Anomalous phylogenetic behavior of ribosomal proteins in metagenome assembled genomes

Abstract

Metagenomic studies have claimed the existence of novel lineages with unprecedented properties never before observed in prokaryotes. Such lineages include Asgard archaea 1-3 , which are purported to represent archaea with eukaryotic cell complexity, and the Candidate Phyla Radiation (CPR), a novel domain level taxon erected solely on the basis of metagenomic data 4 . However, it has escaped the attention of most biologists that these metagenomic sequences are not assembled into genomes by sequence overlap, as for cultured archaea and bacteria. Instead, short contigs are sorted into computer files by a process called binning in which they receive taxonomic assignment on the basis of sequence properties like GC content, dinucleotide frequencies, and stoichiometric co-occurrence across samples.Consequently, they are not genome sequences as we know them, reflecting the gene content of real organisms. Rather they are metagenome assembled genomes (MAGs). Debates that Asgard data are contaminated with individual eukaryotic sequences 5-7 are overshadowed by the more pressing issue that no evidence exists to indicate that any sequences in binned Asgard MAGs actually stem from the same chromosome, as opposed to simply stemming from the same environment. Here we show that Asgard and CPR MAGs fail spectacularly to meet the most basic phylogenetic criterion 8 fulfilled by genome sequences of all cultured prokaryotes investigated to date: the ribosomal proteins of Asgard and CPR MAGs do not share common evolutionary histories. Their phylogenetic behavior is anomalous to a degree never observed with genomes of real organisms. CPR and Asgard MAGs are binning artefacts, assembled from environments where up to 90% of the DNA is from dead cells 9-12 . Asgard and CPR MAGs are unnatural constructs, genome-like patchworks of genes that have been stitched together into computer files by binning.The sequencing of environmental DNA (metagenomics) has become an essential tool of modern science because it allows microbiologists to uncover the existence of genes and species in environments such as marine sediment or the deep biosphere from which representatives cannot readily be cultured 13,14 . Initially an endeavor involving rRNA sequences 15 , metagenomic investigations have led to the binning-assembly and deposition in databases of MAGs, sequences of similar GC content and stoichiometry across samples. Because rRNA has limited phylogenetic resolution, concatenated sequences of ribosomal proteins (r-proteins) and other universally distributed proteins are commonly used for phylogeny. This practice is well established with over 20 years of tradition, whereby the validity of using concatenated rproteins for phylogeny lies in the reproducible crosschecking result that individual r-proteins 3 from the same sequenced genome give the same or very similar trees [16][17][18][19][20][21] . Based in such precedence, it became common practice to use concatenated r-proteins from sequenced genomes for microbial phylogeny without first crosschecking w...

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.…”

Accurate and Complete Genomes from Metagenomes

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A hot spring Asgard archaeon sheds light on the origin of eukaryotic endosomal system.

“…Previous studies have questioned the purity of the MAGs of Asgard archea (Da Cunha et al, 2017;(Garg et al 2019). While the MAG of Odinarchaeceae Tengchong appears to be high quality based on single-copy gene justification, we cannot rule out the possibility that some of the ESPs predicted in this MAG may be exogenous.…”