Primer design for universal amplification of the V4 region of 16S rDNA was based on a protocol published by Caporaso and co-workers (Caporaso et al., 2011 (link)). The forward primer (515F) remained unchanged and the reverse primer was largely similar to the Caporaso V4 indexed reverse primers (806R), but with 0–3 random bases and the Illumina sequencing primer binding site added between the amplification primer and the Illumina adapter sequence. We also used primer pairs targeting the V6–V8 and V7–V8 regions (926F-1392R and 1114F-1392R) (Engelbrektson et al., 2010 (link); Lundberg et al., 2012 (link)). Our primer sequences and staggered sequencing strategy are described in detail in the supplementary methods (Additional File 2 ) and Figure S1 (Additional File 1 ).
For each sample (and each replicate for P. suwonensis and synthetic community), three separate 16S rRNA gene amplification reactions targeting a given hypervariable region were performed, pooled together, cleaned up using AMPureXP (Beckman Coulter) magnetic beads and quantified with the Qubit HS assay (Invitrogen). Some samples were also analyzed with a BioAnalyzer 2100 (Agilent) instrument to confirm appropriate amplicon size. Pooled amplicons were then diluted to 10 nM and quantified by qPCR. Illumina amplicon tag (i.e., Itag) sequencing was performed according to standard DOE Joint Genome Institute procedures. Briefly, a density of 500,000 clusters/mm2 was targeted on each MiSeq lane which was also spiked with ~25% of a PhiX control library. Four hundred and fifty four pyrotag sequencing was performed as described (Kunin et al., 2010 (link)). Basecalling was done using Illumina's Real Time Analysis (RTA) software version 1.14.21. Obtained BCL files were converted into QSeq format using Bcl2Qseq 1.9.3, then converted to fastqs.
For each sample (and each replicate for P. suwonensis and synthetic community), three separate 16S rRNA gene amplification reactions targeting a given hypervariable region were performed, pooled together, cleaned up using AMPureXP (Beckman Coulter) magnetic beads and quantified with the Qubit HS assay (Invitrogen). Some samples were also analyzed with a BioAnalyzer 2100 (Agilent) instrument to confirm appropriate amplicon size. Pooled amplicons were then diluted to 10 nM and quantified by qPCR. Illumina amplicon tag (i.e., Itag) sequencing was performed according to standard DOE Joint Genome Institute procedures. Briefly, a density of 500,000 clusters/mm2 was targeted on each MiSeq lane which was also spiked with ~25% of a PhiX control library. Four hundred and fifty four pyrotag sequencing was performed as described (Kunin et al., 2010 (link)). Basecalling was done using Illumina's Real Time Analysis (RTA) software version 1.14.21. Obtained BCL files were converted into QSeq format using Bcl2Qseq 1.9.3, then converted to fastqs.
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