Chromosome-level, nanopore-only genome and allele-specific DNA methylation of Pallas's cat, Otocolobus manul

Abstract

Pallas's cat, or the manul cat (Otocolobus manul), is a small felid native to the grasslands and steppes of central Asia. Population strongholds in Mongolia and China face growing challenges from climate change, habitat fragmentation, poaching, and other sources. These threats, combined with O. manul’s zoo collection popularity and value in evolutionary biology, necessitate improvement of species genomic resources. We used standalone nanopore sequencing to assemble a 2.5 Gb, 61-contig nuclear assembly and 17097 bp mitogenome for O. manul. The primary nuclear assembly had 56× sequencing coverage, a contig N50 of 118 Mb, and a 94.7% BUSCO completeness score for Carnivora-specific genes. High genome collinearity within Felidae permitted alignment-based scaffolding onto the fishing cat (Prionailurus viverrinus) reference genome. Manul contigs spanned all 19 felid chromosomes with an inferred total gap length of less than 400 kilobases. Modified basecalling and variant phasing produced an alternate pseudohaplotype assembly and allele-specific DNA methylation calls; 61 differentially methylated regions were identified between haplotypes. Nearest features included classical imprinted genes, non-coding RNAs, and putative novel imprinted loci. The assembled mitogenome successfully resolved existing discordance between Felinae nuclear and mtDNA phylogenies. All assembly drafts were generated from 158 Gb of sequence using seven minION flow cells.

The genome of Przewalski’s horse (Equus ferus przewalskii)

“…We detected 21,552 putative genes in EquPr2 with the ab initio gene prediction tool Gene Model Mapper (GeMoMa) [69] using the EquCab3.0 annotation (GCF_002863925.1) as reference. The resulting protein BUSCO score was 85.6% complete (84.6% single copy and 1.0% duplicates); this score appears to be near GeMoMa’s maximum performance based on previous demonstration of similar results for existing high-quality reference genomes [64]. The EquPr2 annotation would likely be significantly improved with by the application of NCBI’s annotation pipeline, which requires the availability of RNA-seq data.…”

“…Oxford Nanopore sequencing can natively detect base modifications including 5-methylcytosine (5mC). This feature has been applied in previous mammalian genome assemblies to evaluate allele-specific DNA methylation and identify known and putative novel imprinted genes [64]. Globally, we found that whole blood leukocyte DNA methylation in Przewalski’s horse was 79.5% methylated and 2.1% hydroxymethylated at CpG sites genome-wide.…”

“…The initial assembly was polished, purged of duplicates, and manually curated to remove contigs with coveage below 15X and 500X as they are unlikely to represent single-copy nuclear regions [64]. This procedure resulted in the removal of 4,012 contigs and 96 Mb of sequence.…”

“…Oxford Nanopore sequencing can natively detect base modifications including 5-methylcytosine (5mC). This feature has been applied in previous mammalian genome assemblies to evaluate allele-specific DNA methylation and identify known and putative novel imprinted genes [64].…”