Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution.
16S rRNA
Gut microbiota
MinION™
Nanopore sequencing
Journal
BMC microbiology
ISSN: 1471-2180
Titre abrégé: BMC Microbiol
Pays: England
ID NLM: 100966981
Informations de publication
Date de publication:
26 01 2021
26 01 2021
Historique:
received:
19
05
2020
accepted:
18
01
2021
entrez:
27
1
2021
pubmed:
28
1
2021
medline:
25
2
2023
Statut:
epublish
Résumé
Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.
Sections du résumé
BACKGROUND
Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples.
RESULTS
We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition.
CONCLUSIONS
Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.
Identifiants
pubmed: 33499799
doi: 10.1186/s12866-021-02094-5
pii: 10.1186/s12866-021-02094-5
pmc: PMC7836573
doi:
Substances chimiques
DNA, Bacterial
0
RNA, Ribosomal, 16S
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
35Subventions
Organisme : Japan Society for the Promotion of Science
ID : JP19K09339
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