Computational protocol: Comparison of methods for fecal microbiome biospecimen collection

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Protocol publication

[…] After three days of storage for the four methods, genomic DNA was extracted from the 24 fecal aliquots using the PowerLyzer PowerSoil DNA Isolation Kit (Mo Bio Laboratory Inc. CA) following the manufacturer’s protocol. DNA concentration was quantified using the Synergy™ H1M microplate reader (Biotech, VM) and corresponding OD 260/280 ratio was used to check DNA purity. 16S rRNA gene amplicon libraries were generated using primers incorporating FLX Titanium adapters and a sample barcode sequence covering variable region V3 to V4 as we described elsewhere []. The amplicon library was sequenced using the 454 Roche FLX Titanium pyrosequencing system following the manufacturer’s instructions.The QIIME pipeline [] was used to process and filter multiplexed sequence reads. The UCLUST method [] was used to cluster the filtered sequences with ≥97% similarity into Operational Taxonomic Unit (OTUs). Chimeric sequences were identified by ChimeraSlayer [] and removed. Representative sequences from each OTU were assigned taxonomy using the Ribosomal Database Project classifier method [] and the IMG/GG GreenGenes database of microbial genomes. A phylogenetic tree was constructed by applying the FastTree method [] to the representative sequences.Rarefactions of 10 to 8,414 [minimum-maximum sequence depth] randomly selected sequences from each sample were used to calculate the Shannon index, a measure of within sample diversity, and to generate rarefaction plots. Pairwise comparisons of Shannon indices by subject and storage condition were obtained by Monte Carlo permutation. All p-values were adjusted by Bonferroni correction. To measure the diversity among subjects or storage conditions, a single rarefaction was performed at a sequencing depth of 4000 so that all samples were included in analyses. Distance matrices containing all pairwise comparisons were created for unweighted (presence/absence) dissimilarity values using the UniFrac phylogenetic method []. Principal coordinates were computed for the unweighted distance matrices and used to generate Principal Coordinate Analysis plots (PCoA). The non-parametric method, adonis [], was used to identify significant differences in phylogenetic distance variation by subjects and by storage condition. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) for clustering of samples was also carried out on the unweighted distance matrices []. A two-sample t-test was used to test for differences between the within and between group variances, with p-values adjusted by Bonferroni correction. Relative abundances of the three major phyla (Bacteroidetes, Firmicutes, Actinobacteria) were compared for the four methods, using the Mann–Whitney-Wilcoxon test, and compared by subject, using the Kruskal-Wallis test (SAS, version 9.3, SAS Institute, Cary, NC). […]

Pipeline specifications

Software tools QIIME, UCLUST, ChimeraSlayer, RDP Classifier, FastTree, UniFrac
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Homo sapiens