Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

Abstract

The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems.

Keywords: MinION; long amplicon sequencing; long-read sequencing; microbial diversity; nanopore sequencer; ribosomal operon.

Figure 1:

Organization of the rrn region in bacteria. (A) Hypothetical transcriptional arrangements expected for rrn and tested experimentally using 2 sets of primer pairs (see small arrows drawn in each configuration). (B) Agarose gel electrophoresis of PCR reactions performed under the 2 hypothetical arrangements of rrn; lanes: (i) 1 kb ruler (Fermentas), (ii) PCR reaction from the top configuration in (A), (iii) PCR reaction from the bottom configuration in (A). The GelAnalyser Java application was used to perform the band size analysis of the 1 kb ruler standard (C) and the amplicons obtained from human faecal DNA (D).

Figure 2:

Variability of the rrn region and its functional domains. The rrn database, compiled after parsing more than 67 000 draft and complete bacterial genomes, was assessed by clustering analysis at different levels of sequence identity: 97% (white bars), 98% (light grey bars), 99% (dark grey bars), and 100% (black bars). For comparative aims, the functional DNA sequences encoded into the rrn region were also individually studied. The normalized diversity (y-axis) resulted from calculating the number of clusters obtained for each analysis, normalized with the median sizes of the respective regions in terms of kb and referenced against the value obtained for 16S sequences clustered at 97%, the canonical threshold for species assignment.

Figure 3:

Microbial structure of the mock communities. (A and B) Microbial species and respective relative proportions determined to be present in the HM782D and D6305 mock communities, respectively, following the analysis of raw data obtained from rrn amplicon sequencing in MinION™ and chemistry R9. (C and D) Comparative analysis of the expected microbial species and proportions against the data obtained after mapping reads generated by a 4.5-kbp amplicon PCR and sequenced in the MinION™ device with R9 and R9.4 chemistries, for HM782D and D6305, respectively. (E) Linear correlation analysis of relative read proportions obtained for all bacterial species present in HM872D and D6305 mock communities with R9 and R9.4 chemistries.