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

Abstract

Background Long 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. Findings We 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. Conclusions We 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.

Accurate and complete genomes from metagenomes

“…Cold Spring Harbor Laboratory Press on July 4, 2020 -Published by genome.cshlp.org Downloaded from Arumugam et al 2019), hybrid assembly (Wick et al 2017), and mock community standards (Nicholls et al 2019) are emerging. Short-read-based assembly strategies often report fragmented contigs caused by repeat elements that exceed short-read lengths, which is an issue long-read sequencing overcomes, improving the quality of genomes from metagenomics (Arumugam et al 2019).…”

Metagenome-Assembled Genome Binning Methods with Short Reads Disproportionately Fail for Plasmids and Genomic Islands

“…These approaches have been instrumental in developing our understanding of the distribution and evolutionary history of AMR genes [3,4,5], as well as tracking pathogen outbreaks [6]. Although long-read DNA technologies (e.g., Oxford Nanopore [7], PacBio [8]) are now being used for metagenomic sequencing [9,10], high-throughput sequencing of relatively short reads (150-250bp) in platforms such as the Illumina MiSeq still dominates metagenomics. These reads can be directly analysed using reference databases and a variety of homology search tools (e.g., [11,12,13,14]).…”

“…While long-read DNA sequencing technology (e.g., Oxford Nanopore [7], PacBio [8]) is now being used for metagenomic sequencing [9,10], high-throughput sequencing of relatively short reads (150-250bp) in platforms such as the Illumina MiSeq currently dominate metagenomic analyses. Inference of taxonomic and functional diversity can be assessed directly from sequenced reads using reference databases and BLAST-based sequence similarity search tools (e.g.…”

“…In accordance with the recommendation in the CAMI challenge [48] the genomes were randomly assigned a relative abundance following a log-normal distribution (μ = 1, σ = 2). Plasmid copy number estimates could not be accurately found for all organisms, therefore, plasmids were randomly assigned a copy number regime: low (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), medium (20-100), or high (500-1000) at a 2:1:1 rate. Within each regime, the exact copy number was selected using an appropriately scaled gamma distribution (α = 4, β = 1) truncated to the regime range.…”

Extensive genomic and transcriptomic variation defines the chromosome-scale assembly ofHaemonchus contortus, a model gastrointestinal worm

“…However, high-throughput sequencing has significantly impacted the rate at which draft genomes are produced and, together with the recent introduction of long-read sequencing from Pacific Biosciences (PacBio) and Oxford Nanopore, the quality and contiguity of assemblies of both large and small genomes has completely changed. Now, the ability to generate highly contiguous and closed prokaryote genomes is becoming routine, even from metagenomic samples containing multiple strains or species [2,3] , and high-quality chromosome-scale genomes for a number of eukaryotic species are rapidly becoming available [4][5][6] . These technologies generally require microgram quantities of high molecular weight DNA, and the success of a genome assembly is highly dependent on the degree of polymorphism present in the DNA that is sequenced.…”