Microarray- and deep sequencing-based profiling approaches: the technological evolution continues…

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• Professor Eric F.P.M. Schoenmakers,. Dr Roland P. Kuiper, Dr. Ioannis Ragoussis,. Professor Stephan Beck,Professor Johan den,Dr. Peter J. Campbell,
• Thursday 09 July 2009, 09:00-17:00
• The BioPark, Hertfordshire,AL7 3AX UK.

If you have a question about this talk, please contact Dr Astrid Englezou.

This one-day meeting aims at providing the audience with a comprehensive overview and in-depth comparison of currently available research tools, including array-, bead- or massive parallel sequencing-based platforms as well as experimental considerations in relation to expression-, genomic-,and epigenetic-profiling. Illustrated by real-life examples, various internationally acknowledged speakers will provide the attendee with critical experimental design parameters. Pitfalls associated with specific technologies as well as their solution will be discussed extensively. This meeting has CPD approval .

The Chair will be Professor Eric F.P.M. Schoenmakers, Radboud University Nijmegen Medical Centre (RUNMC) & Nijmegen Centre for Molecular Life Sciences (NCMLS), Nijmegen, The Netherlands.

The Agenda includes:

Identification of novel biomarkers by high-resolution copy number profiling and homozygosity mapping in hematologic malignancies. Dr Roland P. Kuiper, Microarray Facility Nijmegen, Oncology Research, Radboud University Nijmegen Medical Centre (RUNMC) & Nijmegen Centre for Molecular Life Sciences (NCMLS), Nijmegen, The Netherlands. Recent progress in genomics technology has made detailed characterization of the cancer genome feasible. One example involves the development of high-resolution SNP -based genotyping arrays for detecting regions of genomic amplifications, deletions, and copy-neutral homozygosity. Application of these arrays has revealed major new insights into the field of cancer genomics, particularly in hematologic malignancies, which has led to the discovery of several new biomarkers. In this presentation, examples will be presented for childhood acute lymphoblastic leukemia and myelodisplastic syndrome.

Deciphering the role of miRNAs in hypoxia by Digital Gene Expression profiling Dr. Ioannis Ragoussis, Wellcome Trust Centre for Human Genetics, University of Oxford, UK Hypoxia in tumours may confer resistance to conventional therapies and is associated with a poorer prognosis. MicroRNA expression alterations have been described in cancer and certain microRNAs have shown regulation by hypoxia.

We performed a time course exposition to hypoxia (1% oxygen for 16h, 32h and 48h) using MCF7 cancer cell cultures. We also investigated the effect of VHL suppression in RCC4 renal cancer cells by comparing to RCC4 cells transfected with VHL . The microRNA fraction was isolated from total RNA and sequenced using the GA II analyser. Gene expression profiles were determined using Illumina WG-6 v3 arrays and ImicroRNA arrays v.1. This led to the identification of 376 different microRNAs and microRNA variants in MCF7 samples and 283 in RCC4 and RCC4 +VHL cells. Relative microRNA expression analysis showed a set of 36 microRNAs disregulated in all 3 hypoxia times compared to normoxia. A second set of 62 microRNAs appeared to be disregulated from 32h of hypoxia onwards, suggesting a more severe reaction to hypoxia. Concerning RCC4 cells, 102 microRNAs showed differential levels of expression compared to RCC4 +VHL cell. New microRNAs were identified using a novel machine learning algorithm and are being validated. MicroRNA target sequences were identified among genes differentially expressed in hypoxia and correlated with microRNA expression. All this information will enhance our understanding of hypoxia mediated regulation of gene expression.

Methylome analysis using array and sequencing based approaches . Professor Stephan Beck, Cancer Institute, University College London, UK DNA methylation plays an essential role in biology with wide-ranging implications for human health and disease. To understand the rules governing DNA methylation and the consequences if DNA methylation is perturbed requires genome-wide analysis of its temporal and spatial plasticity. Almost 60 years after the discovery of 5-methyl cytosine and about 25 years since the discovery that altered DNA methylation plays a role in disease aetiology, particularly in cancer, technologies have finally become available for whole-genome DNA methylation profiling (methylome analysis) with ever increasing resolution. I will present data from our efforts using array- and sequencing-based platforms for high-throughput DNA methylation analysis, discuss some of the lessons learnt and give an outlook on how the data may be used in an integrated approach – termed ‘reverse phenotyping’ – to analyse and better understand the (epi)genomics of phenotypic plasticity in health and disease.

A comparison of expression profiling by deep sequencing and microarrays Professor Johan den. Dunnen Center for Human and Clinical Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands

We have done the first large-scale comparison between deep sequencing and microarray-based expression profiling. With the Illumina digital gene expression assay, we obtained ~2.4 million sequence tags per sample, their abundance spanning four orders of magnitude. Results were highly reproducible, even across laboratories. The correlation with five different microarray platforms was modest and most significant for Affymetrix. The changes in expression observed by deep sequencing were larger than observed by microarrays or quantitative PCR . While undetectable by microarrays, antisense transcription was found for 51% of all genes and alternative polyadenylation for 47%. Deep sequencing provides a major advance in robustness, comparability and richness of expression profiling data and is expected to boost collaborative, comparative, and integrative genomics studies.

Use of new sequencing technologies for the annotation of cancer genomes. Dr. Peter J. Campbell, Sanger Institute, Cambridge, UK We are now entering an era in which it will be feasible to catalogue every genetic event in a cancer. Next generation sequencing platforms already offer the capacity to generate gigabases (Gb) of sequence each week at a cost of less than 1 cent per kilobase (kb). Techniques have been developed which allow the detection of genomic rearrangements, copy number changes, point mutations and small insertions and deletions as well as epigenetic alterations on a single instrument. This will be a significant advance on existing approaches to cancer genomics. The analysis will be genuinely genome-wide, cataloguing genetic changes not only in coding sequence but also the other 98% of the human genome including, for example, promoters, enhancers and non-coding RNAs. At the Cancer Genome Project, we have developed protocols for mapping acquired rearrangements to the base-pair level, providing insights into the diversity of aberrant processes sculpting the genome which underlie the evolution and development of cancer.

Abstracts for poster presentation only can be submitted up to two weeks before the event. Please note that there will be a best poster prize.  All accepted abstracts will be published in the meeting proceedings

If you would like to book a place at the meeting, please visit : www.regonline.co.uk/microarrays09

This talk is part of the euroscicon series.

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