Why Pallas's Cat And How Did We Get DNA?

We chose Pallas's Cat (manul cat) because they are locally endangered and under threat from habitat loss, as well as having no genome available. Population strongholds in Mongolia and China face growing challenges from climate change, habitat fragmentation, poaching, and other sources.
In partnership with the Utica Zoo, we asked for blood from Tater, their resident 5 yr old Pallas's cat. During routine veterinary care, Tater's blood was drawn and shipped to Minnesota.

Complete Team (Low Resource DNA sequencing)

Three of us, Chris Faulk (PI), Carrie Walls (lab technician), and Nicole Flack (DVM/PhD student), performed the nanopore sequencing, assembly, bioinformatics, and wrote the paper. 
We generated 158Gb of sequence in 3 weeks using 5 flow cells, for a total cost of $5000. 

Genome Assembly

With only consumer level computers, we assembled the reads into a high quality genome.
We used nanopore-only sequence to assemble a 2.5 Gb, 61-contig nuclear assembly and 17,097 bp mitogenome for Pallas's cat. The primary nuclear assembly had 56x sequencing coverage, a contig N50 of 118 Mb, and a 94.7% BUSCO completeness score.

How Good Is Our Genome?

Our complete Pallas's cat genome is the most contiguous genome for any cat species, including the domestic cat (Felcat9).
High genome colinearity within Felidae permitted alignment-based scaffolding onto the fishing cat (Prionailurus viverrinus) reference genome. Pallas's cat contigs spanned all 19 felid chromosomes with an total missing gap length of less than 400 kilobases. 

How Similar Is The Genome To Other Cats?

The Pallas's cat genome is made up of nearly exactly the same number and type of repetitive elements as other cat genomes. Overall it fits well within the range of other cat species.

DNA Methylation (Epigenetics)

Nanopore reads can capture both genetic and epigenetic variation at the same time. For the Pallas's cat, modified basecalling and variant phasing produced an alternate pseudohaplotype assembly and allele-specific DNA methylation calls; sixty-one differentially methylated regions were identified between haplotypes. Nearest features included classical imprinted genes, non-coding RNAs, and putative novel imprinted loci. 

Mitochondria Are The Powerhouse of Pallas's cat

We assembled the mitochondrial genome with 800X coverage, and included an extra 400 base pairs that were missing from the previously published mitogenome.

Wikipedia Was Wrong

Wikipedia had a problem. One previous study using select nuclear genes across the felid radiation placed Pallas's cat as a sister lineage to the genus Prionailurus. In contrast, a whole-mitogenome phylogeny study placed it closer to the genus Felis, which includes the domestic house cat.
Our improved mitochondria genome built a phylogeny that matches nuclear DNA trees, with Pallas's cat sharing its most recent ancestor with Prionailurus in contradiction of a 2016 mitochondrial phylogeny that placed it closer to Felis. 

Population Genetic Health

We estimated demography, i.e. population genetic variability over time as a measure of genetic health. Our effective population size was relatively large (Ne = 10,000). Results suggest that Pallas's cats experienced a crash in genetic diversity, but that this event was in the relatively distant past (~10,000 years ago); current effective population size has continued to recover in modern times despite recent anthropogenic challenges to the species.