Hybrid, ultra-deep metagenomic sequencing enables genomic and functional characterization of low-abundance species in the human gut microbiome

Abstract

A large number of microbial genomes have already been identified from the human gut microbiome, but the understanding of the role of the low-abundance species at the individual level remains challenging, largely due to the relatively shallow sequencing depth used in most studies. To improve genome assembling performance, a HiSeq-PacBio hybrid, ultra-deep metagenomic sequencing approach was used to reconstruct metagenomic-assembled genomes (MAGs) from 12 fecal samples. Such approach combined third-generation sequencing with ultra-deep second-generation sequencing to improve the sequencing coverage of the low-abundance subpopulation in the gut microbiome. Our study generated a total of 44 megabase-scale scaffolds, achieving four single-scaffolds of complete (circularized, no gaps) MAGs (CMAGs) that were the first circular genomes of their species. Moreover, 475 high-quality MAGs were assembled across all samples. Among them, 234 MAGs were currently uncultured, including 24 MAGs that were not found in any public genome database. Additionally, 287 and 77 MAGs were classified as low-abundance (0.1–1%) and extra-low-abundance (<0.1%) gut species in each individual, respectively. Our results also revealed individual-specific genomic features in the MAG profiles, including microbial genome growth rate, selective pressure, and frequency of chromosomal mobile genetic elements. Finally, thousands of extrachromosomal mobile genetic elements were identified from the metagenomic data, including 5097 bacteriophages and 79 novel plasmid genomes. Overall, our strategy represents an important step toward comprehensive genomic and functional characterization of the human gut microbiome at an individual level.

Reconstruction of Small Subunit Ribosomal RNA from High-Throughput Sequencing Data: A Comparative Study of Metagenomics and Total RNA Sequencing

“…The lack of a correlation between abundance and SSU rRNA completeness was in stark contrast to the observed correlation between abundance and genome completeness for metagenomics since the genome of the most abundant species was near-complete while the completeness of all other species' genomes was close to 0%. This illustrates that the abundance of each species in a community has a significant impact on its genome coverage when applying metagenomics, which is in agreement with other studies showing that successful genome reconstruction of low-abundance species typically requires high sequencing depth (Jin et al, 2022;Merrill et al, 2022). In contrast to that, even the genome completeness of the most abundant species was close to 0% when total RNA-Seq was applied, which further confirms that total RNA-Seq reads are naturally enriched for rRNA and consequently do not provide broad genomic coverage.…”

Ultra-deep Sequencing of Hadza Hunter-Gatherers Recovers Vanishing Gut Microbes

“…1A) . Deeper sequencing improves detection of resident microbes 22 (including microbial eukaryotes 23 ) and provides insight from recently developed techniques including in situ growth rate prediction, high-precision strain-tracking, de novo genome recovery, and microdiversity analysis 24,25 .…”

High-Resolution Metagenomics of Human Gut Microbiota Generated by Nanopore and Illumina Hybrid Metagenome Assembly

“…Currently, short-read sequencing is still one of the most cost-effective approaches to study complex microbial communities. Long-read sequencing methods (PacBio and Oxford nanopore), which have been widely applied in the study of single bacteria genomes, were gradually applied in metagenome studies ( Loman et al, 2015 ; Jin et al, 2022 ). To our knowledge, although research groups have applied the third-generation long-read sequencing in metagenome-related studies ( Frank et al, 2016 ; Tsai et al, 2016 ; Driscoll et al, 2017 ; Kerkhof et al, 2017 ; Bertrand et al, 2019 ; Overholt et al, 2020 ), the feasibility of nanopore sequencing in metagenomic studies remains to be unveiled, and the methods of assembling MAGs depending on a HiSeq-Nanopore hybrid metagenomic approach need to improve.…”

“…Since SMRT sequencing is currently inaccessible to most laboratories because of its high cost and laborious preparation procedure, researchers often work with the portable MinION device available from ONT ( Li et al, 2018 ). Although hybrid approach recovered high-quality MAGs from a complex aquifer system Overholt et al (2020) and Jin et al (2022) used MetaBAT2 to assemble 475 high-quality MAGs by HiSeq-PacBio hybrid, there were few reports on the assessment of different hybrid assemblers and Metagenome-assembled genome binning methods.…”