Profiling bacterial communities by MinION sequencing of ribosomal operons

Abstract

Background: An approach utilizing the long-read capability of the Oxford Nanopore MinION to rapidly sequence bacterial ribosomal operons of complex natural communities was developed. Microbial fingerprinting employs domain-specific forward primers (16S rRNA subunit), reverse primers (23S rRNA subunit), and a high-fidelity Taq polymerase with proofreading capabilities. Amplicons contained both ribosomal subunits for broad-based phylogenetic assignment (~ 3900 bp of sequence), plus the intergenic spacer (ITS) region (~ 300 bp) for potential strain-specific identification.

Results: To test the approach, bacterial rRNA operons (~ 4200 bp) were amplified from six DNA samples employing a mixture of farm soil and bioreactor DNA in known concentrations. Each DNA sample mixture was barcoded, sequenced in quadruplicate (n = 24), on two separate 6-h runs using the MinION system (R7.3 flow cell; MAP005 and 006 chemistry). From nearly 90,000 MinION reads, roughly 33,000 forward and reverse sequences were obtained. This yielded over 10,000 2D sequences which were analyzed using a simplified data analysis pipeline based on NCBI Blast and assembly with Geneious software. The method could detect over 1000 operational taxonomic units in the sample sets in a quantitative manner. Global sequence coverage for the various rRNA operons ranged from 1 to 1951x. An iterative assembly scheme was developed to reconstruct those rRNA operons with > 35x coverage from a set of 30 operational taxonomic units (OTUs) among the Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, and Gemmatimonadetes. Phylogenetic analysis of the 16S rRNA and 23S rRNA genes from each operon demonstrated similar tree topologies with species/strain-level resolution.

Conclusions: This sequencing method represents a cost-effective way to profile microbial communities. Because the MinION is small, portable, and runs on a laptop, the possibility of microbiota characterization in the field or on robotic platforms becomes realistic.

Keywords: Microbiota; MinION; Ribosomal operon; Species/strain-level resolution.

Fig. 1

Schematic of the experimental design to test the MinION’s ability to resolve environmental rRNA operons in a quantitative manner indicating the simplified data analysis pipeline

Fig. 2

a Average frequency of operational taxonomic units (OTUs) by DMegaBlast screening of the 2D sequence reads. Error bars representing the standard deviation are in the positive direction only. b Rarefaction analysis of the MinION reads (closed black triangle) compared with published Illumina (open circles), 454 (open squares), and PacBio (closed orange triangle) studies as indicated for the symbol codes

Fig. 3

Quantitative response of the four most abundant OTU reads (farm soil and bioreactor; y axis) versus the percentage of input DNA from the end-member DNAs (x axis). Error bars represent the standard deviation

Fig. 4

Example of iterative consensus building of rRNA operons using LastZ methods. The number of sequences used to build the consensus, and the disagreements (black bars) within the consensus are indicated

Fig. 5

Phylogenetic tree reconstruction for the Proteobacteria 16S rRNA genes from farm soil, a gray water bioreactor, and closely related matching reference sequences using FastTree for 1292 unambiguously aligned bases. The MinION sequences are indicated by boxes and stars

Fig. 6

Phylogenetic tree reconstruction for the Proteobacteria 23S rRNA genes from farm soil, a gray water bioreactor, and closely related matching reference sequences using FastTree for 1767 unambiguously aligned bases. The MinION sequences are indicated by boxes and stars