5 years ago

Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis

Philip Tsao, Duane Smailus, Rob A. Holt, Martin Hirst, Christian Steidl, Diane Miller, Andrew J. Mungall, Yongjun Zhao, Robin J. Coope, Pawan Pandoh, Simon Haile, Martin Jones, Richard A. Moore, Richard D. Corbett, Heather Kirk, Tina MacLeod, Denise Brooks, Marco A. Marra, David W. Scott, Miruna Bala, Helen McDonald, Yussanne Ma, Steven J. Jones, Steve Bilobram

by Simon Haile, Pawan Pandoh, Helen McDonald, Richard D. Corbett, Philip Tsao, Heather Kirk, Tina MacLeod, Martin Jones, Steve Bilobram, Denise Brooks, Duane Smailus, Christian Steidl, David W. Scott, Miruna Bala, Martin Hirst, Diane Miller, Richard A. Moore, Andrew J. Mungall, Robin J. Coope, Yussanne Ma, Yongjun Zhao, Rob A. Holt, Steven J. Jones, Marco A. Marra

Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95–100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.

Publisher URL: http://journals.plos.org/plosone/article

DOI: 10.1371/journal.pone.0178706

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