SURVEILLANCE OF FOODBORNE PATHOGENS FROM NORTH-EAST INDIA, PHASE II - Zufang
The 16S rRNA gene amplicon libraries were prepared using the Oxford Nanopore Technologies (ONT) 16S Barcoding Kit 24 V14 (SQK-16S024) according to the manufacturers protocol. Briefly, 1050 ng of purified genomic DNA from each sample was used as input for PCR amplification of the full-length 16S rRNA gene using ONTs pre-mixed 16S primers (covering the V1V9 regions). The PCR amplification was carried out using LongAmp Taq 2X Master Mix (New England Biolabs) in a final volume of 50 L per reaction, under the following thermal cycling conditions: initial denaturation at 95C for 3 min; followed by 35 cycles of 95C for 20 sec, 55C for 30 sec, and 65C for 2 min; and a final extension at 65C for 5 min. Barcoding of amplicons was performed using ONTs native barcode primers provided in the kit, and equimolar amounts of barcoded amplicons were pooled. The pooled amplicons were purified using AMPure XP beads (Beckman Coulter) at a 1:1 bead-to-sample ratio and quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The final pooled library was prepared by adding the sequencing adapters using the ONT ligation-based chemistry. Sequencing was performed on PromethION 2 Solo platform (Oxford Nanopore Technologies, Oxford, UK) using R10.4.1 flow cells (FLO-PRO114) on MinKNOW (version 24.06.14, ONT).