Abstract
BackgroundBesides the development of comprehensive tools for high-throughput 16S ribosomal RNA amplicon sequence analysis, there exists a growing need for protocols emphasizing alternative phylogenetic markers such as those representing eukaryotic organisms.ResultsHere we introduce CloVR-ITS, an automated pipeline for comparative analysis of internal transcribed spacer (ITS) pyrosequences amplified from metagenomic DNA isolates and representing fungal species. This pipeline performs a variety of steps similar to those commonly used for 16S rRNA amplicon sequence analysis, including preprocessing for quality, chimera detection, clustering of sequences into operational taxonomic units (OTUs), taxonomic assignment (at class, order, family, genus, and species levels) and statistical analysis of sample groups of interest based on user-provided information. Using ITS amplicon pyrosequencing data from a previous human gastric fluid study, we demonstrate the utility of CloVR-ITS for fungal microbiota analysis and provide runtime and cost examples, including analysis of extremely large datasets on the cloud. We show that the largest fractions of reads from the stomach fluid samples were assigned to Dothideomycetes, Saccharomycetes, Agaricomycetes and Sordariomycetes but that all samples were dominated by sequences that could not be taxonomically classified. Representatives of the Candida genus were identified in all samples, most notably C. quercitrusa, while sequence reads assigned to the Aspergillus genus were only identified in a subset of samples. CloVR-ITS is made available as a pre-installed, automated, and portable software pipeline for cloud-friendly execution as part of the CloVR virtual machine package (http://clovr.org).ConclusionThe CloVR-ITS pipeline provides fungal microbiota analysis that can be complementary to bacterial 16S rRNA and total metagenome sequence analysis allowing for more comprehensive studies of environmental and host-associated microbial communities.
Highlights
Besides the development of comprehensive tools for high-throughput 16S ribosomal RNA amplicon sequence analysis, there exists a growing need for protocols emphasizing alternative phylogenetic markers such as those representing eukaryotic organisms
CloVR implementation The CloVR-internal transcribed spacer (ITS) standard operating procedure (SOP) for ITS amplicon sequence analysis for fungal diversity studies was implemented within the Cloud Virtual Resource (CloVR) framework [18,19]
Implementation of the ITS analysis pipeline into CloVR provides the advantage of portability across multiple platforms, access to pre-installed and pre-configured software tools bundled into an automated analysis pipeline, and seamless support of online academic or commercial cloud computing services
Summary
Besides the development of comprehensive tools for high-throughput 16S ribosomal RNA amplicon sequence analysis, there exists a growing need for protocols emphasizing alternative phylogenetic markers such as those representing eukaryotic organisms. Recent studies have begun to explore fungal diversity through amplification of the internal transcribed spacer (ITS) region of the eukaryotic rRNA operon [6,7,8,9,10,11,12,13,14,15,16], but there remains a need for communityaccepted standards and more rigorous sequence analysis protocols. The “ITS region” refers to the contiguous region of ITS1, the 5.8S gene, and ITS2 (see Additional file 1: Figure S1 for a diagram) Because this region can be amplified across a broad range of fungal organisms, using methodologies developed and tested for 16S rRNA amplicon sequence analysis as a reference bears the potential to introduce comparable tools for the characterization of the fungal microbiota [17]
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