V4V5 amplicon sequencing of stool sample: subject 231, timepoint 3
Amplicon fragments are PCR-amplified from the DNA in duplicate using separate template dilutions using the high-fidelity Phusion polymerase. A single round of PCR was done using "fusion primers" (Illumina adaptors + indices + specific regions) targeting the V4V5 region of the 16S rRNA gene (515FB=GTGYCAGCMGCCGCGGTAA and 926R=CCGYCAATTYMTTTRAGTTT). PCR products were verified visually by running on a high-throughput Hamilton Nimbus Select robot using Coastal Genomics Analytical Gels. The PCR reactions from the same samples are pooled in one plate, then cleaned-up and normalized using the high-throughput Charm Biotech Just-a-Plate 96-well Normalization Kit. Up to 380 samples were then pooled to make one library which was then quantified fluorometrically before sequencing.