Computational protocol: Novel micelle PCR-based method for accurate, sensitive and quantitative microbiota profiling

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Protocol publication

[…] Bidirectional sequencing of the 16S rRNA gene amplicon libraries was performed using the 454 Genome Sequencer (GS) Junior platform (Roche), with Fasta-formatted sequences being extracted from the GS Junior machine and further processed using the mothur v. 1.33.0 software package. Primer sequences were trimmed and sequences that had an ambiguous base call (N) in the sequence or with lengths smaller than 400 were removed from the analysis. Unique sequences were then aligned against a customized reference alignment based on the SILVA reference alignment release 119 (available at: http://www.mothur.org/wiki/Silva_reference_alignment). The reference sequences were trimmed to only include the V3-V4 region of the 16S rRNA gene using the pcr.seqs command. Sequences that did not align to this region were culled from further analysis and the alignments were trimmed so that the sequences fully overlapped the same alignment coordinates. Potentially chimeric sequences were removed using Uchime, as implemented in mothur. The remaining sequences were classified using the classify.seqs command with the customized SILVA alignment release 119 as reference. Next, sequences were clustered into OTUs at 97% similarity using the default settings of the dist.seq and cluster commands respectively. The classify.otu algorithm was used to get a consensus taxonomy for each OTU. Finally, all SMC samples were rarefied to 1,000 sequences per sample and all skin swab samples were rarefied to 5,000 sequences per sample. The sequencing data that are connected to this article are uploaded to the Sequence Read Archive database with accession number SRP076831. […]

Pipeline specifications

Software tools mothur, UCHIME
Application 16S rRNA-seq analysis
Organisms Bacteria