Next generation sequencing on Illumina NextSeq 550 of waterpipe tobacco
Microbial DNA was extracted from eight tobacco samples using the PowerBiofilm kit (Qiagen). To increase yield, 4 x 0.25 g (1 g per sample) replicate samples of each tobacco sample was lysed by bead beating and then pooled on the column for DNA purification and concentration. The gDNA was prepared for whole genome sequencing (WGS) by first fragmenting the gDNA sample by Covaris ultrasonication. Fragment length was confirmed by Agilent Bioanalyzer High Sensitivity DNA Analysis to be on average 150 bp, and a sequencing library prepared using the KAPA HyperPrep Kit with Amplification (Roche). The libraries were quantified by quantitative PCR (qPCR), normalized to 4 nM, multiplexed, and sequenced on an Illumina NextSeq with a NextSeq high output v2.5 300 cycle Cartridge producing 300 bp paired end reads.