Recovery of metagenome-assembled microbial genomes from a full-scale biogas plant of food waste by pacific biosciences high-fidelity sequencing

Abstract

BackgroundAnaerobic digestion (AD) is important in treating of food waste, and thousands of metagenome-assembled genomes (MAGs) have been constructed for the microbiome in AD. However, due to the limitations of the short-read sequencing and assembly technologies, most of these MAGs are grouped from hundreds of short contigs by binning algorithms, and the errors are easily introduced.ResultsIn this study, we constructed a total of 60 non-redundant microbial genomes from 64.5 Gb of PacBio high-fidelity (HiFi) long reads, generated from the digestate samples of a full-scale biogas plant fed with food waste. Of the 60 microbial genomes, all genomes have at least one copy of rRNA operons (16S, 23S, and 5S rRNA), 54 have ≥18 types of standard tRNA genes, and 39 are circular complete genomes. In comparison with the published short-read derived MAGs for AD, we found 23 genomes with average nucleotide identity less than 95% to any known MAGs. Besides, our HiFi-derived genomes have much higher average contig N50 size, slightly higher average genome size and lower contamination. GTDB-Tk classification of these genomes revealed two genomes belonging to novel genus and four genomes belonging to novel species, since their 16S rRNA genes have identities lower than 95 and 97% to any known 16S rRNA genes, respectively. Microbial community analysis based on the these assembled genomes reveals the most predominant phylum was Thermotogae (70.5%), followed by Euryarchaeota (6.1%), and Bacteroidetes (4.7%), and the most predominant bacterial and archaeal genera were Defluviitoga (69.1%) and Methanothrix (5.4%), respectively. Analysis of the full-length 16S rRNA genes identified from the HiFi reads gave similar microbial compositions to that derived from the 60 assembled genomes.ConclusionHigh-fidelity sequencing not only generated microbial genomes with obviously improved quality but also recovered a substantial portion of novel genomes missed in previous short-read based studies, and the novel genomes will deepen our understanding of the microbial composition in AD of food waste.

Highly accurate metagenome-assembled genomes from human gut microbiota using long-read assembly, binning, and consolidation methods

“…2). Our study demonstrates that metagenome assembly with HiFi reads can produce large numbers of highly complete MAGs, corroborating the findings of previous studies (Bickhart et al 2022;Feng et al 2022;Kato et al 2022;Kim et al 2022;Zhang et al 2022;Benoit et al 2023;Jiang et al 2023;Saak et al 2023;Schaerer et al 2023;Masuda et al 2024). Across all method combinations, we found up to 595 total MAGs, 277 HQ-MAGs, and 98 single-contig HQ-MAGs (including 70 circular).…”

Comparative genomic analyses of Cutibacterium granulosum provide insights into genomic diversity

“…The lineage_wf workflow of CheckM v1.0.18 ( Parks et al, 2015 ) was employed to evaluate the quality of metagenomic bins. Only those C. granulosum MAGs with completeness ≥90% and contamination <5% were considered “high quality” ( Singleton et al, 2021 ; Jiang et al, 2022 ) and retained for downstream analyses.…”

“…Following this, all the genomes, including those originally presented in this study and those obtained from public database, underwent evaluation using the “lineage_wf” workflow within CheckM v1.07 ( Parks et al, 2015 ). To standardize the genome assembly quality and minimize potential discrepancies in pan-genome analysis, only those genomes meeting the criteria of near completeness (completeness ≥ 90%) and low contamination (<5%) ( Singleton et al, 2021 ; Jiang et al, 2022 ) were retained for further analysis.…”

Pacbio HiFi sequencing sheds light on key bacteria contributing to deadwood decomposition processes

“…Third-generation (long-read) sequencing allows much more reliable assembly and thus holds the promise for recovering high-quality MAGs or complete/circular prokaryotic genomes from complex microbiome samples (Bickhart et al, 2022;Sereika et al, 2022), enabling an accurate assessment of their ecological roles. The two main long-read platforms include the cost-affordable Oxford Nanopore, albeit with inferior accuracy (Brown et al, 2021;Sereika et al, 2022), and the PacBio platform, offering highly accurate consensus sequencing (HiFi) reads without the need for further polishing (Jiang et al, 2023;Kim et al, 2022).…”