This week, listen to Wahab Khan present our latest discovery using ab initio scFISH at the 2013 meeting of the American Society of Human Genetics:

Title: Non-random, locus-specific differences in DNA accessibility are present in homologous metaphase chromosomes.(168) (10:00AM-10:15AM on Thursday October 24, location: Grand Ballroom CDE, Concourse Level, Westin Boston Waterfront Hotel) (Platform)
Author(s): W. A. Khan, P. K. Rogan, J. H. M. Knoll
Keywords: Cytogenetics, KW025 – chromosomal structure/function, KW062 – FISH, KW082 – genomic structure, KW088 – imprinting, KW162 – structure/function

These differences cannot be detected with conventional large target FISH probes or with labeled pools of oligos.   Only Cytognomix`s ab initio scFISH probes are capable of visualizing differential accessibility in metaphase chromosomes.

Please contact us should you  be interested in purchasing these products.     email: info@cytognomix.com or info@scprobe.info

Cytognomix has released a new version, V2.0, of the Shannon pipeline for mRNA splicing mutation analysis.    It is distributed by CLC bio for use with their Genomics workbench or server. Click here to download the updated documentation.

This release (2.0):

Cytognomix has received a Notice of Allowance from the US Patent and Trademark office for our patent application, “Centromere Detector and Method for Determining Radiation Exposure From Chromosome Abnormalities.”  All claims that were applied for were allowed in the soon to be issued patent.

The application is available at this link: US Patent Application Ser. No 13/822,289

The application was originally filed November 4, 2011 as international patent application: PCT/US11/59257. This application was deemed patentable after examination, and was entered into the Patent Prosecution Highway program. Other applications remain pending in other jurisdictions.

Cytognomix’s Shannon pipeline has been highlighted in a report published describing the 2013 annual scientific meeting of the Human Genome Variation Society (HGVS) in Paris, France. The paper’s authors, Sian Ellard,George P. Patrinos, and William S. Oetting, write:

“Although NGS can identify numerous sequence variants, a major problem is predicting the functional consequence of mutations identified after sequencing. Much work has been done on determining the functionality of protein coding mutations within exon sequences, but significant mutations that affect splicing or gene expression have not been comprehensively assessed. Peter  Rogan, of the Schulich School of Medicine, Western University, London,Canada, has taken on this task and presented some of his results in his talk “Genome-wide prediction and validation of mRNA splicing mutations in cancer.” Using Shannon information theory, software packages were created for genome-scale analysis of variants within splice sites that alter binding site strength [Shirley et al., 2013] and to predict the transcript isoforms generated by splicing mutations [Mucaki et al., 2013]. Variants within the BRCA1 and BRCA2 genes were used to test their model and were grouped into four functional states: wild type, leaky mutations with residual activity, inactivating mutations, and cryptic splicing. Predicted effects of 50% of these calls were confirmed using RNAseq analysis. Additional efforts underway include predicting functional mutations in promoter and intronic regions and the potential effects of mutations in regulatory splicing elements with information theory-based models. The goal is to use these software tools to perform a genome-scale mutation analysis to identify functional variants outside the coding regions affecting either splicing or expression levels.”

Citation:   Ellard S, Patrinos GP, and Oetting WS, Clinical Applications of Next-Generation Sequencing: The 2013 Human Genome Variation Society Scientific Meeting, Human Mutation, DOI: 10.1002/humu.22400, 2013.

Letter to the editor: “Best practices for evaluating mutation prediction methods”

Available online: Human Mutation, in press.

Peter K. Rogan(1,2) and GuangYong Zou(3)

(1) Biochemistry, (2) Computer Science, (3) Epidemiology and Biostatistics

Schulich School of Medicine and Dentistry, Western University, London ON

[available upon request. Contact: info@cytognomix.com]

Session 27: Causes and Consequences of Chromosomal Variations.

Title: Non-random, locus-specific differences in DNA accessibility are present in homologous metaphase chromosomes.

Authors: Wahab Khan, Peter Rogan, and Joan Knoll.

Thursday, October 24, 10 AM. Boston Convention & Exhibition Center, Grand Ballroom CDE, Concourse Level

This study examines locus specific DNA accessibility during metaphase using Cytognomix’s single copy FISH probes imaged by superresolution microscopy.

Cytognomix has been invited to present applications of our ab initio sc probe targeted capture array technology and the Shannon software pipeline this fall at several venues in Italy.

18th Congress of the Italian Society of Human Genetics (SIGU) in Rome 25-28 September 2013 (platform) :   IDENTIFICATION, PREDICTION, AND PRIORITIZATION OF NON-CODING VARIANTS OF UNCERTAINSIGNIFICANCE IN HERITABLE BREAST CANCER.   P.K. Rogan1, E.J. Mucaki2, A. Stuart2, E. Dovigi1, C. Viner2, B. Shirley3, J.H. Knoll1, P. Ainsworth2.   1. Univ. Western Ontario, London, N6A 2C1 Canada and Molecular Bases of Genetic Risk and Genetic Testing, FondazioneIRCCS Inst. Nazionale dei Tumori,20133 Milano, Italy  2. Univ. Western Ontario    3. Cytognomix Inc., London ON Canada

Department of Biology, University of Padua, 3-October 2013 (host Stefania Bortoluzzi):  Deciphering variants of unknown significance that affect gene expression in inherited and somatic breast cancer

Institute of European Oncology, Milan, 30-October 2013 (hosts  Ugo Cavallaro and  Bernardo Bonanni),   “Clinical Science in Oncology Seminars” Program:  Deciphering variants of unknown significance that affect gene expression in inherited and somatic breast cancer

Invited presentation: “Deciphering variants of unknown significance in inherited breast cancer genes and exomes,” to the Clinical Cancer Genetics group of the Italian Society of Human Genetics (18th Incontro di Genetica Oncologica Clinica, SIGU), that will be held at the Policlinico S. Orsola in Bologna, Italy on July 19 2013 at 11 AM. We will describe predicted splicing, UTR and transcription factor binding site mutations, confirmed by RNASeq transcriptome data, and pathway analysis of the genes containing these mutations.

OGI’s Early Stage Investment in Cytognomix Contributes to the Success of the Shannon Human Splicing Pipeline

IFOM-IEO. May 10, 02:00 PM

“Discovery and predicted consequences of non-coding sequence variants affecting gene expression on a genome-scale and in inherited breast cancer.”

(http://www.semm.it/seminars.php)

The IFOM-IEO Campus is a new biomedical research centre, created by the joint efforts of the FIRC Institute of Molecular Oncology Foundation (IFOM) and the Department of Experimental Oncology of the European Institute of Oncology (IEO), which have expanded and integrated their research activities on a common campus.

The IFOM-IEO Campus is located in Milan, the heart of the commercial/industrial north of Italy, where it benefits from numerous interactions with other scientific and medical organizations in the area (see participants below). The Campus is also home to the Ph.D programs of the European School of Molecular Medicine (SEMM), which it is running in collaboration with the University of Milan, the University of Naples “Federico II” and the Italian Institute of Technology (IIT) in Genoa.

Host: Dr. Paolo Peterlongo

Fondazione IRCCS Istituto Nazionale dei Tumori, May 15, 2013. 02:00 PM

“Discovery and predicting impact of non-coding sequence variants that affect gene expression on a genome scale and in inherited breast cancer”

Since its  founding of the objectives of the Istituto Nazionale dei Tumori via Venezian were defined as follows:

• Research in the field of cancer to better understand the biological mechanisms and systems to continuously improve diagnosis and treatment.
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• The diagnosis and treatment of cancer, with the methods that are recognized internationally as the most valid.
• The oncology information of citizens and the training of medical and nursing staff involved in the treatment of cancer.

Host: Dr. Paolo Radice – S.C. Medicina Predittiva: basi molecolari del rischio genetico e TestGenetici, Dipartimento di Medicina Predittiva e per la Prevenzione.

Ben Shirley, Chief Software Architect of the Shannon pipeline for human mRNA splicing mutation analysis presented a webinar today including a live demonstration of the software’s capabilities. We received a number of favorable comments on this LinkedIn Network Discussion.

The presentation was recorded and will be available in the future for viewing. Please bookmark this webpage. We will share the URL of the recorded presentation when it becomes available.

The presentation was recorded in Adobe Connect. The URL to watch it is: http://ogi.adobeconnect.com/p6u1uglzuos/

Peter Rogan is giving a platform presentation on: “Genome-wide prediction and validation of mRNA splicing mutations in cancer” at the HGVS meeting in Paris, France on June 8, 2013.  The meeting will be held at the Hotel’Concorde’La’Fayette, just preceding the annual European Society of Human Genetics conference, at which Dr. Rogan will also be presenting a paper.

The Ontario Genomics Institute (OGI) and Cytognomix Inc. are hosting a 30 minute webinar about the Shannon Human Splicing Pipeline – software for genome-scale splicing mutation analysis on April 24th at 1 PM EST.  We require all attendees to register for the webinar on April 24, 2013 at 1:00 PM (EST) at the OGI website:  https://ogi.factorial.ca/Forms/fm_forms.jsp?token=HwkGRx0GZl5aSxdR

The Shannon Human Splicing Pipeline is an information theory-based tool for splicing mutation detection and analysis on a genome-scale. It offers high-throughput variant analysis and can examine – for example – 211,049 variants in 1 hour 12 minutes on an I7-based CPU. The software has been implemented for the CLC-Bio Genomics Workbench platform. Variants are annotated to indicate the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Variants determined to be potentially deleterious can be viewed in tabular format, as plots, or genome browser tracks. A post-hoc filtering process can further reduce the number of resulting variants. Some pipeline results will be examined, pipeline implementation will be described, and the post-hoc filtering process will be discussed.  Check out our recently published paper:   Shirley BC, Mucaki EJ, Whitehead T, Costea PI, Akan P, Rogan PK. Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences. Genomics Proteomics Bioinformatics. 2013 Mar 14. doi:pii: S1672-0229(13)00029-6. 10.1016/j.gpb.2013.01.008. [Epub ahead of print] PubMed PMID: 23499923

The presentation will be led by Ben Shirley, the chief developer of the Shannon Human Splicing Pipeline. The webinar will be appropriate for all levels of researchers interested in using the software to reduce a genome-scale list of splicing variants to a tractable set of potentially deleterious mutations, suitable for further analysis in the laboratory.

On the day of the webinar, you will be able to access the webinar by clicking here and entering as a guest.

Please be sure to provide your name, title, institution and valid email address so that we can send you an email with webinar details and instructions.

Cytognomix is presenting the following paper  at the Joint HGM 2013 and 21st International Congress of Genetics meeting in Singapore  in the April 15 poster session (9:30-10 AM, 12:30-1:30 PM) on Cancer Genetics and Genomics (T05):

STRATEGY FOR IDENTIFICATION, PREDICTION, AND PRIORITIZATION OF NON-CODING VARIANTS OF UNCERTAIN SIGNIFICANCE IN HERITABLE BREAST CANCER.

P. Rogan 1,*, E. Mucaki 1, A. Stuart 2, E. Dovigi 1, C. Viner 3, B. Shirley 3, J. Knoll 2, P. Ainsworth 2  1Departments of Biochemistry, 2Pathology, and  3Computer Science, University of Western Ontario, London, Canada

Objectives: Non-coding sequence variants have been proven to significantly contribute to the phenotypes of high penetrance disorders. We develop an approach to predict pathogenic non-coding variants of uncertain significance(VUS) based on information theory-based analysis of changes in DNA and RNA sequences bound by regulatory factors.

Methods: Complete gene sequences are captured, enriching for non-coding variants in genes known to harbor mutations that increase breast cancer risk. Oligo baits covering the complete coding and intergenic regions 10kb up- and downstream of ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2 and TP53 were used in solution hybridization. Probe design captures both repeat-free and divergent repeat sequences that are effectively single copy. After Illumina sequencing of 21 high risk patient samples lacking coding mutations, information analysis prioritized non-coding variants within sequence elements recognized by proteins or protein complexes. VUS are being screened for mutations affecting essential binding sites recognized in mRNA splicing, by transcription factors (TFBS), and by protein interacting with untranslated regions (UTR). Information models for exon recognition predict the relative abundance of natural, cryptic, and mutant splice isoforms resulting from predicted mutations. A similar approach is introduced to detect mutations that alter strengths of TFBS and UTR binding sites. Information weight matrices were determined by entropy minimization of ENCODE ChIP-seq regions for 60 transcription factors embedded within DNAse I hypersensitive domains.

Results: The matrices were used to evaluate novel variants discovered by sequence analysis of breast cancer patients for alteration the TFBS binding strengths. This analysis prioritized 9 splicing, 8 TFBS, and 2 UTR variants as most likely to affect gene expression, potentially affecting 6 protein coding genes in the patient samples (from 7,909 variants in 7 genes).

Conclusion: This strategy more comprehensively covers non-coding regions in breast cancer genes than repeat masking, and introduces a unified framework for systematic interpretation of VUS that affect expression.

Khan W.A., Chisholm R., Tadayyon S., Subasinghe A., Norton P., Samarabandu J., Johnston L.J., Knoll J.H., Rogan P.K. Relating Centromeric Topography in Fixed Human Chromosomes to α-Satellite DNA and CENP-B Distribution. Cytogenetics and Genome Research, 2013 (DOI: 10.1159/000348744).

Abstract

Despite extensive analyses on the centromere and its associated proteins, detailed studies of centromeric DNA structure have provided limited information about its topography in condensed chromatin. We have developed a method with correlative fluorescence light microscopy and atomic force microscopy that investigates the physical and structural organization of α-satellite DNA sequences in the context of its associated protein, CENP-B, on human metaphase chromosome topography. Comparison of centromeric DNA and protein distribution patterns in fixed homologous chromosomes indicates that CENP-B and α-satellite DNA are distributed distinctly from one another and relative to observed centromeric ridge topography. Our approach facilitates correlated studies of multiple chromatin components comprising higher-order structures of human metaphase chromosomes.

US Pat. No.8,407,013 issued: “Ab initio generation of single copy probes”.

This patent covers single copy probes that also contain divergent repetitive sequences, which under certain conditions, hybridize like single copy sequences. This expands the human genomic real estate accessible for single copy genomic analysis techniques. Cytognomix has used this approach to produce FISH probes in regions that could not be accessed by repeat masking, genomic microarrays, and capture arrays for next generation sequencing.

If you are interested in licensing any of these fields of use, please contact us at info@cytognomix.com.

Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences.

B.C. Shirley, E.J. Mucaki, T. Whitehead, P.I. Costea, P. Akan, P.K. Rogan (2013), Genomics, Proteomics and Bioinformatics.

DOI:10.1016/j.gpb.2013.01.008

(http://www.sciencedirect.com/science/article/pii/S1672022913000296)

Intensity Integrated Laplacian Based Thickness Measurement for Detecting Human Metaphase Chromosome Centromere Location.
Arachchige AS, Samarabandu J, Knoll J, Rogan P.
IEEE Trans Biomed Eng. 2013 Feb 20.
PMID: 23434602

Abstract