Managing PI: Diether Lambrechts
Life Technologies OpenArray® RT PCR Platform
The OpenArray® RTPCR Instrument and OpenArray® AccuFill™ system combination enables the processing of hundreds to thousands of samples per day for high-throughput RT-PCR gene expression, microRNA expression or genotyping experiments. The systemís flexible assay design allows researchers to choose different options to match specific experimental needs, ranging from 256 assays x 12 samples up to 16 assays x 144 samples per chip.
The OpenArray® system uses plates containing 3,072 through-holes (33 nL each) in which separate PCR reactions are run. Over 1 million predesigned gene expression, 3 million predesigned SNP genotyping and 1 million predesigned copy number assays are available from Life Technologies. Custom assay design is also possible.
The OpenArray® RTPCR platform has been used in the VIB Center for Cancer Biology (CCB) in the past to perform pharmacogenetic studies and genotype large sets of SNPs in randomized clinical studies (jointly with Hoffman-La-Roche, Basel, Switserland). For instance, we have developed a tag SNP panel covering genetic variability in >10 key VEGF pathway genes and have evaluated whether any of these SNPs predict outcome to anti-VEGF (bevacizumab) therapy. These studies have led to the discovery of a genetic locus predictive for bevacizumab treatment outcome, as recently published in Lambrechts and Claes, et al. (Lancet Oncology 2012). We are also performing pharmacogenetic studies of anti-angiogenic therapies in the context of a European Framework Programme (FP7- Angiopredict).
Illumina iSCAN system
The Illumina iSCAN system is a high throughput array scanner that supports a wide range of applications. The platform is mainly used for genome-wide and custom designed single nucleotide polymorphism (SNP) genotyping. However, it can also be used to detect high-resolution single copy number variations (CNV) and to perform DNA methylation and gene expression analyses.
SNP genotyping can, for instance, be performed with the Infinium CytoSNP Assay. From 300,000 to 4.000 000 SNPs can be determined at once, making this assay perfectly suited to perform GWA studies and CNV analyses. In addition, the arraysí content can be customized, for instance by using the Custom GoldenGate Genotyping panels, allowing researchers to tailor assays to their specific needs.
At the CCB, we are currently using Infinium SNP arrays and the ASCAT software suite for the analysis of copy number variation and tumor fraction estimation. We successfully used this technique to locate frequently amplified and deleted regions in endometrial and ovarian cancer and identify genomic regions associated with platinum-resistant chemotherapy in ovarian cancer (jointly with Profs. F Amant and I Vergote, UZLeuven).
The recently developed Infinium HumanMethylation 450K Beadchip allows the interrogation of over 450,000 cytosine sites spread across the entire genome, hereby covering 99% of all known genes and 96% of all known CpG islands. Hence, this technology, which is based on genotyping of bisulfite-treated DNA, is ideal for performing epigenome-wide association studies (EWAS). For instance, at the CCB, we are currently performing epigenome-wide profiling of lung tumors: by comparing the methylation pattern in tumor tissue and adjacent normal lung tissue from lung cancer patients, we aim to identify tumor-specific methylation signatures (jointly with the Respiratory Oncology Unit, UZLeuven). This technology, which works for blood and tissue samples (DNA from FFPE and fresh frozen material can be used), can also be used to investigate the contribution of methylation changes to various non-malignant diseases.
An overview of all possible BeadArray contents from the company Illumina can be found here.
Sequenom MassARRAY® Platform
The Sequenom MassARRAY® is a mass-spectrometry based platform used for high-throughput SNP genotyping and oncogene mutation profiling of targeted candidate variations. It is additionally capable of methylation mapping in selected CpG-rich regions. Its iPLEXÆ technology efficiently multiplexes up to 40 different genotyping assays in one PCR reaction and thereby provides very flexible, effective, rapid and accurate genotypes. Our current set-up allows us to genotype 1152 patients per day, thereby generating up to 46,000 genotypes in a single experiment. In recent years, we have analyzed an average of over 300 Sequenom chips per year.
The platform has contributed to numerous complex genetic studies in large clinical cohorts, which have in part been performed in the context of international consortia, such as BCAC (breast cancer), OCAC (Ovarian cancer) and ECAC (Endometrial cancer). Projects include, but are not limited to:
- Genetic susceptibility to cancer and associations with clinical cancer subtypes (Antoniou et al, Nat Genet 2010; Goode et al, Nat Genet 2010; Yang et al, JNCI 2011; Spurdle et al, Nat Genet 2011; Haiman et al, Nat Genet 2011; Lambrechts et al, Hum Mutat 2012).
- Genetics of cardiovascular diseases (Buysschaert, Eur Heart J 2010; Schunkert et al, Nat Genet 2011 Voight et al, Lancet 2012).
- Pharmacogenetic studies of bevacizumab (Lambrechts, Claes et al, Lancet Oncology 2012), tamoxifen (DieudonnÈ et al, Breast Cancer Res Treat 2009) and carboplatin (Huang et al, Clin Cancer Res 2011).
- Genetics of other diseases: COPD (Wauters et al, Eur Respir J. 2011), hemolytic-uremic syndrome (Delvaeye et al, NEJM 2009), ALS (Van Es et al, Amyotroph Lateral Scler. 2009).
The platform has also frequently been used for somatic hot-spot mutation profiling. The advantage of using Sequenom for oncogene profiling experiments is that it uses only little amounts of DNA (±-10 ng DNA per reaction) and provides reliable results on Formalin Fixed Parraffin Embedded (FFPE)-extracted tumor DNA. Most notable studies performed thus far include:
- Effect of EGF pathway mutations on cetuximab efficacy in metastatic CRC (De Roock et al, Lancet Oncology 2010).
- Effect of KRAS mutations on cetuximab efficacy in the phase III COIN trial in metastatic CRC (Maughan et al, Lancet 2011).
- Validation of somatic mutations identified by cancer genome sequencing (Reumers et al, Nat Biotech 2011).
As mentioned, Sequenom is also capable of doing quantitative methylation analysis using the Sequenom EpiTyper technology, which delivers individual methylation ratios for CpGs within target sequences of up to 600bp. Pre-validated EpiPanels can be used or custom DNA Methylation Assays can be designed by using the EpiDesigner Software. This technology is therefore suitable for the epigenetic analysis of large promoter regions. So far, the technology has mainly been used by K Freson (Center for Molecular and Vascular Biology, KULeuven) to detect imprinting defects in the GNAS gene cluster (Izzi et al, Clin Chim Acta 2010).
Illumina HiSeq 2000
The HiSeq 2000 Illumina sequencing platform (funded by Stichting Tegen Kanker, jointly with Prof. J. Cools) has been operational since November 2011 in the KULeuven. This state of the art next-generation short read sequencer can be used for various sequencing applications, such as whole genome sequencing of human or mouse genomes, targeted re-sequencing (e.g., exome or custom amplicon sequencing), expression profiling (full transcriptome analysis, small RNA profiling and discovery) and ChIP-Seq (sequencing specific binding sites of DNA-associated proteins). One single run of the HiSeq 2000 is capable of generating 600 Gbases in only 12 days. This ultra-high output and speed enables the simultaneous sequencing of 5 human genomes at ~30x coverage, 80 full exomes at ~50x coverage or the generation of 192 gene expression profiles in one run.
The CCB possesses high throughput computing servers with adequate large storage capacity to assemble and store millions of reads generated by the HiSeq 2000. The results are further processed by in-house developed filters (Reumers et al. Nat Biotechnol. 2011) and can be validated on the Sequenom MassARRAY Platform. Combining targeted re-sequencing and expression profiling from the HiSeq 2000 with copy-number and methylation profiling obtained with the Illumina iSCAN system provides us with a powerful comprehensive approach to locate driver genes and recurrently affected pathways in various diseases, and especially in cancer.
At the CCB, this technology has been successfully applied to DNA derived from both FFPE and fresh frozen samples. So far, we have sequenced >200 human cancer exomes, mainly from ovarian tumors (jointly with I Vergote, UZ Leuven and in the context of the OCTIPS-FP7-project), endometrial tumors (jointly with F. Amant, UZ Leuven), microsatellite unstable colorectal tumors, as well as several breast tumors (collaborations with J De Greve, VUB and C Sotiriou, Bordet Institute). Furthermore, RNA-sequencing has successfully been applied to RNA derived from lung cancer patients to study the effect of altered DNA methylation on gene expression levels. Finally, we are also using the sequencer to assess the influence of hypoxia on the methylome by applying quantitative sequencing methods of 5-methylcytosine and 5-hydroxymethylcytosine.
- All technologies are accessible collaboratively (contact: Diether Lambrechts) or on a fee-for-service basis (https://www.genomicscore.be/).