Short-read next-generation sequencing of 16s rRNA gene amplicons for characterizing amplicon sequence variants (ASVs) and determination of gene copy numbers using ion Torrent platform.

Abstract

The short-amplicon sequencing of hypervariable regions of 16S rRNA gene is the widely used method for bacterial identification and microbiota profiling. Bacteria possess multiple copies of 16S rRNA gene and may contain single nucleotide variations (SNPs) or amplicon sequence variants (ASVs). The ASVs based determination of microbial taxa can be better representation over operational taxonomic units (OTUs). Illumina based NGS platforms are mostly used to define the ASVs whereas Ion-torrent platform is commonly used for diagnostic purposes. We aimed to identify bacterial isolates having ASVs and infer the copy numbers of16S rRNA gene using short read sequencing performed on the Ion Gene Studio S5 NGS platform. The V2-V3 regions of 16S rRNA gene were amplified from the bacterial isolates and subjected to NGS. Further, the sequences produced by NGS were compared with those generated from Sanger sequencing. The bacterial isolates were identified and characterize during ASVs. The copy number of the 16S rRNA gene was established in Gram-negative isolates. Assigning bacterial taxa based on ASVs would provide a more accurate representation of the variant data.