Copy number variation of individual cattle genomes using next-generation sequencing

Abstract

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one Holstein, and one Hereford) and one indicine (Nelore) cattle. Within mapped chromosomal sequence, we identified 1265 CNV regions comprising ~55.6-Mbp sequence--476 of which (~38%) have not previously been reported. We validated this sequence-based CNV call set with array comparative genomic hybridization (aCGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH), achieving a validation rate of 82% and a false positive rate of 8%. We further estimated absolute copy numbers for genomic segments and annotated genes in each individual. Surveys of the top 25 most variable genes revealed that the Nelore individual had the lowest copy numbers in 13 cases (~52%, χ(2) test; P-value <0.05). In contrast, genes related to pathogen- and parasite-resistance, such as CATHL4 and ULBP17, were highly duplicated in the Nelore individual relative to the taurine cattle, while genes involved in lipid transport and metabolism, including APOL3 and FABP2, were highly duplicated in the beef breeds. These CNV regions also harbor genes like BPIFA2A (BSP30A) and WC1, suggesting that some CNVs may be associated with breed-specific differences in adaptation, health, and production traits. By providing the first individualized cattle CNV and segmental duplication maps and genome-wide gene copy number estimates, we enable future CNV studies into highly duplicated regions in the cattle genome.

Figure 1.

Individualized cattle CNV map. The Btau_4.0 assembly is represented as black bars with assembly gaps indicated by white boxes on the chromosomes. Larger bars intersecting the chromosomes represent the previously discovered WSSD (red), WGAC (blue), and WSSD/WGAC joint-prediction (purple) regions. Tracks underneath the chromosomes represent the CNV data sets (in order from top to bottom) for DTTRACE, merged CNVRs from all data sets, BINE, BTAN1, BTAN2, BTAN3, and BTHO. The colors for each bar in the animal data set tracks represent the average estimated CN for each CNV as shown in the legend. The merged CNVR track does not have CN information and is uniformly colored brown.

Figure 2.

Correlation between computational predictions and experimental validations. (A) A good agreement of lengths (r = 0.904) exists between previously discovered WSSD+, WGAC+, and predicted DTTRACE duplications. (B) Calculated digital aCGH probe values (BTAN2_ngs) were compared with probe log₂ ratios from a whole-genome aCGH (BTAN2_whole). Digital aCGH values were estimated using a log₂ ratio of the 1-kbp CN windows from BTAN2 divided by CN estimates from DTTRACE. A moderate correlation (r = 0.524) was found for aCGH probe values and digital aCGH values within CNV intervals >20 kbp that had fewer than 80% of their lengths occupied by common repeats.

Figure 3.

Computational predictions and aCGH validations of segmental duplication copy number differences for six cattle genomes. Depth-of-coverage tracks for DTTRACE, BINE, BTAN2, and BTHO are below a UCSC track for each investigated gene region. Regions colored in red on the plot indicate excessive read depth (> mean + 4 × STDEV), whereas gray regions indicate intermediate read depth (> mean + 3 × STDEV). Normal read depth values are colored green (mean ± 2 × STDEV). Digital aCGH tracks show the log₂ ratio of the copy number of each listed animal compared to DTTRACE, with high values listed in green (>0.5); low values: red (<−0.5); and nominal values: gray (0.5 > x > −0.5). Whole-genome CGH array experiments, using Dominette as a reference sample in all cases, are listed below the digital aCGH experiments. Color schemes for the aCGH plots are the same as for the digital aCGH. Previously detected segmental duplications (SDs) are shown below the UCSC plot, if present in the region. (A) CNVs intersecting the BPIFA2A (BSP30A) locus (chr13:63364661-63487495). A duplication of this region was predicted for all animals and was confirmed by whole-genome aCGH. (B) In the ULBP17 locus (chr9:90209622-90499803), BINE was predicted to have a higher copy number than DTTRACE across the region from both read depth and aCGH experiments. (C) The promoter region of FABP2 (chr6:6701747-6888288) was a predicted duplication in Dominette (Hereford; beef breed), BTAN2 (Angus; beef), and BINE (Nelore; dual-purpose) but not in BTHO (Holstein; milk).

Figure 4.

Cluster analysis of copy number variable genes in individual cattle. (A) Copy number values for each animal were plotted within the AOX1 locus (chr2:93376314-93484307) using the color scheme depicted in the legend. Heatmap boxes represent 1-kbp sliding, nonoverlapping windows in the region. The dendrogram indicates the hierarchical ordering of animals based on a Pearson's hierarchical clustering of the CN values within the region. Within AOX1, the last exons are predicted to have a higher CN in BINE than in any other animal. This observation was confirmed using aCGH and qPCR. (B) A heatmap of APOL3 reveals significantly higher CN in the three Angus animals (BTAN3, BTAN2, and BTAN1) for the first APOL3 transcript (NM_001100297) than in the other breeds (chr5:80158821-80417344).