Full length 16S rRNA gene sequencing (V1-V9)
To identity microbial community in our defined gnotobiotic zebrafish larvae (6 dpf), near full length V1-V9 16S rDNA Single Molecule Real Time (SMRT) sequencing was performed on the Revio system (Pacific Biosciences) on extracted DNA samples. Samples were prepared using Zymo Researchs Quick-16STM Full-Length Library Prep Kit using the following primers: 27f AGRGTTYGATYMTGGCTCAG and 1492r RGYTACCTTGTTACGACTT. Samples were prepared via 1-step PCR and were pooled by equal volume. A MagBead cleanup was performed on the pooled library which was then prepared for sequencing using the PacBio HiFi prep kit 96 with the SMRTbell barcoded adapter plate 3.01. Final libraries underwent the PacBio binding and cleanup steps using the Revio SPRQ Polymerase kit. Prepared libraries were sequenced on the PacBio Revio with a 12hr run time. SMRTbell barcoded adapters were removed on instrument during CCS. Post-CCS HiFi reads underwent secondary demultiplexing via Lima (v2.12.0). Generated BAM files from the secondary demultiplexing were converted to fastq.gz files using samtools (v1.17) and pigz (v2.6).