16S sequencing of Mus musculus: cecal content
Library Preparation: 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 various sub-regions of the 16S (Bacteria/Archaea), 18S (Eukarya, incl. Fungi) or ITS2 (Fungi only) genes with multiplexing which allows up to 380 samples to be run. Preparation for PacBio Sequel 2 is essentially the same, except full-length 16S/18S/ITS fusion primers (PacBio adaptors + barcodes + specific regions) are used instead. PCR products are 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 (Illumina) or 190 (PacBio) samples are then pooled to make one library which is then quantified fluorometrically before sequencing.  Next-Generation Sequencing: Amplicon samples and small genomes are run on our Illumina MiSeq using 300+300 bp paired-end V3 chemistry which allows for overlap and stitching togther of paired amplicon reads into one full-length read of higher quality. Output is generally ~20-22 million raw reads and ~13 Gb of sequence = ~50,000 reads per sample for 380 amplicons. Long amplicons and de novo genomes are run on our PacBio Sequel 2 using new 8M chips which output roughly 240-320 Gb of sequence per cell of variable (long) lengths. For larger metagenomic/metatranscriptomic projects, we run on our Illumina NextSeq 550 using 150+150 bp paired-end "high output" chemistry generating up to ~400 million raw reads and ~120 Gb of sequence.