Gs run processor

Amplicon libraries were obtained using barcoded pyrosequencing primers 909F and 1492R, pooled, purified and quantified as described before (Berry et al., 2012 (link)). Pyrosequencing was performed with Titanium reagents on a 454 genome sequencer FLX (Roche, Vienna, Austria). All 20 samples collected on 5 sampling days were subjected to 16S rRNA amplicon sequencing, but one sample from day 1 was excluded from analysis due to low sequencing depth. Reads were quality-filtered using the amplicon pipeline of the GS Run Processor (Roche) and the Pyronoise algorithm in mothur (Schloss et al., 2011 (link)). Operational taxonomic units were generated at 97% sequence identity using UCLUST, as described (Berry et al., 2012 (link)). Amplicon sequencing libraries, which had a mean number of 17 999 reads (range [4415–26 495]) of an average length of 269 nt were re-sampled at 3500 reads for α- and β-diversity analyses using QIIME (Caporaso et al., 2010 (link)).

Total RNA was extracted from stem, rhizome, root, or callus tissue using a modified CTAB method [38 (link)]. RNA quality was based on UV absorption ratios, where only samples with ratios above 2.0 (260/280 nm) and 2.2 (260/230 nm) were used. Poly(A) + RNA purification, cDNA library synthesis, emulsion-based PCR (emPCR) and NGS was performed at the McGill University and Génome Québec Innovation Center (Montréal, Québec) as described [53 (link)]. Briefly, RNA quality and quantity was assessed using NanoDrop ND-1000 (Thermo Scientific, Waltham, Massachusetts) and BioAnalyzer 2100 (Agilent Technologies, Santa Clara, California) instruments, and Poly(A) + RNA purification was done using either a Dynabeads mRNA Purification kit (Invitrogen) or TrueSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, California). cDNA synthesis was performed using either a cDNA Rapid Library kit (Roche, Basel, Switzerland) or TruSeq Stranded mRNA Sample Prep kit (Illumina) depending on the downstream NGS method. For Roche-454 GS-FLX Titanium pyrosequencing, data processing was done using GS Run Processor (Roche) to generate Standard Flowgram Format (SFF) files. For Illumina GA and HiSeq sequencing, HCS 1.4 and CASAVA 1.6-1.8 software suites (Illumina) were used to generate raw fastq reads.

Genes encoding for 16S rRNA were amplified with primers f563-577: 5′-AYTGGGYDTAAAGNG-3′ (Cole et al., 2009 (link)) and r1406-1392: 5′- ACGGGCGGTGTGTRC-3′ (Lane, 1991 ). Each primer contained a 5′-end sequencing adapter (454 Sequencing Application Brief No. 001-2009, Roche); the forward primer also bore different tags (454 Sequencing Technical Bulletin No. 005-2009, Roche) for different samples. The 20-μL PCR mixture contained 0.2 mM dNTPs (Finnzymes, Finland), 0.25 μM primers (Generi Biotech, Czech Republic), 0.1 mg.mL^-1 bovine serum albumin (New England BioLabs, Great Britain), 0.4 U of Phusion Hot Start II DNA Polymerase (Finnzymes, Finland) with the corresponding buffer, and template DNA (10–50 ng). The reaction conditions were as follows: 98°C for 30 s, 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 60 s with final extension at 72°C for 10 min. Obtained PCR products were pooled to approximately the same concentrations of DNA and purified with AMPure XP Beads (Agencourt, Beckman Coulter, USA) following the manufacturer’s instructions in order to remove fragments shorter than 200 bp. Pooled amplicons were sequenced from the forward primer using GS FLX+ chemistry and results were analyzed with gsRunProcessor (Roche).