Similar protocols

Pipeline publication

[…] nd the V6-V8 region for archaea) were amplified as previously described by Kittelmann et al. () and sequenced using 454 GS FLX Titanium chemistry at Eurofins MWG Operon (Ebersberg, Germany). For the RNA Amplicon-seq, total RNA was first reverse-transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen) with random primers following procedures for first-strand cDNA synthesis. Then, partial 16S rRNA amplicons of bacteria and archaea were generated using the same primers as for the DNA Amplicon-seq and sequenced using a 454 pyrosequencing platform at McGill University and Génome Québec Innovation Centre (Montreal, QC, Canada)., The sequence data quality was checked using the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The program Trimmomatic (version 0.32; Bolger et al., ) was used to trim residual artificial sequences, cut bases with quality scores below 20, and remove reads shorter than 50 bp. The filtered reads were then sorted to enrich 16S rRNA fragments for taxonomic identification and mRNA reads for functional analysis (not reported in this study) using SortMeRNA (version 1.9; Kopylova et al., ) by aligning with the rRNA reference databases SILVA_SSU (release 119), SILVA_LSU (release 119; Quast et al., ), and Rfam 11.0 (Burge et al., ). After the 16S rRNA sequences were enriched, downstream analyses were performed using the mothur program (version 1.31.2; Schloss et al., ) according to the procedures (http://www.mothur.org/wiki/MiSeq_SOP) described by Kozich et al. (), with modifications. For taxonomic identification, regionally enriched reference datasets were built for bacteria and archaea (mothur command: pcr.seqs). Specifically, sequences belonging to the V1-V3 region (mean length: 466 bp) were extracted from the aligned Greengenes 16S […]

Pipeline specifications

Software tools FastQC, Trimmomatic, SortMeRNA