Accurate and complete genomes from metagenomes

Abstract

Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes.

Microbial genomes from non-human primate gut metagenomes expand the primate-associated bacterial tree of life with over 1000 novel species

“…Despite the genomes assembled from metagenomes are not free from assembly problems [64,65] and should be considered for complementing rather than substituting those obtained from isolate sequencing, large-scale metagenomic assembly efforts to mine available metagenomic data showed to be crucial to uncover the whole diversity of environment-specific microbiomes [11,66,67], especially in these under-investigated hosts. Indeed, given the efficiency of metagenomic assembly pipelines [67,68] and the availability of complementary tools to explore the microbial diversity in a microbiome [69,70], the limiting factor appears to be the technical difficulties in sampling primates in the wild.…”

Advantages and Limits of Metagenomic Assembly and Binning of a Giant Virus

“…Of note, depending on the binning approach, contigs with a combined length of 15 kb (MetaBAT 2, without differential coverage binning) to 351 kb (CONCOCT) were wrongly assigned to the Fadolivirus MAG. This so-called "misbinning" is clearly a limitation of the metagenomic approach (38) and demands careful downstream evaluation based on other criteria, such as gene content (39). In contrast to the Fadolivirus reference genome assembly, our metagenomic workflow did not yield a closed genome.…”

A method for achieving complete microbial genomes and improving bins from metagenomics data

“…To demonstrate this method, we have obtained 34 circular CPR genomes, one circular Margulisbacteria genome, and two circular megaphage genomes from 19 public and published metagenomics datasets. To our knowledge, only 41 other CPR circularized genomes have been published from 9 studies [14][15][16][17][18][19][20][21][22], so we believe this to be the largest presentation of circularized CPR genomes in a single study. With this set, we demonstrate findings that would likely be difficult without a large number of unique circularized genomes, including that ribosomal genes are likely not operonic in the majority of CPR and finding diverged forms of RNase P RNA in CPR species.…”

“…Candidate Phyla Radiation (CPR) species are often classified as having 60-80% completeness by these tools, even for circular genomes. To overcome challenges of binning, scientists have started to assemble circular, complete genomes from metagenomes [14][15][16][17][18][19][20][21][22][23], which are also called CMAGs (complete metagenomic-assembled genomes) [24]. In comparison to genome bins, a high quality reference collection that controls for misassemblies and is composed of circular genomes (1) provides more accurate inference of identity and estimation of capabilities of uncultured microbes within complex microbiomes, (2) allows more accurate taxonomic assessment of the composition of these microbiomes through better linkage of marker genes in single organisms, (3) provides high-quality scaffolds on which reads can be assembled, both to allow measures of strain variation within a microbiome study and to aid in better assembly of reads across many microbiome samples, and (4) affords the ability to study synteny and genomic context of genes in these organisms.…”

“…Circularization of genomes helps assure that there is likely no contamination in the assemblies. Despite the advantages of circularizing genomes, very few metagenomics studies to date (<30) have published circular genomes [14,15,24,[16][17][18][19][20][21][22][23].…”