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Oligoarray comparative genomic hybridisation-mediated mapping of suppressors of an essential receptor tyrosine kinase generated in a deletion-biased mutagenesis screen

ABSTRACT: We have used oligoarray comparative genomic hybridization (aCGH) to identify novel suppressors generated in a UV-TMP based suppressor screen. This approach is suitable for detecting single gene mutations as well as copy number variations. Keywords: C.elegans Suppressor screen array CGH [1] 2 test samples from homozygous rol-3 suppressor strains mapped to chromosome II individually hybridized to reference DNA from BC6637 (rol-3(s1040ts) conditional strain), using a high density tiling array for chromosome II. [2] 4 test samples from homozygous rol-3 suppressor strains individually hybridized to reference DNA from BC6637 (rol-3(s1040ts) conditional strain).

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Martin Jones

PROVIDER: E-GEOD-15224 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Oligoarray comparative genomic hybridization-mediated mapping of suppressor mutations generated in a deletion-biased mutagenesis screen.

Jones Martin R MR Rose Ann M AM Baillie David L DL

G3 (Bethesda, Md.) 20120601 6

Suppressor screens are an invaluable method for identifying novel genetic interactions between genes in the model organism Caenorhabditis elegans. However, traditionally this approach has suffered from the laborious and protracted process of mapping mutations at the molecular level. Using a mutagen known to generate small deletions, coupled with oligoarray comparative genomic hybridization (aCGH), we have identified mutations in two genes that suppress the lethality associated with a mutation of ...[more]

PMID: 22690375

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Project description:BACKGROUND: Microarray comparative genomic hybridization (CGH) is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans) the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. RESULTS: We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10 C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data quality when varying the oligonucleotide length between 50 and 70, and even when using an isothermal design strategy. CONCLUSIONS: We have determined experimentally the effects of varying several key oligonucleotide microarray design criteria for detection of deletions in C. elegans and humans with NimbleGen's CGH technology. Our oligonucleotide design recommendations should be applicable for CGH analysis in most species. One-copy deletions were studied in both the C. elegans (2 samples) and human (1 sample) genomes. There were no replicate or dye-swap hybridizations.

Project description:Gene copy-number variation, which provides the raw material for the evolution of novel genes, is surprisingly widespread in natural populations. Experimental evolution studies have demonstrated an extremely high spontaneous rate of origin of gene duplications. When organisms are suboptimally adapted to their environment, gene duplication may compensate for reduced fitness by amplifying promiscuous activity of a gene, or increasing dosage of a suboptimal gene. The overarching goal of this study is to inverstigate whether CNVs constitute a common mechanism of adaptive genetic change during compensatory evolution and to further characterize the role of natural selection in dictating their evolutionary spread at a population-genomic level. Outcrossing populations of C. elegans with low fitness were evolved for >200 generations and the frequencies of CNVs in these populations were analyzed by oligonucleotide array comparative genome hybridization, quantitative PCR, and single-worm PCR. Multiple duplications and deletions were detected in intermediate to high frequencies and several lines of evidence suggest that the changes in frequency were adaptive. 1) Many copy-number changes reached high frequency, were near fixation, or were fixed in a short time. 2) Many independent duplications and deletions in high frequency harbor overlapping regions which likely include genes that are under selection for either higher or lower rates of expression. 3) The size spectrum of deuplications and deletions in the adaptive recovery populations is significantly larger than that of spontaneous copy-number variants in mutation accumulation experiments. This is expected if larger CNVs are more likely to encompass genes that are being selected for altered gene dosage. Out results validate the great potential borne by gene copy-number changes for compensatory evolution and adaptation. Experimental genome evolution of copy-number variants in 25 experimental lines compared to 5 ancestral control lines.

Dataset Information

Oligoarray comparative genomic hybridisation-mediated mapping of suppressors of an essential receptor tyrosine kinase generated in a deletion-biased mutagenesis screen

Publications

Oligoarray comparative genomic hybridization-mediated mapping of suppressor mutations generated in a deletion-biased mutagenesis screen.

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