16SrRNA of Bacteria
Amplicons were normalized at 5ng/L, pooled and purified with 1.3X SMRTbell cleanup beads. The SMRTbell library was prepared using a SMRTbell prep kit 3.0, following the Preparing multiplexed amplicon libraries using SMRTbell prep kit 3.0 protocol. A Femto Pulse (Agilent Technologies, Santa Clara, CA, USA) assay was used to assess the fragments size distribution. Pools were carried into the enzymatic reactions to remove the single-strand overhangs and to repair any damage that may be present on the DNA backbone. An A-tailing reaction followed by the overhang adapter ligation was conducted to generate the SMRTbell template. After nuclease treatment, pools were cleanup with 1.3X SMRTbell cleanup beads. The SMRTbell libraries were quality inspected and quantified on a Femto Pulse (Agilent Technologies) and a Qubit fluorimeter with Qubit dsDNA HS reagent Assay kit (Life Technologies). A ready-to-sequence SMRTbell Polymerase Complex was created using a Binding Kit 3.1 (PacBio). The PacBio Sequel II instrument was programmed to load a 150pM library and sequenced in CCS mode on a PacBio SMRTcell 8M, with the Sequencing Plate 2.0 (Pacific Biosciences, Menlo Park, CA, USA) with 0.5 hour of pre-extension time and acquiring one movie of 10 hours.