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

Background The miniaturised and portable DNA sequencer MinIONTM 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 MinIONTM 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 MinIONTM 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.

[1]  Michael Wagner,et al.  probeBase—an online resource for rRNA-targeted oligonucleotide probes: new features 2007 , 2006, Nucleic Acids Res..

[2]  Petri Auvinen,et al.  Inconsistent Denoising and Clustering Algorithms for Amplicon Sequence Data , 2014, J. Comput. Biol..

[3]  Miriam L. Land,et al.  Evaluation and validation of de novo and hybrid assembly techniques to derive high-quality genome sequences , 2014, Bioinform..

[4]  Joshua Quick,et al.  Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella , 2015, Genome Biology.

[5]  P. Ashton,et al.  MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island , 2014, Nature Biotechnology.

[6]  Ribosomal RNA (rRNA) , 2004 .

[7]  Paul Horton,et al.  Parameters for accurate genome alignment , 2010, BMC Bioinformatics.

[8]  Aaron R. Quinlan,et al.  Poretools: a toolkit for analyzing nanopore sequence data , 2014, bioRxiv.

[9]  Aaron R Quinlan,et al.  A reference bacterial genome dataset generated on the MinION™ portable single-molecule nanopore sequencer , 2014, GigaScience.

[10]  Eoin L. Brodie,et al.  Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB , 2006, Applied and Environmental Microbiology.

[11]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[12]  Alexander S. Mikheyev,et al.  A first look at the Oxford Nanopore MinION sequencer , 2014, Molecular ecology resources.

[13]  Christian von Mering,et al.  Limits to robustness and reproducibility in the demarcation of operational taxonomic units. , 2015, Environmental microbiology.

[14]  Christian von Mering,et al.  Ecological Consistency of SSU rRNA-Based Operational Taxonomic Units at a Global Scale , 2014, PLoS Comput. Biol..

[15]  William A. Walters,et al.  Erratum to: Stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity , 2015, Microbiome.

[16]  Pelin Yilmaz,et al.  The SILVA ribosomal RNA gene database project: improved data processing and web-based tools , 2012, Nucleic Acids Res..

[17]  Elmar Pruesse,et al.  SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes , 2012, Bioinform..

[18]  Alvin T. Liem,et al.  Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer , 2015, GigaScience.

[19]  Mick Watson,et al.  poRe: an R package for the visualization and analysis of nanopore sequencing data , 2015, Bioinform..

[20]  A. Klindworth,et al.  Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies , 2012, Nucleic acids research.