Ultra-deep, long-read nanopore sequencing of mock microbial community standards.

Samuel M Nicholls,Nicholas J Loman,Shuiquan Tang,Joshua C Quick

doi:10.1093/gigascience/giz043

Abstract

BackgroundLong sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition.FindingsWe sequenced 2 commercially available mock communities containing 10 microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Both communities and the 10 individual species isolates were also sequenced with Illumina technology. We generated 14 and 16 gigabase pairs from 2 GridION flowcells and 150 and 153 gigabase pairs from 2 PromethION flowcells for the evenly distributed and log-distributed communities, respectively. Read length N50 ranged between 5.3 and 5.4 kilobase pairs over the 4 sequencing runs. Basecalls and corresponding signal data are made available (4.2 TB in total). Alignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning.ConclusionsWe present ultra-deep, long-read nanopore datasets from a well-defined mock community. These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines.

Highlights

Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and have great potential for the study of microbial communities
These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines
We present four nanopore sequencing datasets of two microbial community standards, providing a state-of-theart benchmark to accelerate the development of methods for analysing long-read metagenomics data

Summary

Introduction

Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and have great potential for the study of microbial communities. The best approaches for analysis of long-read metagenomic data are unknown. Rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition. The ZymoBIOMICS Microbial Community Standards (CS and CSII) are each composed of ten microbial species: eight bacteria and two yeasts (Table 1). The organisms in CS (hereafter referred to as ‘Even’) are distributed (12%), with the exception of the two yeasts which are each present at 2%. Cell counts from organisms in CSII (‘Log’) community are distributed on a log scale, ranging from 89.1% (Listeria monocytogenes), down to 0.000089% (Staphylococcus aureus)

Methods

Results

Discussion

Conclusion