Improved microbial genomes and gene catalog of the chicken gut from metagenomic sequencing of high-fidelity long reads

Abstract

Background Due to the importance of chicken production and the remarkable influence of the gut microbiota on host health and growth, tens of thousands of metagenome-assembled genomes (MAGs) have been constructed for the chicken gut microbiome. However, due to the limitations of short-read sequencing and assembly technologies, most of these MAGs are far from complete, are of lower quality, and include contaminant reads. Results We generated 332 Gb of high-fidelity (HiFi) long reads from the 5 chicken intestinal compartments and assembled 461 and 337 microbial genomes, of which 53% and 55% are circular, at the species and strain levels, respectively. For the assembled microbial genomes, approximately 95% were regarded as complete according to the “RNA complete” criteria, which requires at least 1 full-length ribosomal RNA (rRNA) operon encoding all 3 types of rRNA (16S, 23S, and 5S) and at least 18 copies of full-length transfer RNA genes. In comparison with the short-read-derived chicken MAGs, 384 (83% of 461) and 89 (26% of 337) strain-level and species-level genomes in this study are novel, with no matches to previously reported sequences. At the gene level, one-third of the 2.5 million genes in the HiFi-derived gene catalog are novel and cannot be matched to the short-read-derived gene catalog. Moreover, the HiFi-derived genomes have much higher continuity and completeness, as well as lower contamination; the HiFi-derived gene catalog has a much higher ratio of complete gene structures. The dominant phylum in our HiFi-assembled genomes was Firmicutes (82.5%), and the foregut was highly enriched in 5 genera: Ligilactobacillus, Limosilactobacillus, Lactobacillus, Weissella, and Enterococcus, all of which belong to the order Lactobacillales. Using GTDB-Tk, all 337 species-level genomes were successfully classified at the order level; however, 2, 35, and 189 genomes could not be classified into any known family, genus, and species, respectively. Among these incompletely classified genomes, 9 and 49 may belong to novel genera and species, respectively, because their 16S rRNA genes have identities lower than 95% and 97% to any known 16S rRNA genes. Conclusions HiFi sequencing not only produced metagenome assemblies and gene structures with markedly improved quality but also recovered a substantial portion of novel genomes and genes that were missed in previous short-read-based metagenome studies. The novel genomes and species obtained in this study will facilitate gut microbiome and host–microbiota interaction studies, thereby contributing to the sustainable development of poultry resources.

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

“…Recently, the use of highly accurate HiFi long reads and the related assemblers have greatly improved the assembly quality of MAGs, which even generated hundreds of circular genomes ( Bickhart et al, 2022 ; Feng et al, 2022 ). Besides, high proportion of novel genomes were generated, which recovered 89 novel genomes (26% of the total assembled genomes) from the chicken gut microbiomes by using 332 Gb of HiFi reads ( Zhang Y. et al, 2022 ). In addition, HiFi long reads also enabled the analysis of full-length 16S rRNA genes covering all hypervariable regions, which could provide more accurate taxonomic classification for microorganisms ( Liang et al, 2022 ).…”

A natural ANI gap that can define intra-species units of bacteriophages and other viruses

“…Presumably due to the large amount of DNA required to perform long-read sequencing, we did not find any viral long-read metagenomes in public databases at the time of this writing. Instead, we used 19 PacBio HiFi cellular metagenomes from human and chicken guts as well as seawater samples described previously (42)(43)(44)(45)(46). Sequences were quality filtered using filtlong v0.2.1 (https://github.com/rrwick/Filtlong) with a minimum read length of 10 kbp and a minimum window quality of 99.…”

Comprehensive Assessment of Elevende novoHiFi Assemblers on Complex Eukaryotic Genomes and Metagenomes

“…To build the first synthetic datasets, we selected 382 bacterial genomes from the GTDB database covering 90 genera of Bacteroidetes, Actinobacteria, Firmicutes, Proteobacteria, and Fusobacteria phyla, and merged them into a metagenome by assigning different abundances to them. The abundance profile was obtained by sampling coverage values from a real chicken gut metagenome assembly (48). PBsim v1.0.4 was used to simulate the HiFi reads with the error model learned on the real sheep gut dataset.…”