Computational protocol: Regional Mucosa-Associated Microbiota Determine Physiological Expression of TLR2 and TLR4 in Murine Colon

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Protocol publication

[…] The 16S rRNA gene sequences were analyzed as described previously . Briefly, raw sequence data were processed using the RDP pipeline server at the Ribosomal Database Project II (RDP-II) website (http://rdp.cme.msu.edu/pipeline) by base-calling, quality-trimming and alignment. Potential chimeric sequences were checked using the SimRank 2.7 package available through the RDP and excluded . The classifier analysis tool of RDP-II and NEBI BLAST tool were used to assign 16S rRNA sequences to the taxonomical hierarchy at different levels. The program DOTUR with the furthest neighbor algorithm was used to group sequences into operational taxonomical units (OTUs) or phylotypes which represented the number of 16S rRNA sequence similarity groupings. A 97% cutoff value was used such that sequences with more than 97% similarity were considered the same. For principal coordinate analysis (PCA), all 16S rRNA gene sequences were imported into the ARB software package and aligned into a phylogenetic tree which was used to perform clustering analysis using online UniFrac without abundance weighting . The P-Test in the UniFrac was performed to determine whether each sample was significantly different from others. All sequences were deposited in the GenBank nucleotide sequence databases under the accession numbers HQ318942-HQ322000. […]

Pipeline specifications

Software tools Simrank, DOTUR, ARB, UniFrac
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Mus musculus