V6 Bacterial community composition in river samples

The hypervariable Bv6 region of the bacterial 16S rRNA gene (~60 bp) was amplified in two separate PCR reactions. The first amplified the Bv6 rRNA region with a set of primers that target all major bacterial groups using 25 cycles of amplification. DNA for sequencing was pooled from three independent PCR reactions, cleaned, and quality checked. Triplicate 33 uL reactions of the first PCR were pooled and the amplicon and negative control evaluated using a Caliper LabChip GX. Pooled amplicons were cleaned using a 96-well QIAquick PCR purification kit (Qiagen, Valencia CA) to remove primer dimer, salts and nucleotides. Following evaluation (as above), 5 uL of cleaned product were used as the template in the second PCR, where unique custom fusion primers (Illumina adaptor and 12 different inline barcodes for the forward primers plus 8 specific indices for reverse primers annealed to conserved regions flanking the Bv6 sequence) were added to the conserved region flanking the Bv6 sequence using an additional 5 cycles following the same condition as above. The PCR product and negative control for each sample were evaluated (as above) and each product was quantified using PicoGreen DNA assay (Life Technologies, Carlsbad CA). Products were pooled in equimolar fashion based on the PicoGreen quantity and the resulting pool was size selected to a range of 200-250bp using a 1.5% agarose Pipin prep cassette (SageScience, Beverly MA). The resulting product of size selection was cleaned with a Minelute column (Qiagen, Valencia CA) and quantified with a qPCR assay (Kapa Biosystems, Woburn MA). Samples were sequenced using v3 chemistry on an Illumina HiSeq 1000 with a custom 108-cycle paired-end read and six cycle index read recipe. Samples were part of a 96-sample multiplex run on the same lane with 50% of lane capacity dedicated to the 96 samples.