TY - JOUR

T1 - Long-read MinION™ sequencing of 16S and 16S-ITS-23S rRNA genes provides species-level resolution of Lactobacillaceae in mixed communities

AU - Olivier, Sandra A.

AU - Bull, Michelle K.

AU - Strube, Mikael Lenz

AU - Murphy, Robert

AU - Ross, Tom

AU - Bowman, John P.

AU - Chapman, Belinda

N1 - Publisher Copyright: Copyright © 2023 Olivier, Bull, Strube, Murphy, Ross, Bowman and Chapman.

PY - 2023

Y1 - 2023

N2 - 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 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 Lactobacillaceae 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 Lactobacillaceae using in-silico 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 Lactobacillaceae mock community in practice. In-silico analysis identified more instances of allele/species overlap with V3–V4 amplicons (n = 43) compared to the 16S rRNA gene (n = 11) and partial (n = up to 15) or complete (n = 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 (n = 3) and 16S-ITS-23S rRNA region (n = 9) amplicons compared with V3–V4 amplicons (n = 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.

AB - 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 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 Lactobacillaceae 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 Lactobacillaceae using in-silico 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 Lactobacillaceae mock community in practice. In-silico analysis identified more instances of allele/species overlap with V3–V4 amplicons (n = 43) compared to the 16S rRNA gene (n = 11) and partial (n = up to 15) or complete (n = 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 (n = 3) and 16S-ITS-23S rRNA region (n = 9) amplicons compared with V3–V4 amplicons (n = 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.

KW - amplicon sequencing

KW - Lactobacillaceae

KW - long read sequencing

KW - metataxonomics

KW - microbiome

KW - nanopore

KW - rRNA

U2 - 10.3389/fmicb.2023.1290756

DO - 10.3389/fmicb.2023.1290756

M3 - Journal article

C2 - 38143859

AN - SCOPUS:85180439628

VL - 14

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

M1 - 1290756

ER -