Near-complete Lokiarchaeota genomes from complex environmental samples using long and short read metagenomic analyses

Abstract

Asgard archaea is a recently proposed superphylum currently comprised of five recognised phyla: Lokiarchaeota, Thorarchaeota, Odinarchaeota, Heimdallarchaeota and Helarchaeota. Members of this group have been identified based on culture-independent approaches with several metagenome-assembled genomes (MAGs) reconstructed to date. However, most of these genomes consist of several relatively small contigs, and, until recently, no complete Asgard archaea genome is yet available. Large scale phylogenetic analyses suggest that Asgard archaea represent the closest archaeal relatives of eukaryotes. In addition, members of this superphylum encode proteins that were originally thought to be specific to eukaryotes, including components of the trafficking machinery, cytoskeleton and endosomal sorting complexes required for transport (ESCRT). Yet, these findings have been questioned on the basis that the genome sequences that underpin them were assembled from metagenomic data, and could have been subjected to contamination and other assembly artefacts. Even though several lines of evidence indicate that the previously reported findings were not affected by these issues, having access to high-quality and preferentially fully closed Asgard archaea genomes is needed to definitively close this debate. Current long-read sequencing technologies such as Oxford Nanopore allow the generation of long reads in a high-throughput manner making them suitable for their use in metagenomics. Although the use of long reads is still limited in this field, recent analyses have shown that it is feasible to obtain complete or nearcomplete genomes of abundant members of mock communities and metagenomes of various level of complexity. Here, we show that long read metagenomics can be successfully applied to obtain near-complete genomes of low-abundant members of complex communities from sediment samples. We were able to reconstruct six MAGs from different Lokiarchaeota lineages that show high completeness and low fragmentation, with one of them being a nearcomplete genome only consisting of three contigs. Our analyses confirm that the eukaryote-like features previously associated with Lokiarchaeota are not the result of contamination or assembly artefacts, and can indeed be found in the newly reconstructed genomes.Recent advances in long-read DNA sequencing technologies have made long read-based metagenomic sequencing efforts a feasible option (Hao et al., 2018;Nicholls et al., 2019). Long DNA sequencing reads improve the ability to resolve repetitive regions of sequences in de novo assembly, improving the contiguity of genomic assemblies. In metagenomes, repetitive sequences can be present within a single genome, as well as within several genomes that share similar regions (e.g., closely related strains, horizontally transferred genes). Therefore, if reads are long enough to span across repetitive regions, and also cover regions that are unique to a particular genome, their inclusion in metagenome assemblies can help to separate the genomic sequenc...

Visualization of Lokiarchaeia and Heimdallarchaeia (Asgardarchaeota) by Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition (CARD-FISH)

“…The recent success in cultivating the first representative of Lokiarchaeia (“ Candidatus Prometheoarchaeum syntrophicum” MK-D1 [ 14 ]) was a breakthrough in an ongoing discussion about the reliability of metagenome-assembled genomes (MAGs) of Asgardarchaeota ( 15 – 18 ), as it proved the existence of these enigmatic microbes. “ Ca .…”

Complete and validated genomes from a metagenome

“…It was therefore necessary to filter the alignments to ensure only the retention of correctly mapped alignments. We found that filtering long reads with the following criteria (similar to (Caceres et al 2019)) achieved minimal loss of true mapping, while removing almost all of the mismapped reads.…”

“…Furthermore, multiply mapped and chimeric reads required additional filtering to remove. Others have used similar filtering strategies (Caceres et al 2019), but we propose an additional criterion to help reduce the number of chimeric reads. When using the default scoring parameters of minimap2, we propose that a minimum read length of 1000 and a read length to alignment score ratio of less than 0.9 is used for filtering, in addition to percent query coverage of each read.…”

“…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).…”

A lab in the field: applications of real-time, in situ metagenomic sequencing

“…All the protocols were carried out in the lab, and the authors were able to achieve up to ∼5.5 M 16S rRNA full-length sequences with exclusive barcode assignments in a single MinION run. Other groups have used MinION devices for characterizing river water [ 45 ], seawater [ 46 ], and marine sediments [ 47 ] through metagenomic sequencing. Even though these experiments were not implemented in the field, they demonstrated the possibility of obtaining higher sequencing yields ( Table 1 ).…”