Efficient high-resolution deletion discovery in Caenorhabditis elegans by array comparative genomic hybridization

Abstract

We have developed array Comparative Genomic Hybridization for Caenorhabditis elegans as a means of screening for novel induced deletions in this organism. We designed three microarrays consisting of overlapping 50-mer probes to annotated exons and micro-RNAs, the first with probes to chromosomes X and II, the second with probes to chromosome II alone, and a third to the entire genome. These arrays were used to reliably detect both a large (50 kb) multigene deletion and a small (1 kb) single-gene deletion in homozygous and heterozygous samples. In one case, a deletion breakpoint was resolved to fewer than 50 bp. In an experiment designed to identify new mutations we used the X:II and II arrays to detect deletions associated with lethal mutants on chromosome II. One is an 8-kb deletion targeting the ast-1 gene on chromosome II and another is a 141-bp deletion in the gene C06A8.1. Others span large sections of the chromosome, up to >750 kb. As a further application of array Comparative Genomic Hybridization in C. elegans we used the whole-genome array to detect the extensive natural gene content variation (almost 2%) between the N2 Bristol strain and the strain CB4856, a strain isolated in Hawaii and JU258, a strain isolated in Madeira.

Figure 1.

Detection of a 50-kb homozygous viable deletion in gkDf2. (A) Normalized log₂ ratios (gkDf2/WT) of the average fluorescent intensities for each of the 92,209 forward and reverse pairs of probes to the X chromosome are represented by circles. The deletion is identified by negative log₂ ratios and indicated by an arrow. (B) A higher resolution view of fluorescence ratios for probe pairs targeting the 50-kb deletion. Horizontal bars indicate the positions of the nine genes targeted by the deletion. Duplications of sequences flanking the deletion are indicated by positive log₂ ratios. Adjacent 50-mer probes in this region overlap by as much as 44 bp.

Figure 2.

Detection of a 1047-bp homozygous viable deletion in ceh-39 (gk329). (A) Normalized log₂ ratios (gk329/WT) for the average fluorescence intensities for all probe pairs to the X chromosome are shown. The arrow indicates the deletion. (B) Intensity ratios for probes to ceh-39, ceh-21, and ceh-41 are shown with WormBase gene models to illustrate probe coverage in exons near the deletion. Sequenced deletion breakpoints are indicated by dotted lines. CGH accurately identified the left breakpoint between exons 3 and 4 of ceh-39.

Figure 3.

Comparison of the normalized average fluorescence ratios (XO male/XX hermaphrodite) for all probe pairs to chromosomes II and X. The graph in the top right corner plots the probe density versus the log₂ fluorescence ratio for probes to chromosomes II and X. The curve peaking on the left is for the X chromosome and the curve peaking on the right is for chromosome II. The distributions overlap by ∼4%.

Figure 4.

Detection of the 1202-bp deletion in dab-1 (gk291) in a wash-sampled balanced heterozygous population. (A) The normalized log₂ ratios [(dab-1(-)/mIn1)/WT] of the average fluorescence intensities for probe pairs to chromosome II are plotted. The arrow indicates the dab-1 deletion (other features are discussed in the text). (B) Normalized fluorescence ratios for probe pairs targeting dab-1 are shown. Sequenced deletion breakpoints are indicated by dotted lines and were accurately predicted by CGH. The left breakpoint lies within the second intron. Overlapping probes targeting the right breakpoint span just 73 bp, allowing resolution of the right breakpoint to within fewer than 50 bp.

Figure 5.

Deletions detected in a screen for homozygous lethal mutations in six wash-sampled balanced heterozygous populations. The following normalized log₂ ratios are shown: (A) (gk463/mIn1)/WT; (B) (gk460/mIn1)/WT; (C) (gk462/mIn1)/WT; (D) (gk465/mIn1)/WT; (E) (gk488/mIn1)/WT; and (F) (gk487/mIn1)/WT.

Figure 6.

Whole-genome array CGH comparing Hawaiian (CB4856) and Bristol N2 (VC196) hermaphrodites. Large-scale copy number polymorphism is evident between these two wild-type isolates. Normalized log₂ fluorescence ratios (CB4856/N2) for all probes on the chip are shown.

Figure 7.

A homozygous viable deletion identified on chromosome V in the Hawaiian strain (CB4856). Normalized log₂ ratios (CB4856/N2) indicate that the deletion targets the genes C49G7.1 and D1065.3.