PacBio sequencing of ITS region of ectomyocrrhizae: isolated from spruce and fir roots
Taken directly from the Intergrated Microbiome Resource website: Amplicon fragments are PCR-amplified from the DNA in duplicate using separate template dilutions (generally 1:1 & 1:10) using the high-fidelity Phusion Plus polymerase. A single round of PCR is done using full-length ITS  "fusion primers" (PacBio barcodes + specific regions) genes 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. 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 190 (PacBio) samples are then pooled to make one library which is then quantified fluorometrically before sequencing.