title: Full Length 16S Sequencing: Cat Rectal Microbiome
design: DNA was extracted using the QIAamp PowerFecal Pro. Full-length 16S amplicons were generated with primers from PacBio (forward 5-AGRGTTYGATYMTGGCTCAG-3 and reverse 5-RGYTACCTTGTTACGACTT-3) and the 2x Roche KAPA HiFi Hot Start Ready Mix. The amplicons were concatenated 12-fold with the PacBio Kinnex Concatenation Kit and Kinnex PCR 12-Fold Kit and libraries evaluated with Qubit and a Fragment Analyzer. Libraries were sequenced on the Pacbio Revio instrument with a 30 hr movie time. Circular Consensus analysis (CCS), de-concatenation, and demultiplexing were done using SMRTlink 13.0 using reads processed with 99.9% accuracy. Note, samples with LR7 designation were part of an attempt to sequence a fungal ITS_LR7 amplicon on a Kinnex 16S run, which did not work.