Computational protocol: Sjögren-Like Lacrimal Keratoconjunctivitis in Germ-Free Mice

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Protocol publication

[…] The MiSeq pipeline in Mothur was used to process sequence data []. The MiSeq pipeline for Mothur (essentially as described [] and the MiSEQ SOP version 28 March 2013 (http://www.mothur.org/wiki/MiSeq_SOP) was used to process sequence data. Following alignment of forward and reverse reads, sequences were quality-trimmed and aligned to the Silva 16S rRNA gene reference database formatted for mothur. Sequences were then trimmed to overlap the same region of the 16S rRNA gene, pre-clustered to clusters with ≥99% identity, and potentially chimeric sequences were identified and removed using the mothur-impelementation of uchime. Sequences were classified according to the mothur-formatted ribosomal database project version 9 (August 2013) using the Bayesian classifier in Mothur, and those sequences classified as Eukarya, Archaea, Chloroplast, Mitochondria or unknown were removed. The sequence data was then filtered to remove any sequences present only once in the dataset. After building a distance matrix from the remaining sequences with the default parameters in mothur, sequences were clustered into operational taxonomic units (OTUs) with ≥97% using the average-neighbor algorithm in Mothur. Taxonomic assignments for each OTU are the majority consensus taxonomic assignment for each sequence within the OTU. Before analysis with the phyloseq package of R, additional filtering of the OTU table was done to remove rare OTUs. Namely, those OTUs present in less than three samples and that contained less than 25 sequences were removed. These filtering steps reduced the number of OTUs from 6306 to 923 but only decreased the number of sequences per sample by a mean of 0.7 ± 0.4% (range, 0.1–2.3%). Analysis and visualization of microbiome communities were conducted in R utilizing the phyloseq package to import sample data and calculate α- and β-diversity metrics []. […]

Pipeline specifications

Software tools mothur, UCHIME, phyloseq
Application 16S rRNA-seq analysis
Organisms Bacteria, Mus musculus, Homo sapiens
Diseases Keratoconjunctivitis, Dry Eye Syndromes