Metagenomic assembly through the lens of validation: recent advances in assessing and improving the quality of genomes assembled from metagenomes

Abstract

Metagenomic samples are snapshots of complex ecosystems at work. They comprise hundreds of known and unknown species, contain multiple strain variants and vary greatly within and across environments. Many microbes found in microbial communities are not easily grown in culture making their DNA sequence our only clue into their evolutionary history and biological function. Metagenomic assembly is a computational process aimed at reconstructing genes and genomes from metagenomic mixtures. Current methods have made significant strides in reconstructing DNA segments comprising operons, tandem gene arrays and syntenic blocks. Shorter, higher-throughput sequencing technologies have become the de facto standard in the field. Sequencers are now able to generate billions of short reads in only a few days. Multiple metagenomic assembly strategies, pipelines and assemblers have appeared in recent years. Owing to the inherent complexity of metagenome assembly, regardless of the assembly algorithm and sequencing method, metagenome assemblies contain errors. Recent developments in assembly validation tools have played a pivotal role in improving metagenomics assemblers. Here, we survey recent progress in the field of metagenomic assembly, provide an overview of key approaches for genomic and metagenomic assembly validation and demonstrate the insights that can be derived from assemblies through the use of assembly validation strategies. We also discuss the potential for impact of long-read technologies in metagenomics. We conclude with a discussion of future challenges and opportunities in the field of metagenomic assembly and validation.

Accurate and Complete Genomes from Metagenomes

“…However, these genomes are readily adopted by the scientific community for a wide variety of investigations, and errors will propagate. In fact, the proposal of a new nomenclature for large swaths of the tree of life based largely on MAGs (Parks et al 2018) brings a potential crisis into focus. We conclude that it is imperative that complete, curated genomes are recovered for all major lineages (including those that lack any isolated representative).…”

“…In combination, these considerations improve predictions of metabolic capacities. Further, assembled sequences provide information about gene synteny and better resolve taxonomic profiles (e.g., by providing sets of proteins for taxonomy based on concatenated proteins encoded in the same genome (e.g., Parks et al 2018) ). These improvements can overcome misleading interpretations of short-read data (Ackelsberg et al 2015;Afshinnekoo et al 2015) .…”

“…Fragmentation is doubly problematic because small fragments are hard to bin accurately and gaps result in incomplete gene inventories. Fragmentation can arise due to the presence of duplicated sequences (e.g., transposases, rRNA operons), but the most pronounced problems usually are the result of coexisting closely related strains that confuse de Bruijn graph based assemblers (Olson et al 2017) . For example, although Prochlorococcus and SAR11 are among the most abundant bacteria in ocean habitats, the co-occurence of closely related strains (Giovannoni 2017) leads to very fragmented MAGs and poor representation in the final datasets (Delmont et al 2018;Tully et al 2018) .…”

“…For example, although Prochlorococcus and SAR11 are among the most abundant bacteria in ocean habitats, the co-occurence of closely related strains (Giovannoni 2017) leads to very fragmented MAGs and poor representation in the final datasets (Delmont et al 2018;Tully et al 2018) . Of the three commonly used metagenomic assemblers, IDBA_UD, MEGAHIT, metaSPAdes (Greenwald et al 2017) , metaSPAdes was best designed to handle micro-variations between fragments from related strains to generate longer composite sequences (Olson et al 2017) . However, care should be taken when undertaking detailed analyses (e.g., biochemical testing) of open reading frames generated in this way as they may be chimeric.…”

Long read-based de novo assembly of low complex metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system

“…This was illustrated by a recent study, which implicated that long repeat regions of prokaryotic genomes can harbor genes that may confer fitness advantages for the strain [8]. While the major challenge of complete de novo genome assembly of individual strains is the resolution of all genomic repeats [8,9], this situation becomes even more complex for metagenomics: here, the reads do not only have to span intragenomic repeats but also intergenomic repeats, i.e., genomic segments shared by different strains [10]. So far, WMGS studies have mainly relied on short read next-generation sequencing (NGS) technologies, which are not able to span intra-and intergenomic repeats.…”