Accurate and Complete Genomes from Metagenomes

Abstract

51Genomes are an integral component of the biological information about an organism and, logically, the more 52 complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were 53 reconstructed from pure (monoclonal) cultures and the first reported sequences were manually curated to 54 completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights 55 for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as 56 community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation 57 by obtaining metagenome-assembled genomes (MAGs), but gaps, local assembly errors, chimeras and 58 contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome 59 curation to improve and in some cases achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few 60 CMAGs have been generated, although notably some are from very complex systems such as soil and 61 sediment. Through analysis of ~7000 published complete bacterial isolate genomes, we verify the value of 62 cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. 63Interestingly, analysis of cumulative GC skew identified potential mis-assemblies in some reference genomes 64 of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could 65 be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary 66 analyses can be based on very high-quality genomes. 67 68 69 70 2017), and even human blood (Moustafa et al. 2017) amenable to shotgun metagenomic surveys and recovery 87 of MAGs. Although incomplete, draft MAGs represent a major advance over knowing nothing about the genes 88 and pathways present in an organism, and led to the discovery of new metabolisms. For example, the complete 89 oxidation of ammonia to nitrate via nitrite (i.e., comammox) was determined by the detection of necessary 90 genes in a single MAG (Daims et al. 2015; van Kessel et al. 2015). MAGs are often derived from uncultivated 91 organisms that can be quite distantly related to any isolated species, which is a clear advantage of MAGs 92 (Becraft et al. 2017; Garg et al. 2019). For this reason, genome-resolved metagenomics has been critical for 93 more comprehensive descriptions of bacterial and archaeal diversity and the overall topology of the Tree of 94

Large Freshwater Phages with the Potential to Augment Aerobic Methane Oxidation

“…The confirmed phage scaffolds were predicted for protein-coding genes using Prodigal 58 and searched against the HMM databases of proteins involved in methane metabolisms. The phage scaffolds with pmoC genes and also a minimum sequencing coverage of 20X were manually curated to completion and/or to fix any assembly errors following the pipelines as described previously 39 . Manual fixation of assembled errors and extension to completion of phage genomes are time-consuming but essential to reveal their metabolic potentials.…”

“…PmoC-phages have been overlooked in previous studies, in part because of focus on high level patterns such as global distribution, diversity and host specificity rather than gene inventories 38 and in part because of the high similarity between phage-associated and bacterial PmoC can fragment assemblies. The reconstruction of pmoC-phage genomes from multiple distinct habitats highlights the power of genome-resolved metagenomics and also the necessity of manual genome curation for accuracy 39 .…”

SkewIT: Skew Index Test for detecting mis-assembled bacterial genomes

“…Although GC skew has been used as an indicator of the replication strand in thousands of bacterial genomes, it is rarely used as a means to validate genome assemblies. However, the association between GC skew and replication is strong enough that when a genome has a major mis-assembly such as a translocation or inversion, the GC skew plot is clearly disrupted [19]. We decided to use GC skew to probe the 15,000+ complete bacterial genomes in NCBI's Refseq library.…”

Complete and validated genomes from a metagenome

“…Generating complete genomes from metagenomes is often difficult or impossible with Illumina-only sequencing because of gaps, local assembly errors, and contamination by fragments from other genomes. Of the thousands of MAGs that have been deposited to public databases, only approximately 60-70 have been completed (Chen et al 2020), many of these belonging to Candidate Phyla Radiation due to their smaller genome size.…”

“…Recent studies have been successful at improving contiguity using long reads (Caceres et al 2019), and some groups have been reported completing more than a single genome (Stewart et al 2019;Moss, Maghini, and Bhatt 2020) using Nanopore sequencing. However, there is still a very small percentage of MAGs that have been reported in the literature as complete (~60-70 from 7000 submitted MAGs) (Chen et al 2020). Chen et al, 2020 also proposed a general workflow for validating circularized genomes.…”