Structural variation in the chicken genome identified by paired-end next-generation DNA sequencing of reduced representation libraries

Abstract

Background: Variation within individual genomes ranges from single nucleotide polymorphisms (SNPs) to kilobase, and even megabase, sized structural variants (SVs), such as deletions, insertions, inversions, and more complex rearrangements. Although much is known about the extent of SVs in humans and mice, species in which they exert significant effects on phenotypes, very little is known about the extent of SVs in the 2.5-times smaller and less repetitive genome of the chicken.

Results: We identified hundreds of shared and divergent SVs in four commercial chicken lines relative to the reference chicken genome. The majority of SVs were found in intronic and intergenic regions, and we also found SVs in the coding regions. To identify the SVs, we combined high-throughput short read paired-end sequencing of genomic reduced representation libraries (RRLs) of pooled samples from 25 individuals and computational mapping of DNA sequences from a reference genome.

Conclusion: We provide a first glimpse of the high abundance of small structural genomic variations in the chicken. Extrapolating our results, we estimate that there are thousands of rearrangements in the chicken genome, the majority of which are located in non-coding regions. We observed that structural variation contributes to genetic differentiation among current domesticated chicken breeds and the Red Jungle Fowl. We expect that, because of their high abundance, SVs might explain phenotypic differences and play a role in the evolution of the chicken genome. Finally, our study exemplifies an efficient and cost-effective approach for identifying structural variation in sequenced genomes.

Figure 1

Sequence coverage of the RRL. On the x-axis are the obtained sequence coverages of RRL-fragments estimated by read-pair clusters and on the y-axis the frequency in which they occurred (10 log scale)

Figure 2

Distribution of fragment sizes for concordantly mapping reads in the four sequenced chicken breeds. For unclear reasons, broiler 2 had remarkably higher representation of smaller fragments (left long shoulder), whereas fragments in base pairs of the size range 180-200 were two magnitudes less abundant compared to the three other breeds.

Figure 3

PCR-based genotyping on a breed level (A) and individual level (B). A) Genotyping for the presence of SVs in breeds, represented by pooled samples. Except for SV50 and SV51, a small (see Table 4 for approximate sizes and breed encoding) PCR fragment that was absent in the reference was expected in some of the breeds that have the deletion. In SV50 and SV51, a slightly larger PCR fragment than that observed in the reference was expected in breeds that have the insertion. B) Genotyping for the presence of SVs in eight individuals of breeds in which the SV was detected in pooled samples. Except for SV50 and SV51, a small PCR fragment was expected in individuals homozygous for the deletion and SVs in which the reference genotype is too long for PCR. Heterozygous individuals in which both genotypes can be spanned (see Table 4) by PCR show two bands. In SV50 and SV51, both PCR fragments, which differ slightly in size, are expected in heterozygous individuals, whereas only the larger fragment is expected in individuals homozygous for the insertion.

Figure 4

Distinguishing putative deletions from false positives in genotyping validation results obtained by PCR. Predicted deletions in the initial validation study that were confirmed are in green; those that could not be confirmed are in red. The black line represents the discrimination rule (span-size difference)×n >500, which is valid for 220-720 bp. The SV predictions that were selected based on the model and confirmed are in blue.

Figure 5

Venn diagrams representing the distribution of predicted deletions in the four chicken breeds at mapping constraints 60 (left) and 35 (right). The number of structural variants is proportionally represented per breed, and line colors were assigned as follows: green = brown egg layer; blue = broiler 1; red = broiler 2; and purple = white egg layer. For example, the area that is surrounded by the blue line in the left diagram represents SVs found in broiler 1. Of these, 23 were specific for broiler 1 (yellow area), and 28 were shared with broiler 2 (dark yellow area surrounded by both the blue and red lines). The orange area surrounded by the blue, red, and green line represent 18 SVs shared by broiler 1, broiler 2, and brown egg layers. The red area in the middle of the diagram surrounded by the four line colors represents 20 SVs shared by the four breeds analyzed.

Figure 6

Size distribution of predicted deletions at two mapping constraints.

Figure 7

Distribution of predicted SVs over the chicken chromosomes. Shown are chicken chromosomes in which 186 deletions (red) and 2 insertions (blue) were identified.

Figure 8

Microhomologies detected in sequenced SVs. Shown are the three SVs in which microhomology (grey boxes) was detected at the SV breakpoints.