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Chromosomal microarray analysis, or comparative genomic hybridization: A high throughput approach.

ABSTRACT: Pathological copy number variants (CNVs) and point mutations are major genetic causes of hundreds of disorders. Comparative genomic hybridization (CGH) also known as chromosomal microarray analysis (CMA) is the best available tool to detect copy number variations in chromosomal make up. We have optimized several different protocols and introduce a high-throughput approach to perform a cost-effective, fast, high-throughput and high-quality CMA. We managed to reach to high quality arrays with 17 ± 0.04 (mean ± SD, n = 90) Derivative Log Ratio (DLR) spread, a measure of array quality (<0.20 considered as excellent) for our arrays. High-throughput and high-quality arrays are gaining more attention and the current manuscript is a step forward to this increasing demand.•This manuscript introduces a low cost, fast, efficient, high throughput and high-quality aCGH protocol;•This protocol provides specific instructions and crucial detail for processing up to 24 slides which is equal to 48, 96, or 192 arrays by only one person in one day;•This manuscript is accompanied with a step-by-step video.

SUBMITTER: Haeri M

PROVIDER: S-EPMC4707176 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Chromosomal microarray analysis, or comparative genomic hybridization: A high throughput approach.

Haeri Mohammad M Gelowani Violet V Beaudet Arthur L AL

MethodsX 20151202

Pathological copy number variants (CNVs) and point mutations are major genetic causes of hundreds of disorders. Comparative genomic hybridization (CGH) also known as chromosomal microarray analysis (CMA) is the best available tool to detect copy number variations in chromosomal make up. We have optimized several different protocols and introduce a high-throughput approach to perform a cost-effective, fast, high-throughput and high-quality CMA. We managed to reach to high quality arrays with 17 ± ...[more]

PMID: 26862485

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Project description:BackgroundMicroarray-based Comparative Genomic Hybridization (M-CGH) has been used to characterize the extensive intraspecies genetic diversity found in bacteria at the whole-genome level. Although conventional microarray analytical procedures have proved adequate in handling M-CGH data, data interpretation using these methods is based on a continuous character model in which gene divergence and gene absence form a spectrum of decreasing gene conservation levels. However, whereas gene divergence may yet be accompanied by retention in gene function, gene absence invariably leads to loss of function. This distinction, if ignored, leads to a loss in the information to be gained from M-CGH data. We present here results from experiments in which two genome-sequenced strains of C. jejuni were compared against each other using M-CGH. Because the gene content of both strains was known a priori, we were able to closely examine the effects of sequence divergence and gene absence on M-CGH data in order to define analytical parameters for M-CGH data interpretation. This would facilitate the examination of the relative effects of sequence divergence or gene absence in comparative genomics analyses of multiple strains of any species for which genome sequence data and a DNA microarray are available.ResultsAs a first step towards improving the analysis of M-CGH data, we estimated the degree of experimental error in a series of experiments in which identical samples were compared against each other by M-CGH. This variance estimate was used to validate a Log Ratio-based methodology for identification of outliers in M-CGH data. We compared two genome strains by M-CGH to examine the effect of probe/target identity on the Log Ratios of signal intensities using prior knowledge of gene divergence and gene absence to establish Log Ratio thresholds for the identification of absent and conserved genes.ConclusionThe results from this empirical study validate the Log Ratio thresholds that have been used in other studies to establish gene divergence/absence. Moreover, the analytical framework presented here enhances the information content derived from M-CGH data by shifting the focus from divergent/absent gene detection to accurate detection of conserved and absent genes. This approach closely aligns the technical limitations of M-CGH analysis with practical limitations on the biological interpretation of comparative genomics data.

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 degrees 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. CONCLUSION: 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.

Project description:BackgroundIt has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH).MethodsTumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested.ResultsThe mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers.ConclusionsThus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A-bomb radiation may affect the increased amount of CNA as a hallmark of GIN and, subsequently, be associated with a higher histologic grade in breast cancer found in A-bomb survivors.

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Chromosomal microarray analysis, or comparative genomic hybridization: A high throughput approach.

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Chromosomal microarray analysis, or comparative genomic hybridization: A high throughput approach.

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