Dataset Information

Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer.

ABSTRACT: BACKGROUND:The miniaturised and portable DNA sequencer MinION™ has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinION™ system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences. RESULTS:Using R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90 % of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using 'universal' primers. CONCLUSIONS:Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinION™ DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.

SUBMITTER: Benitez-Paez A

PROVIDER: S-EPMC4730766 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer.

Benítez-Páez Alfonso A Portune Kevin J KJ Sanz Yolanda Y

GigaScience 20160128

<h4>Background</h4>The miniaturised and portable DNA sequencer MinION™ has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinION™ system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length ...[more]

PMID: 26823973

Similar Datasets

Project description: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.

Project description:BackgroundAccurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1-V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control.ResultsBased on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92.ConclusionsThese findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification.

Dataset Information

Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer.

Publications

Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets