and taxonomic profiles of murine microbial communities.ResultsThe here developed de novo assembly metatranscriptome pipeline combined rRNA removal, IDBA-UD assembler, functional annotation and taxonomic classification. Different assemblers were tested and validated using RNA-Seq data from an in silico generated mock community and in vivo RNA-Seq data from a restricted microbial community taken from a mouse model colonized with Altered Schaedler Flora (ASF). Precision and recall of resulting gene expression, functional and taxonomic profiles were compared to those obtained with a standard alignment method. The validated pipeline was subsequently used to generate expression profiles from non-restricted cecal communities of four C57BL/6J mice fed on a high-fat high-protein diet spiked with an RNA-Seq data set from a well-characterized human sample. The spike in control was used to estimate precision and recall at assembly, functional and taxonomic level of non-restricted communities.ConclusionsA generic de novo assembly pipeline for metatranscriptome data analysis was designed for microbial ecosystems, which can be applied for microbial metatranscriptome analysis in any chosen niche.

Comparative Transcriptomic Approaches Exploring Contamination Stress Tolerance in Salix sp. Reveal the Importance for a Metaorganismal de Novo Assembly Approach for Nonmodel Plants

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Comparative proteomics ofRhizopus delemarATCC 20344 unravels the role of amino acid catabolism in fumarate accumulation

“…Following the wokflow suggested and validated by Davids et al (2016), the RNA seq reads were filtered using SortMeRNA v1.9 (Kopylova, Noé & Touzet, 2012), cutadapt v1.2.1 (Martin, 2011) and PRINSEQ v0.20.2 (Schmieder & Edwards, 2011). De novo assembly of the reads that passed the quality filtering was performed using the IDBA-UD assembler v1.1 (Peng et al, 2012).…”

Interactive functional networks in microbiota

“…Today obtaining a high quality single genome can still be a challenge [21], and with a community genome assembly approach these challenges can multiply. Examples are chimeric assemblies between genomes due to presence of multiple strains of the same species (although miss-assemblies should not occur very often [182]), and a low coverage of low abundant species. At this point, it is also important to consider the mapping rate after the assembly.…”

“…The bioinformatics workflow was adapted from Davids et al [182]. SortMeRNA v1.9 [209] software was used to screen the metatranscriptome data against all databases deployed with the program and to remove rRNA reads.…”

“…The general workflow for data quality assessment and filtering was adapted from [182]. rRNA reads were removed with SortMeRNA v1.9 [209] and all included databases.…”