Tag Archives: FISH probes

September 19. Abstract on metaphase epigenetics: platform presentation at American Society of Human Genetics meeting

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Non-random, locus-specific differences in DNA accessibility are present in homologous metaphase chromosomes. W. A. Khan1,3, P. K. Rogan2,3,4, J. H. M. Knoll1,3,4 1) Department of Pathology; 2) Departments of Biochemistry and Computer Science; 3) University of Western Ontario, London, Ontario, Canada; 4) Cytognomix, London, Ontario, Canada.
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Condensation differences between heterochromatin and euchromatin along the lengths of homologous, mitotic metaphase chromosomes are well known. This study describes differences in metaphase compaction between homologous euchromatic loci. We report molecular cytogenetic data showing local differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed at ~10% of 450 distinct genomic regions mapped by single copy fluorescence in situ hybridization (scFISH). Fourteen short (1.5-5kb) sc and low copy (lc) FISH probes (from chromosomes 1, 5, 9, 11, 15, 16, 17, 22) targeting genic and non-genic regions with and without DA were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization were non-random for 6 genomic regions (RGS7, CACNAB1, HERC2, PMP22:IVS3, ADORA2B:IVS1, ACR) and were significantly-biased towards the same homolog (p< 0.01; n = 355 cells). The imprinted paternal chromosome 15 in a three-generation pedigree also showed non-random bias in DA. DNA probes within CCNB1, C9orf66, ADORA2B:Ex 1-IVS1, PMP22:IVS4-Ex 5, and a nongenic region within 1p36.3 did not show DA, while OPCML showed unbiased DA. A subset of probes was mapped onto chromosome topography by FISH-correlated atomic force microscopy (AFM). To quantify DA and pinpoint probe locations, we performed 3D-structured illumination super-resolution microscopy (3D-SIM). 3D anaglyph videos showed genomic regions with DA having nearly 5-fold larger differences in volumetric integrated probe intensities between homologs. Additional non-DA probes (NOMO1, NOMO3) hybridized to grooves in chromosome topography and exhibited a narrow range of probe depths (average: 0.08 μm) along axial and lateral axes of the 2 homologs. In contrast, probe for targets with DA (HERC2, PMP22:IVS3, ACR) significantly differed in probe depth (average: 0.77 μm) and volume (p < 0.05) between each homolog. Interestingly, genomic regions without DA are enriched in epigenetic marks (DHS, H3K27Ac, H3K4me1) of accessible interphase chromatin to a greater extent than regions with DA, suggesting these differences may be correlated with epigenetic marks established during the previous interphase. In summary, we present several lines of evidence that regional differences in condensation between homologs are programmed during metaphase chromosome compaction.

Click here for presentation details:  session, location, time.

June 26, 2012. New US Patent

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FISH of short cancer related genesUS Patent 8,209,129 has issued on single copy DNA probes which include divergent repetitive sequences, thus significantly extending the portions of the genome that can be used for such probes beyond traditional single copy sequences.

The technology also increases the density of genomic DNA probes for higher resolution genetic analysis beyond what is used in FISH, genomic microarrays for array comparative genomic hybridization, and solution capture hybrdization arrays for sequence enrichment in deep sequencing. It is licensed to Cytognomix.

Cytognomix receives new US Patent

US patent (8,209,129) was issued today on Single Copy DNA probes that include divergent repetitive sequences, thus extending the portions of the genome that can be used for such probes beyond traditional single copy sequences. The technology also increases the density of genomic DNA probes for higher resolution genetic analysis beyond what is used in FISH, genomic microarrays for array comparative genomic hybridization, and solution capture hybrdization arrays for sequence enrichment in deep sequencing. The patent has been licensed to Cytognomix.

Applications

Mutation interpretation software.

Our MutationForecaster suite is designed to analyze individual variants, gene panels, complete exomes or genomes.  One component is our Shannon pipeline for splicing mutation analysis, the only product on the market capable of comprehensive prediction of mRNA splicing mutations in exomes or complete genomes.  This product finds mutations that no other software, public domain or commercial, detects. It is based on a proven technology backed up by more than 400 peer-reviewed publications, and thousands of users. Aside from the online cloud application, it is available as an Application on Illumina’s BaseSpace system.  Veridical is another genome-scale product for the automatic validation of DNA sequencing variants that alter mRNA splicing. It is companion software to the article “Validation of predicted mRNA splicing mutations using high-throughput transcriptome data”, Viner et al. published in F1000research.  Veridical performs statistically valid comparisons of the normalized read counts of abnormal RNA species in mutant versus non-mutant tissues using RNASeq data. Veridical uses output from the Shannon human splicing pipeline as input.  The Automated Splice Site and Exon Definition Analysis server predicts the effects of sequence changes that alter mRNA splicing in human diseases. We designed the system to evaluate changes in splice site strength based on information theory-based models of donor and acceptor splice sites. It has been updated with the latest genomic coordinates (HG19), the newest mRNA tables (UCSC), known SNPs (dbSNP135), and updated donor and acceptor weight matrices. ASSEDA predicts potential normal and mutant isoform structures and their relative abundance.

Solution capture hybridization reagents.

Oligonucleotide pools covering unique sequences present in promoter, intragenic, and downstream regions have been designed for a series of genes. The first product has been validated for 7 hereditary breast and ovarian cancer genes. These reagents enable targeted enrichment of specific genes in any next generation, high throughput sequencing platform.The coverage using ab initio technology extends beyond products available from others, since Cytognomix covers both repeat-masked and divergent repetitive sequences with properties of unique sequences. This means that high multiplicity coverage is available for regions that were previously avoided – regions potentially containing mutations that would have been missed by other commercially available capture libraries.

Radiation biodosimetry software.

The Automated Dicentric Chromosome Identifier and Dose Estimator (ADCI) completely automates the interpretation of cytogenetic biodosimetry data. The performance of the system fulfills International Atomic Energy Agency criteria for triage biodosimetry. Both workstation and high performance computing versions can accurately estimate exposures using image data obtained from any commercial metaphase system on the market, without user curation.  See our biodosimetry product page for more details.  You can also test our algorithm for discriminating dicentric from monocentric chromosomes.

Laboratory-validated SCFISH probes.

The applicability and precision of the SC-FISH has been established for over twenty different chromosomal disorders, covered by over seventy different probes, and all currently available.  This set includes probes for congenital syndromes and leukemias, for conditions such as Monosomy 1p36, Wolf-Hirschorn, Cri-du-Chat, Chronic Myelogenous Leukemia (CML), Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Prader-Willi & Angelman, Smith-Magenis Syndromes, and many others. A complete set of subtelomeric SCFISH probes has also been validated for detection of chromosome imbalances close to the termini of individual chromosomes.

Diagnostic results from a patient with acute myelogenous leukemia

The Cytognomix SC Genome Browser.

The Company’s SC Genome Browser contains design information on over 220,000 ab initio single-copy intervals for which SC FISH probes have been pre-designed or are being used for microarray content development. The probes can be selected by chromosomal location (band or genomic coordinate), by gene locus, and by disease or phenotype. Each probe is unique, and is representative of a single–copy interval, as assigned by the Company’s proprietary ab initio processes. Please visit the browser website or request an account for access to these products.

Microarray product designs for array comparative genomic hybridization.

Array CGH is used to identify chromosome abnormalities by genome-wide assessment of sequence copy number.   Fundamental questions remain about data quality, evident by inter-platform comparisons monitored by the US FDA.  Coefficients of variation remain high between platforms. We have shown that noise in genomic hybridization is related to contaminating SC sequences present in repeat-enriched (C0t-1 DNA) used to block repetitive sequences, and repetitive sequences in local context to single copy probes on those arrays. Systematic noise in microarray data can be reduced with  ab initio SC designs, eliminate the requirement for C0t-1 DNA and obviate its effects.

Repeat-masked probes.

Cytognomix’s founders previously invented and carried out studies based on repeat-masked approaches* for SCFISH probe design. In most instances, these probes perform as expected, however, we have found that only ab initio technology can assure true genome-wide SC designs.