PacBio sequencing of healthy white shrimp microbiota
Total DNA was extracted from each of these samples (Group 1 - C1, C2 and C3; Group 2 - Ex1, Ex2 and Ex 3) using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA)). Quality and quantity of isolated DNA was checked using Nanodrop spectrophotometer 2000 (Thermofisher Scientific, Massachusetts, USA). Further, integrity of DNA was confirmed by using 1% agarose gel electrophoresis, and DNA concentration of about 200 ng was used for the study. The following primers were used to amplify the full-length sequence (~1500 bp) of the 16S rRNA gene: Forward primer 5'-AGRGTTYGATYMTGGCTCAG-3' (27F) and reverse primer 5'-RGYTACCTTGTTACGACTT-3' (1492R) which were barcoded. PCR was performed using KAPA HiFi HotStart ReadyMix PCR Kit to amplify 1.5 kb fragment. Then, equal concentration of amplified products was pooled and SMRTbell library was prepared following the manufacturers protocol. To eliminate non-specific fragments and adapter dimers, library was purified using AMPure PB beads (Pacific Biosciences, California, USA). Further, library was quantified using Agilent FEMTO pulse analyser (Agilent Technologies, California, USA) and Qubit 3.0 fluorometer (Thermofisher #Q33238, Thermofisher Scientific, Massachusetts, USA) using DNA HS assay kit (Thermofisher #Q32851, Thermofisher Scientific, Massachusetts, USA). A single peak of ~1.9kb (insert + adaptor) was observed and verified by FEMTO pulse analyser which shows electropherogram of amplicon, base yield density, polymerase read length, and subreads lengths. Next, we proceeded for sequencing of SMRT library using V4 primer and 2.1 binding kit in PacBio sequel II.  Movie times for all SMRTcells set for 3 hrs. The RS Remote PacBio and RS Dashboard software were used to configure and monitor PacBio sequencing runs. The full-length 16S rRNA amplicons were sequenced on PacBio Sequel II platform in circular consensus sequence (CCS) mode which resulted in PacBio sequenced reads (subreads.bam files) which was further converted to high fidelity CCS reads (HiFi) and demultiplexed using PacBio SMRT Link tools ccs (version 5.0.0) and lima (version 2.0.0) respectively.