Inclusion of Oxford Nanopore long reads improves all microbial and phage metagenome-assembled genomes from a complex aquifer system

Abstract

Assembling microbial and phage genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. In order to compare the recovery of genomes from microorganisms and their phages from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore sequencing platform. A ssembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from Illumina-only assemblies and a hybrid assembly approach. Strikingly, the hybrid approach more than doubled the number of mid to high-quality MAGs (> 50% completion, < 10% redundancy), generated nearly four-fold more phage genomes, and improved all associated genome metrics relative to

Metagenomics workflow for hybrid assembly, differential coverage binning, transcriptomics and pathway analysis (MUFFIN)

“…Not surprisingly, the vast majority of tools and workflows for the analysis of metagenomic samples are designed around short reads. However, long-read sequencing technologies such as provided by PacBio or Oxford Nanopore Technologies (ONT) retrieve genomes from metagenomic datasets with higher completeness and less contamination 6 . The long-read information bridges gaps in a short-read-only assembly that often occur due to intra- and interspecies repeats 6 .…”

“…However, long-read sequencing technologies such as provided by PacBio or Oxford Nanopore Technologies (ONT) retrieve genomes from metagenomic datasets with higher completeness and less contamination 6 . The long-read information bridges gaps in a short-read-only assembly that often occur due to intra- and interspecies repeats 6 . Complete viral genomes can be already identified from environmental samples without any assembly step via nanopore-based sequencing 7 .…”

“…In particular, with the MinION, ONT offers mobile and cost-effective sequencing device for long reads that paves the way for the real-time analysis of metagenomic samples. Currently, the combination of both worlds (long reads and high-precision short reads) allows the reconstruction of more complete and more accurate metagenome-assembled genomes (MAGs) 6 .…”

What the Phage: A scalable workflow for the identification and analysis of phage sequences

“…This facilitates phage genome recovery in their natural habitat, without the need to culture their hosts to isolate the phages (2) . However, the identification of phages from metagenomes in general and their differentiation from prophages remains a challenge as there is no established computational gold standard (9) .…”

“…However, predicting phages from metagenomes in general and their differentiation from prophages remains a challenge as there is no established computational gold standard (11). Existing prediction tools rely on direct comparison of sequence similarity (12,13), sequence composition (14,15), and models based on these features derived through learning algorithms (12,13,16,17).…”