16S rRNA sequencing of Mus musculus: adult female gut microbiota and malaria
Amplicon fragments are PCR-amplified from the DNA in duplicate using separate template dilutions (generally 1:1 & 1:10) using the high-fidelity Phusion polymerase. A single round of PCR is done using "fusion primers" (Illumina adaptors + indices + specific regions) targeting either the 16S V6-V8  regions with multiplexing which allows up to 380 samples to be run. PCR products are verified visually by running on a high-throughput Hamilton Nimbus Select robot using Coastal Genomics Analytical Gels. Any samples with failed PCRs (or spurious bands) are re-amplified by optimizing PCR conditions to produce correct bands in order to complete the sample plate before continuing. 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. The (up to) 380 samples are then pooled to make one library which is then quantified fluorometrically before sequencing (Protocol from cgeb-imr.ca/protocols.html).