CloVR-16S: Phylogenetic microbial community composition analysis based on 16S ribosomal RNA amplicon sequencing – standard operating procedure, version1.0

Abstract

The CloVR-16S pipeline employs several well-known phylogenetic tools and protocols for the analysis of 16S rRNA sequence datasets:A) Mothur [1] – a C++ - based software package used for clustering 16SrRNA sequences into operational taxonomic units (OTUs). Mothur creates OTUs using a matrix that describes pairwise distances between representative aligned sequences and subsequently estimates within-sample diversity (alpha diversity);B)The Ribosomal Database (RDP) naive Bayesian classifier [2] assigns each 16S sequence to a reference taxonomy with associated empirical probabilities based on oligonucleotide frequencies;C) Qiime [3] – a python-based workflow package, allowing for sequence processing and phylogenetic analysis using different methods including phylogenetic distance (UniFrac [4]) for within- (alpha diversity) and between- (beta diversity) sample analysis;D) Metastats [5] and custom R scripts used to generate additional statistical and graphical evaluations.Though some of the different protocols used in CloVR-16S overlap in purpose (e.g. OTU clustering), the end-user benefits from their overall complementary nature as they focus on different aspects of the phylogenetic analysis. CloVR-16S accepts as input raw multiplex 454-pyrosequencer output, i.e. pooled pyrotagged sequences from multiple samples, or alternatively, pre-processed sequences from multiple samples in separate files. This protocol is available in CloVR beta versions 0.5 and 0.6.