Annotated bacterial chromosomes from frame-shift-corrected long-read metagenomic data

Abstract

Background Short-read sequencing technologies have long been the work-horse of microbiome analysis. Continuing technological advances are making the application of long-read sequencing to metagenomic samples increasingly feasible. Results We demonstrate that whole bacterial chromosomes can be obtained from an enriched community, by application of MinION sequencing to a sample from an EBPR bioreactor, producing 6 Gb of sequence that assembles into multiple closed bacterial chromosomes. We provide a simple pipeline for processing such data, which includes a new approach to correcting erroneous frame-shifts. Conclusions Advances in long-read sequencing technology and corresponding algorithms will allow the routine extraction of whole chromosomes from environmental samples, providing a more detailed picture of individual members of a microbiome. Electronic supplementary material The online version of this article (10.1186/s40168-019-0665-y) contains supplementary material, which is available to authorized users.

Analysis procedures for assessing recovery of high quality, complete, closed genomes from Nanopore long read metagenome sequencing

“…Here we extend our previous work [16, 22] on recovering metagenome-assembled genomes from long read data obtained from enrichment (continuous culture) reactors inoculated with activated sludge microbial communities. Enrichment reactor communities [23, 24] offer a moderate level of complexity compared to the inoculum communities [25] and so are realistic, yet tractable, systems to use for developing approaches for recovery and validation of MAG analysis using long read data.…”

“…The reactor was sampled on day 267 of the operation, with both long read (Nanopore MinION) and short read (Illumina Miseq 301bp PE) sequencing data from the same DNA aliquot. These data have been previously published by us [16] and are available via NCBI BioProject accession .This data set is referred to below as the PAO1 data. A lab–scale sequencing batch reactor, inoculated with activated sludge from a full scale wastewater treatment plant (Public Utilities Board, Singapore), was operated using alternative carbon sources to enrich for polyphosphate accumulating organisms (PAO). The reactor was sampled on April 6, 2018, gDNA extracted and both long read (Nanopore MinION) and short read (Illumina HiSeq2500 251bp PE) data obtained from the same DNA aliquot.…”

“…The reactor was sampled on day 267 of the operation, with both long read (Nanopore MinION) and short read (Illumina Miseq 301bp PE) sequencing data from the same DNA aliquot. These data have been previously published by us [16] and are available via NCBI BioProject accession .This data set is referred to below as the PAO1 data.…”

“…Genomic DNA in the case of the samples from PAO1, PAO3A, PAO3B and PAO4 was extracted from the sampled biomass as described previously by us [16], briefly, we used the FastDNA™SPIN Kit for Soil (MP Biomedicals), using 2× bead beating with a FastPrep homogenizer (MP Biomedicals). Extracted gDNA from the PAO1, PAO3A and PAO3B samples was then size–selected on a BluePippin DNA size selection device (SageScience) using a BLF–7510 cassette with high pass filtering with a 8 kbp cut–off.…”

“…New long read analysis methods [13, 14] and binning algorithms designed for long read metagenome data [15] have also appeared, anticipating the future expansion of metagenome data generated from these new instruments. More recent studies [16, 17, 18, 19, 20, 21, 22] have collectively established that full length (or near full length genomes) can be recovered from long read sequencing of complex communities, which sets the stage for further development of genome–resolved long read metagenomics.…”