Rumen ciliates modulate methane emissions in ruminants

The rumen ciliate genome (RCG) catalog was constructed using three omics approaches. First, we manually isolated ciliates based on their morphology from the rumen of cattle, sheep, goat, and deer, conducting single-cell next-generation and HiFi sequencing to generate 436 SAGs. Second, we compiled 2,007 metagenomic datasets from 50 studies across 16 ruminant species worldwide and performed co-assembly and binning to obtain 108 rumen ciliate MAGs. Third, we incorporated previously published data from 69 single-cell sequencing datasets of rumen ciliates and from the <i>Entodinium caudatum</i> genome. Genomic data of rumen ciliates often contain significant contamination from prokaryotes, plant materials, fungi, and endo- or ectosymbionts. The main challenge in obtaining ciliate genomes is distinguishing the ciliate sequences from the abundant contaminating sequences. We developed a new genome decontamination pipeline (iGDP, https://github.com/CodeFeiX/iGDP-rc) optimized for rumen ciliate genome purification, which effectively removed these contaminants. On average, 91.9% of the assembled sequences were identified as contaminants and subsequently removed, which only reduced the rumen ciliate genome BUSCO completeness by 11.2%, demonstrating the high effectiveness of iGDP. In total, we obtained 450 genomes that met medium-quality standards (completeness &gt; 50%), including 233 genomes considered high-quality (completeness &gt; 80%; 215 SAGs, 17 MAGs, and 1 genome from a ciliate monoculture). Notably, 87% of these genomes are novel, highlighting the previously unexplored representation of rumen ciliates. Genome evaluation showed that there were no discordant peaks for the GC content in these genomes, and an average of 72.7% of contigs in all genomes that contained telomeres specific to rumen ciliates or were supported by telomere sequences. These constitute the largest high-purity rumen ciliate genomes (RCGs) reported to date, providing novel guidelines and resources for studying the rumen microbiome. The entire RCG catalog and associated information is available at https://ngdc.cncb.ac.cn/rcg_rbag.<b>The RCG-associated gene prediction information is available here:</b>RCG_450.cds.zip: Gene files corresponding to 450 rumen ciliate genomes.RCG_450.pep.zip: Protein files corresponding to 450 rumen ciliate genomes.RCG_450.gtf.zip: GTF files corresponding to 450 rumen ciliate genomes.<br>