Long-read MinION™ sequencing of 16S and 16S-ITS-23S rRNA genes provides species-level resolution of Lactobacillaceae in mixed communities

• DOI: 10.3389/fmicb.2023.1290756
• Publication Date: 2023-12-07T15:20:04Z
• Authors: Sandra A. Olivier; Michelle K. Bull; Mikael Lenz Strube; Robert Murphy; Tom Ross; John P. Bowman; Belinda Chapman
• Keywords: 23S ribosomal RNA; Amplicon

The Lactobacillaceae are lactic acid bacteria harnessed to deliver important outcomes across numerous industries, and their unambiguous, species-level identification from mixed community environments is an endeavor. Amplicon-based metataxonomics using short-read sequencing of partial 16S rRNA gene regions widely used support this, however, the high genetic similarity among species restricts our ability confidently describe these communities even at genus level. Long-read (LRS) whole or near complete operon (16S-ITS-23S) has potential improve this. We explored ambiguity amongst in-silico tool RibDif2, which identified allele overlap when various 16S-ITS-23S were amplified. subsequently implemented LRS by MinION™ compare capacity V3-V4, amplicons accurately diversity a 20-species mock in practice. In-silico analysis more instances allele/species with V3-V4 (n = 43) compared 11) up 15) 0) amplicons. With subsequent DNA community, 80% target whilst region resulted 95 100% being identified. A considerable reduction false-positive identifications was also seen 3) 9) 43). Whilst affected sequenced predicted unpredicted observed communities. Considering average nucleotide identity (ANI) between ambiguous (~97%) basecall accuracy protocol (96.4%), misassignment reads closely related taxa be expected. exceeding 99% for recent releases, increased differentiating power promised longer like region, may soon fully realized.