Dear Reader,
Please note, this page contains templates of our various Next Generation Sequencing protocols. You’re welcome to copy and appropriately adjust for your purposes and/or publications.
=== PacBio Full Length 16s protocol ====
Soil samples were sent to Inqaba Biotechnical Industries, a commercial NGS service provider, for sequencing.
Genomic DNA was extracted using the ZymoBiomics kit.
Briefly, genomic DNA samples were polymerase chain reaction (PCR) amplified using barcoded universal primer pair 27F and 1492R targeting the V1 -V9 region of the bacterial 16S rRNA gene. The resulting barcoded amplicons were individually quantified, pooled equimolar and Ampure PB bead-based purification step was performed on the pool. The pooled sample was then used to prepare a SMRTbell library following manufacture protocol starting at step 3-Pool and Cleanup, page 9 of the protocol (link to PacBio protocol). The SMRTbell library was qualified using Fragment Analyzer (Agilent) and quantified using Qubit HS dsDNA Assay (Thermo Fisher).
The annealing of the sequencing primer and polymerase binding steps were performed following PacBio SMRT link v13.1 software protocol to prepare the library for sequencing on PacBio Revio system using SMRT cell 25M.
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