AI Article Synopsis
- - Lactic acid bacteria play crucial roles in various industries, and accurately identifying their species from mixed environments is essential but challenging due to genetic similarities among species.
- - Traditional amplicon-based methods, like short-read sequencing of the 16S rRNA gene, often struggle with species-level resolution, which can be improved by using long-read sequencing (LRS) techniques.
- - In experiments comparing different amplicons, LRS using V3-V4 yielded lower accuracy in species identification and higher false positives compared to complete 16S rRNA gene and 16S-ITS-23S rRNA region amplicons, emphasizing the need for careful method selection in microbial community analysis.
Article Abstract
The are lactic acid bacteria harnessed to deliver important outcomes across numerous industries, and their unambiguous, species-level identification from mixed community environments is an important endeavor. Amplicon-based metataxonomics using short-read sequencing of partial 16S rRNA gene regions is widely used to support this, however, the high genetic similarity among species restricts our ability to confidently describe these communities even at genus level. Long-read sequencing (LRS) of the whole 16S rRNA gene or the near complete rRNA operon (16S-ITS-23S) has the potential to improve this. We explored species ambiguity amongst using tool RibDif2, which identified allele overlap when various partial and complete 16S rRNA gene and 16S-ITS-23S rRNA regions were amplified. We subsequently implemented LRS by MinION™ to compare the capacity of V3-V4, 16S and 16S-ITS-23S rRNA amplicons to accurately describe the diversity of a 20-species mock community in practice. analysis identified more instances of allele/species overlap with V3-V4 amplicons ( = 43) compared to the 16S rRNA gene ( = 11) and partial ( = up to 15) or complete ( = 0) 16S-ITS-23S rRNA amplicons. With subsequent LRS of a DNA mock community, 80% of target species were identified using V3-V4 amplicons whilst the 16S rRNA gene and 16S-ITS-23S rRNA region amplicons resulted in 95 and 100% of target species being identified. A considerable reduction in false-positive identifications was also seen with 16S rRNA gene ( = 3) and 16S-ITS-23S rRNA region ( = 9) amplicons compared with V3-V4 amplicons ( = 43). Whilst the target species affected by allele overlap in V3-V4 and 16S rRNA gene sequenced mock communities were predicted by RibDif2, unpredicted species ambiguity was observed in 16S-ITS-23S rRNA sequenced communities. Considering the average nucleotide identity (ANI) between ambiguous species (~97%) and the basecall accuracy of our MinION™ sequencing protocol (96.4%), the misassignment of reads between closely related taxa is to be expected. With basecall accuracy exceeding 99% for recent MinION™ releases, the increased species-level differentiating power promised by longer amplicons like the 16S-ITS-23S rRNA region, may soon be fully realized.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10740194 | PMC |
| http://dx.doi.org/10.3389/fmicb.2023.1290756 | DOI Listing |
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