Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes

Abstract

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long non-coding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3’ end of LINE-1_Cfs (i.e., LINE-1_Cf 3’-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.SignificanceAdvancements in long-read DNA sequencing technologies provide more comprehensive views of genomes. We used long-read sequences to assemble a Great Dane dog genome that provides several improvements over the existing reference derived from a Boxer dog. Assembly comparisons revealed that gaps in the Boxer assembly often occur at the beginning of protein-coding genes and have a high-GC content, which likely reflects limitations of previous technologies in resolving GC-rich sequences. Dimorphic LINE-1 and SINEC retrotransposon sequences represent the predominant differences between the Great Dane and Boxer assemblies. Proof-of-principle experiments demonstrated that expression of a canine LINE-1 could promote the retrotransposition of itself and a SINEC_Cf consensus sequence in cultured human cells. Thus, ongoing retrotransposon activity may contribute to canine genetic diversity.

The Australian dingo is an early offshoot of modern breed dogs

“…We compared our de novo assembly with five domestic breeds that span the domestic dog genealogy, boxer ( 12 ), GSD ( 13 ), basenji ( 14 ), Great Dane ( 15 ), and Labrador retriever ( 16 ), as well as Greenland wolf (table S1) ( 18 ). As expected, the dingo assembly is highly concordant with the five domestic dog assemblies.…”

“…The basenji is considered the most primitive breed ( 7 , 17 ), and we included the near-complete CanFam_BAS ( 14 ). The Great Dane and Labrador retriever both have contiguous long-read de novo assemblies ( 15 , 16 ), with a contig N50 of >1 Mb. As an outgroup, we include a recently released assembly of the Greenland wolf (also known as the Polar wolf, Canis lupus orion ) ( 18 ).…”

Long-read sequencing reveals rapid evolution of immunity- and cancer-related genes in bats

“…We present a comprehensive analysis of these genome sequences together with 13 previously assembled bats and other mammalian genomes. Importantly, long-read assemblies enable dramatic improvements in the characterization of gene duplications and losses, and of genomic repeats [29][30][31] . These benefits are of particular value in studies of mammalian immunity-related genes, many of which fall in highly repetitive genomic regions including large arrays of duplicated…”

Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome

“…Sequencing and assembly efforts are increasingly moving from species reference genomes to breed-specific assemblies, such as those recently published for Great Dane [52], Labrador Retriever [53], and a second GSD [54]. Together, our data suggest that a single high-quality reference should be sufficient for most general analyses, but the generation of breed-specific genomes is likely to be important for canine nutrition and disease studies.…”