Author Correction: Development and performance of a targeted whole exome sequencing enrichment kit for the dog (Canis Familiaris Build 3.1).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Improved canine exome designs, featuring ncRNAs and increased coverage of protein coding genes.

By limiting sequencing to those sequences transcribed as mRNA, whole exome sequencing is a cost-efficient technique often used in disease-association studies. We developed two target enrichment designs based on the recently released annotation of the canine genome: the exome-plus design and the exome-CDS design. The exome-plus design combines the exons of the CanFam 3.1 Ensembl annotation, more recently discovered protein-coding exons and a variety of non-coding RNA regions (microRNAs, long non-coding RNAs and antisense transcripts), leading to a total size of ≈ 152 Mb. The exome-CDS was designed as a subset of the exome-plus by omitting all 3' and 5' untranslated regions. This reduced the size of the exome-CDS to ≈ 71 Mb. To test the capturing performance, four exome-plus captures were sequenced on a NextSeq 500 with each capture containing four pre-capture pooled, barcoded samples. At an average sequencing depth of 68.3x, 80% of the regions and well over 90% of the targeted base pairs were completely covered at least 5 times with high reproducibility. Based on the performance of the exome-plus, we estimated the performance of the exome-CDS. Overall, these designs provide flexible solutions for a variety of research questions and are likely to be reliable tools in disease studies.

Development and performance of a targeted whole exome sequencing enrichment kit for the dog (Canis Familiaris Build 3.1).

Whole exome sequencing is a technique that aims to selectively sequence all exons of protein-coding genes. A canine whole exome sequencing enrichment kit was designed based on the latest canine reference genome (build 3.1.72). Its performance was tested by sequencing 2 exome captures, each consisting of 4 pre-capture pooled, barcoded Illumina libraries on an Illumina HiSeq 2500. At an average sequencing depth of 102x, 83 to 86% of the target regions were completely sequenced with a minimum coverage of five and 90% of the reads mapped on the target regions. Additionally, it is shown that the reproducibility within and between captures is high and that pooling four samples per capture is a valid option. Overall, we have demonstrated the strong performance of this WES enrichment kit and are confident it will be a valuable tool in future disease association studies.

The prevalence of nine genetic disorders in a dog population from Belgium, the Netherlands and Germany.

The objective of this study was to screen a dog population from Belgium, the Netherlands and Germany for the presence of mutant alleles associated with hip dysplasia (HD), degenerative myelopathy (DM), exercise-induced collapse (EIC), neuronal ceroid lipofuscinosis 4A (NCL), centronuclear myopathy (HMLR), mucopolysaccharidosis VII (MPS VII), myotonia congenita (MG), gangliosidosis (GM1) and muscular dystrophy (Duchenne type) (GRMD). Blood samples (K3EDTA) were collected for genotyping with Kompetitive Allele Specific PCR (n = 476). Allele and genotype frequencies were calculated in those breeds with at least 12 samples (n = 8). Hardy-Weinberg equilibrium was tested. Genetic variation was identified for 4 out of 9 disorders: mutant alleles were found in 49, 15, 3 and 2 breeds for HD, DM, EIC and NCL respectively. Additionally, mutant alleles were identified in crossbreeds for both HD and EIC. For HD, DM, EIC and NCL mutant alleles were newly discovered in 43, 13, 2 and 1 breed(s), respectively. In 9, 2 and 1 breed(s) for DM, EIC and NCL respectively, the mutant allele was detected, but the respective disorder has not been reported in those breeds. For 5 disorders (HMLR, MPS VII, MG, GM1, GRMD), the mutant allele could not be identified in our population. For the other 4 disorders (HD, DM, EIC, NCL), prevalence of associated mutant alleles seems strongly breed dependent. Surprisingly, mutant alleles were found in many breeds where the disorder has not been reported to date.

The effect of a technical quality assessment of hip-extended radiographs on interobserver agreement in the diagnosis of canine hip dysplasia.

Experienced and inexperienced observers evaluated the assessability of 50 radiographs (25 dogs) and determined the hip status (dysplasia/nondysplasia and final scoring according Fédération Cynologique Internationale [FCI]-criteria) individually. A radiographic technical quality assessment was performed in a separate reading session. Interobserver agreement in determining dysplasia/nondysplasia and FCI-scoring did not significantly increase with the increasing quality of a radiograph, irrespective whether these observers are experienced or not. There was a significant agreement between the technical quality assessment and assessability (P < 0.0005). Despite the effort to objectify radiographic quality and to present high-quality radiographs to observers, interobserver agreement on dysplasia/nondysplasia and final scoring, remains low, even in the experienced group. Although increased radiographic quality narrows the range of scoring, the range remains unacceptably high.

Interobserver agreement on the assessability of standard ventrodorsal hip-extended radiographs and its effect on agreement in the diagnosis of canine hip dysplasia and on routine FCI scoring.

Insufficient agreement on scoring hip quality might be caused by differences in the assessability of a radiograph (exposure, contrast, positioning, and diagnostic quality). We studied the agreement in assessability of standard ventrodorsal hip-extended radiographs by experienced (nine) and inexperienced (21) observers, using the standard subjective method of quality control, currently applied in screening programs. The effect of assessability on the agreement of scoring hip quality [dysplastic vs. nondysplastic and the final Federation Cinologique International (FCI) score] was also investigated. There was a significant difference (P < 0.0001) in agreement on assessability between the experienced and inexperienced observers. In 68% of evaluations, experienced observers stated that the radiograph was assessable. Inexperienced observers evaluated the radiographs as being assessable in only 46.5% of evaluations. Increased interobserver agreement on assessability of a radiograph did not increase the overall interobserver agreement in the diagnosis of hip dysplasia, nor did it result in consistent scoring of the hip status from that radiograph, despite a significant (P < 0.05) increase in agreement of FCI scoring with an increasing agreement on assessability at a one to five ratio in the experienced group. The inconsistent evaluation of radiographic quality, as well as the inconsistent evaluation of the hip quality, caused differences in diagnosing hip dysplasia and FCI scoring in the same dog ranging from excellent hips to moderate hip dysplasia. Therefore, the credibility of the FCI screening method for canine hip dysplasia, using the standard hip-extended radiographic view, as currently applied in most European countries, is questionable.