— Since “clovr-1.0 RC1” this protocol has been replaced with CloVR-16S version 1.1 —
The CloVR-16S sequence analysis protocol supports microbial community composition analysis based on the 16S ribosomal RNA short multiplex amplicon 454 pyrosequencing protocol. It processes sequence reads derived from 454 GS FLX pyrosequencing of pooled PCR products from the amplification of short 16S ribosomal RNA gene fragments with specifically tagged or barcoded primers. Sequence reads are processed using several common phylogenetic tools and protocols:
A) Mothur is used for the distance matrix-based sequence clustering, assignment of representative reads to operational taxonomic units (OTUs), and estimation of within-sample diversity (alpha-diversity).
B) The classifier tool from the Ribosomal Database Project is used for the taxonomic classification of all sequence reads.
C) Qiime is used for sequence processing and phylogenetic analysis based on different different methods, including phylogenetic distance calculations with UniFrac, to estimate the within- (alpha-diversity) and between- (beta-diversity) sample diversity.
D) Metastats and CloVR-implemented R scripts are applied for additional statistical and graphical evaluations of the pipeline results.
CloVR-16S generates a number of text and figure files as output that give an overview of the sample community compositions using common richness and diversity estimators as well as principal coordinate analysis (PCoA) plots, rarefaction curves, OTU heatmaps, statistical comparisons, and clustering diagrams. See SOP for details.