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.

Keywords: 16S rDNA amplicon sequencing; Long-read sequencing; Microbial diversity; MinION; Nanopore sequencer.

Fig. 1

Species abundance in the mock community detected by MinION™. Species coverage was calculated by obtaining the fold-change (Log₂) of species-specific read counting against the expected average for the entire community. A coverage bias was assumed when coverage deviation was lower than −1 or higher than 1

Fig. 2

Sequencing coverage versus copies of respective 16S rRNA genes present in the starting material. Scatter plot of the coverage deviation calculated for B. vulgatus, C. beijerinkii, and E. coli against the calculated number of respective 16S copies present in the PCR sample used as starting material of the sequencing reaction

Fig. 3

Per-base accuracy of the mapped reads. a Scatter plot of the GC content observed in mapped reads against the GC obtained from the reference sequences. The dashed line indicates a correlation with Pearson’s r = 1. b Correlation between the GC content observed in mapped reads and coverage bias observed in Fig. 1. c Influence of the GC content observed in mapped reads on mismatch rates calculated after mapping. d Scatter plot of the observed GC content of mapped reads and indel rates calculated after mapping. In all cases the Pearson’s r coefficients and p values supporting such correlations are presented inside the scatter plots and solid lines indicate the tendency of correlations