Abstract B14: A high-throughput allelic copy-number platform utilizing a 75-ng DNA input in a 330,000 molecular inversion probes (MIP) assay on microarrays with FFPE samples

Autor: Yuker Wang, Tom Asbury, Victoria Carlton, Ron Sapolsky, Jen Wilkins, Farooq Siddiqui
Rok vydání: 2010
Předmět:
Zdroj: Clinical Cancer Research. 16:B14-B14
ISSN: 1557-3265
1078-0432
Popis: Background: Copy number (CN) studies of cancer hold great promise for the discovery of reliable biomarkers that can predict clinical outcomes such as prognosis and response to medication. One of the greatest challenges in the study of cancer CN (CCN) is that the vast majority of banked samples are formalin-fixed paraffin- embedded (FFPE). Due to DNA degradation, FFPE samples generally perform poorly with most CN technologies. However, MIP technology works well on FFPE samples, requiring only small stretches of intact genomic DNA (40-60 bp) in as little as a 75-ng input. We have applied this 330,000-plex platform on thousands of FFPE samples from various tumor tissue types, including breast, colon, ovary and brain, with more than 90% overall pass rate. This new platform allows an improved whole genomic coverage to an average of 10 kb-per-probe with a dense coverage of cancer-relevant coding regions. Experimental Design: MIP probes were synthesized, pooled and screened for their performance. A select set of 368,000 probes were chosen and used in the final chip design. The selection criteria were: (1) good quantitative performance, in terms of reproducibility with small variation in experiment metrics and in terms of the responsive dynamic range; (2) nonredundant whole genome coverage with minimum gaps (the maximum gap is limited to 100 kb) that includes additional dense coverage for relevant oncogenic regions. Our platform employs a measurement comparing neighboring markers across the entire genome: this median of absolute pairwise distribution, or MAPD, is a reliable metric for assessing tumor sample quality in the MIP-CCN assay. For array design, tag sequences were switched to genomic sequences with minimal impact on CN assessment. The best sequence of each interrogated SNP region was chosen based on empirical screening data. For the protocol, since each MIP probe hybridizes independently, the assay design should scale up for the larger 300K-plex probe panel. Indeed, the 330,000 probes worked well within the existing protocol with only minor modifications. Results: Using only 75 ng of input genomic DNA, we have obtained both good genotyping and CN data with the current 330K- probe platform. For cell-line DNA, CN quantitation performs with the same high quality as proven by 1X to 5X titration experiments. For FFPE samples, a dynamic range of up to 100 copies has been achieved. 0.6, a 2000-FFPE sample project has an overall pass rate £ Good concordance between normal and tumor sample pairs is also observed. Using an arbitrary cutoff of MAPD > 92%. In addition to CN information, data for hundreds of single-base somatic mutations are also generated. These markers are currently under validation. Conclusion: We have developed a powerful 330K MIP-CCN platform that works well on both fresh frozen/cell-line and archival FFPE samples. Currently we are applying the new platform to dozens of custom FFPE projects. References: 1. Wang Y et al. Performance of molecular inversion probes (MIP) in allele copy number determination. Genome Biol. 2007;8(11):R246. 2. Wang Y et al. High-quality copy number and genotype data from FFPE samples using molecular inversion probe (MIP) microarrays. BMC Med Genomics. 2009 Feb 19;2:8. 3. http://www.affymetrix.com/index.affx; search for “MIP” Citation Information: Clin Cancer Res 2010;16(14 Suppl):B14.
Databáze: OpenAIRE
Externí odkaz: