Dataset Information

Efficient High-Resolution Discovery in C. elegans by Array Comparative Genomic Hybridization

ABSTRACT: The Males Experiment: This experiment was done to test the sensitivity of our array in detecting single copy losses of probes to the X chromosome. Males (XO) have just one X chromosome, while hermaphrodites (XX) have two. CB4856 and JU258: This experiment was done to determine the natural copy number variation that exists in a wild C. elegans isolate. Extensive natural copy number variation exists in this strain. gkDf2 (VC100): This control experiment was done to confirm that we could reliably detect a large 50-kb homozygous deletion on chromosome X using this array. gk329 (VC766): This control experiment was done to confirm that we could reliably detect a homozygous 1-kb single-gene deletion on the X chromosome. This deletion targets the gene ceh-39. For gk291 (VC615): This control experiment was done to confirm that we could reliably detect a heterozygous (single copy) single-gene 1-kb deletion on chromsome II. This deletion targets the gene dab-1. All other experiments: This experiment was done in an effort to detect novel balanced single copy losses on chromosome II in mutagenized strains. Mutants were previously screened for homozygous lethal mutations on chromosome II, and these mutations were then balanced with the mIn1 balancer chromosome to generate the mutant strain screened in this experiment. Keywords: comparative genomic hybridization

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE6294 | GEO | 2006/11/18

SECONDARY ACCESSION(S): PRJNA100607

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:DNA copy number analysis of myxoinflammatory fibroblastic sarcomas, and related lesions, using 32k BAC array CGH. All four cases analyzed with array CGH showed amplification of the region 86.30-87.74 Mb in 3p11.1-12.1. No additional aberration was detected in more than two tumors, except for the deletion distal to the amplification on chromosome 3. Case 1 showed deletion affecting the region from the centromere of chromosome 1 to the 5'-end of TGFBR3. Homozygous deletion was found in one case (case 6) affecting the region 21.08-22.15 Mb on chromosome arm 9p harboring the CDKN2A and CDKN2B genes. DNA copy numbers in cases 1 and 6-8 were analyzed using tiling microarrays containing more than 32 000 partly overlapping BAC clones, generating complete coverage of the human genome (JÃ¶nsson et al., 2007, Genes Chromosomes Cancer 46: 543-58.). The arrays were produced at the Swegene DNA Microarray Resource Center, Department of Oncology, Lund University (http://swegene.onk.lu.se), as previously described (JÃ¶nsson et al., 2007), using BAC clones mapped to the hg17 genome build. Extraction, labeling and hybridization of genomic DNA from freshly frozen tumor biopsies, as well as pretreatment and washing of slides were performed as described (Heidenblad et al., 2006, Oncogene 25: 7106-16). As a control for normal copy number, a DNA pool derived from multiple healthy male donors was used (Promega, Madison, WI, USA). Primary array CGH data were collected using the GenePix Pro 4.0 (Axon Instruments Inc., Foster City, CA, USA). The quantified data matrix was deposited into the web-based database BioArray Software Environment (BASE) (Saal et al., 2002, Genome Biol 3: software0003.1-0003.6), and data analyses were done as described (Hallor et al., 2008, Br J Cancer 98: 434-42). In brief, following background correction the log(2) ratios were calculated for each spot, and unreliable features and spots not showing signal-to-noise ratios ?5 for both channels were eliminated. Normalization of data was performed using pin-based Lowess normalization and single outlier probes were removed. Thereafter, the log(2) ratios for each sample were segmented, followed by elimination of segments less than 500 kb in size. Copy number alterations were determined by comparing the segmented log(2) ratios to gain/loss thresholds obtained by an adaptive scaling method (Staaf et al., 2007, BMC Genomics 8: 382).

Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high resolution chromosome-wide CGH. Specifically, HR-CGH with 5 ng-input gDNA-derived amplicon detected all previously known chromosomal segmental aberrations in chromosome 22 in samples from two different probands, and was indistinguishable from the HR-CGH result with native gDNA from the same probands (Fig. 4, Fig. S3 and S4). The break points were also precisely demonstrated. These include a heterozygous genomic segmental duplication (3 copies, 3 Mb in size, sample 05-050, Fig. 4) and 2 different heterozygous deletions (1 copy, 1.4 Mb and 18 kb respectively, sample 04-018, Fig. S4), all of which are located in or bounded by regions of low copy repeats (LCRs). In addition, a previous known homozygous deletion of 975 bp (in 04-018 and 05-050) was again accurately demonstrated (05-050 data showed in Fig. 4c), although sometimes (04-018) the data was a little noisier than with unamplified DNA (Fig. S4d). In contrast, the Wpa-40oC resulted in abundant signal noise and failed in detection of these copy number aberrations (Fig. 4, Fig. S3, S4). Impressively, HR-CGH with 0.5 ng gDNA-derived amplicons via Wpa also clearly detected the known CNVs, although noisier (Fig. S4). The 0.1ng gDNA derived amplicons via Wpa could not unambiguously show CNVs because of higher variability of signals, but the CNVs’ patterns were mostly well maintained (Fig. S4 for 04-018). We did also notice some locus-imbalance in the amplicon, however this was minimized, and was reproducible when the input was above a certain threshold amount, and it could be well compensated if the same amplified reference sample was applied in parallel as showed above. Keywords: Whole-pool amplification, high resolution comparative genome hybridization (HR-CGH)

Dataset Information

Efficient High-Resolution Discovery in C. elegans by Array Comparative Genomic Hybridization

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure