Computational protocol: Microbial communities of the Lemon Creek Glacier show subtle structural variation yet stable phylogenetic composition over space and time

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Protocol publication

[…] Qiime v. 1.6.0 software () was used to parse pyrosequencing reads by barcode, and screen by quality with minlength = 180 and number of homopolymers set to eight. The majority (95%) of resulting reads were 250 bases in size. Reads were error corrected with Ampliconnoise (). Operational taxonomic units (OTUs) were defined de novo with Uclust () using a 0.97 cutoff and the following alterations to the default settings minsize = 1, optimal = T, and pick = T. Representative OTU sequences were picked based on highest abundance. Uchime () was used to de novo detect and remove chimeric OTU sequences. All OTUs were classified to the Silva v.111 database () using the RDP naïve Bayesian method (). To confirm the original classifications, a BLASTn of the top OTUs was performed using the Silva v.111 database. Before calculating alpha and beta diversity measures, the sequence library subsampled to a uniform depth of 1354 sequences per sample (based on the sample with the least number of sequences), and a phylogenetic tree was calculated with FastTree (). Alpha diversity metrics, Shannon, Simpson, Good’s coverage, and phylogenetic diversity were calculated in Qiime. Rare OTUs (<2 occurrences) were removed prior to calculation of Bray–Curtis dissimilarities. Beta diversity patterns were visualized with principal coordinate analysis (PCoA).Alternative measures of phylogenetic distance, SES.mpd (SE size of mean pairwise distances) and SES.mntd (SE size of the mean nearest taxon distance), were calculated with the Picante package () in . Correlations between geochemical measurements and microbial community structure were calculated using mantel tests. Geochemical distance matrices were calculated with vegdist using the vegan package () in R with the Bray–Curtis dissimilatory metric. Mantel tests utilizing Spearman product-moment correlation coefficient were performed with environmental distance matrices and Bray–Curtis derived microbial community patterns from PCoA analysis. In addition, an adonis model (analogous to a non-parametric MANOVA) was used to verify mantel tests and to assess whether changes in microbial communities and geochemistry were independent of sampling location. Beta dispersion analysis (betadisper in vegan) was used to test the intergroup homogeneity between sampling sites. Differences in homogeneity were tested using a tukey post hoc significance test in R. […]

Pipeline specifications

Software tools QIIME, AmpliconNoise, UCLUST, UCHIME, BLASTN, FastTree, Picante
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Hemisus marmoratus
Chemicals Sodium