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RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing

Despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use Illumina to sequence small (~400 bp) hypervariable regions of the 16S rRNA SSU for phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biase...

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Autores principales: Petrone, Joseph R., Rios Glusberger, Paula, George, Christian D., Milletich, Patricia L., Ahrens, Angelica P., Roesch, Luiz Fernando Wurdig, Triplett, Eric W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10402275/
https://www.ncbi.nlm.nih.gov/pubmed/37547696
http://dx.doi.org/10.3389/fmicb.2023.1201064
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author Petrone, Joseph R.
Rios Glusberger, Paula
George, Christian D.
Milletich, Patricia L.
Ahrens, Angelica P.
Roesch, Luiz Fernando Wurdig
Triplett, Eric W.
author_facet Petrone, Joseph R.
Rios Glusberger, Paula
George, Christian D.
Milletich, Patricia L.
Ahrens, Angelica P.
Roesch, Luiz Fernando Wurdig
Triplett, Eric W.
author_sort Petrone, Joseph R.
collection PubMed
description Despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use Illumina to sequence small (~400 bp) hypervariable regions of the 16S rRNA SSU for phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biases of sequencing small portions can be removed, allowing for more accurate classification with deeper taxonomic resolution. With Nanopore sequencing now providing raw simplex reads with quality scores above Q20 using the kit 12 chemistry, the ease, cost, and portability of Nanopore play a leading role in performing differential bacterial abundance analysis. Sequencing the near-entire rrn operon of bacteria and archaea enables the use of the universally conserved operon holding evolutionary polymorphisms for taxonomic resolution. Here, a reproducible and validated pipeline was developed, RRN-operon Enabled Species-level Classification Using EMU (RESCUE), to facilitate the sequencing of bacterial rrn operons and to support import into phyloseq. Benchmarking RESCUE showed that fully processed reads are now parallel or exceed the quality of Sanger, with median quality scores of approximately Q20+, using the R10.4 and Guppy SUP basecalling. The pipeline was validated through two complex mock samples, the use of multiple sample types, with actual Illumina data, and across four databases. RESCUE sequencing is shown to drastically improve classification to the species level for most taxa and resolves erroneous taxa caused by using short reads such as Illumina.
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spelling pubmed-104022752023-08-05 RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing Petrone, Joseph R. Rios Glusberger, Paula George, Christian D. Milletich, Patricia L. Ahrens, Angelica P. Roesch, Luiz Fernando Wurdig Triplett, Eric W. Front Microbiol Microbiology Despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use Illumina to sequence small (~400 bp) hypervariable regions of the 16S rRNA SSU for phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biases of sequencing small portions can be removed, allowing for more accurate classification with deeper taxonomic resolution. With Nanopore sequencing now providing raw simplex reads with quality scores above Q20 using the kit 12 chemistry, the ease, cost, and portability of Nanopore play a leading role in performing differential bacterial abundance analysis. Sequencing the near-entire rrn operon of bacteria and archaea enables the use of the universally conserved operon holding evolutionary polymorphisms for taxonomic resolution. Here, a reproducible and validated pipeline was developed, RRN-operon Enabled Species-level Classification Using EMU (RESCUE), to facilitate the sequencing of bacterial rrn operons and to support import into phyloseq. Benchmarking RESCUE showed that fully processed reads are now parallel or exceed the quality of Sanger, with median quality scores of approximately Q20+, using the R10.4 and Guppy SUP basecalling. The pipeline was validated through two complex mock samples, the use of multiple sample types, with actual Illumina data, and across four databases. RESCUE sequencing is shown to drastically improve classification to the species level for most taxa and resolves erroneous taxa caused by using short reads such as Illumina. Frontiers Media S.A. 2023-07-20 /pmc/articles/PMC10402275/ /pubmed/37547696 http://dx.doi.org/10.3389/fmicb.2023.1201064 Text en Copyright © 2023 Petrone, Rios Glusberger, George, Milletich, Ahrens, Roesch and Triplett. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Petrone, Joseph R.
Rios Glusberger, Paula
George, Christian D.
Milletich, Patricia L.
Ahrens, Angelica P.
Roesch, Luiz Fernando Wurdig
Triplett, Eric W.
RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing
title RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing
title_full RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing
title_fullStr RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing
title_full_unstemmed RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing
title_short RESCUE: a validated Nanopore pipeline to classify bacteria through long-read, 16S-ITS-23S rRNA sequencing
title_sort rescue: a validated nanopore pipeline to classify bacteria through long-read, 16s-its-23s rrna sequencing
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10402275/
https://www.ncbi.nlm.nih.gov/pubmed/37547696
http://dx.doi.org/10.3389/fmicb.2023.1201064
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