A novel canis lupus familiaris reference genome improves variant resolution for use in breed-specific GWAS

(A) Total extracted DNA. (B) DNA quality; green line indicates the ideal 260/280 ratio for DNA purity at 1.80. Extractions from the Nanobind kit had the most consistently high yield and quality.

Each point represents a distinct assembly resulting from one of 144 unique miniasm parameter combinations. Sequence data from eight ONT flow cells are represented in the plot, four from each of the Ligation and Rapid library preparation kits (SQK-LSK109 and SQK-RAD004, respectively). See Table 3 for details linking “estimated depth” to sequencing run and library kit. The estimated depth of 22.65 is a combination of reads from all eight flow cells (black boxed region in upper left, see Fig 4 for details regarding parameters). Estimated coverage is based on the total bps in the read set divided by the total length of CanFam3.1 assembly including Ns. Total bps of assembly approaches estimated total genome size as depth approaches 20×. Horizontal dashed red line—size of CanFam3.1 with N’s (2,327,604,993 bp); vertical dashed red line—contig count (19,555) of CanFam3.1 chromosomal scaffolds broken at every occurrence of N. The following “Estimated Depth(s)” are from: the rapid kit only (5.99, 6.65, and 12.64); the ligation kit only (3.96, 6.66, 10.02); and a combination of the two (10.60, 12.05, and 22.65).

The estimated genome depth of the data is 22.65×. Contig count calculated from counting number of headers in resulting assembly FASTA files, and total length calculated from non-header character count. (A, B, C, D) Zoomed in view of the top-left group of assemblies from Fig 3, colored by parameter value and broken down by miniasm parameter type: (A) i, ignore mappings with identity less than INT identity; (B) s, drop mapping less than INT total bps; (C) I, minimap overlap ratio; and (D) e, contig is removed if it is generated from less than INT reads. Note that miniasm parameter “m” (for dropping read mappings with less than INT matching bps) is left out, as all points for the three values used (25, 50, and 100) are all overlapping (i.e., “m” has no effect on contig count or total bps). Default parameters for miniasm are: m = 100, i = 0.05, s = 1,000, I = 0.8, e = 4. The blue diamond indicates the down-selected assembly (v0.0 in Table 4) used for polishing and final scaffolding, miniasm parameters used: m = 100, i = 0.05, s = 500, I = 0.8, e = 3. The red dashed line indicates the genome size (with N’s) of CanFam3.1.

Accessions and additional metrics can be found in Table S1. (A) Reads alignment rates to CF (GCF_000002285.3, CamFam3.1, Boxer breed), GS (GCA_008641245.1, German Shepherd breed), and YA (Yella v1.0; Labrador Retriever breed) reference genomes (paired t test; CF versus YA P-value = 2.457 × 10⁻⁶, GS versus YA P-value = 1.397 × 10⁻³). (B) Total variants detected at Q > 29 in references (paired t test; CF versus YA P-value = 4.744 × 10⁻⁶, GS versus YA P-value = 3.931 × 10⁻⁶).