A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA
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Published on: July 12, 2012
Jackie L Ludgate1, James Wright1,2, Peter A Stockwell3
1Department of Pathology, Dunedin School of Medicine, University of Otago, 270 Great King Street, P.O. Box 56, Dunedin, 9054, New Zealand.
Background:
Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis.
Methods:
Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing.
Results:
The main features and advantages of this protocol are: An optimized method for extracting good quality DNA from FFPE tissues. An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue. Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing.
Conclusions:
We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.
Insights
This study presents an optimized workflow for DNA methylation analysis from challenging Formalin-fixed paraffin-embedded (FFPE) tumor tissues. The streamlined protocol ensures efficient bisulfite conversion and library preparation for next-generation sequencing, facilitating epigenetic research.
Area of Science:
- Genomics
- Epigenetics
- Molecular Biology
Background:
- Formalin-fixed paraffin-embedded (FFPE) tumor samples are crucial for cancer research but present challenges for DNA extraction and analysis due to macromolecular damage.
- FFPE tissues pose specific difficulties for methylation analysis and the preparation of sequencing libraries, particularly those requiring bisulfite conversion.
Purpose of the Study:
- To develop and present a complete, streamlined workflow for preparing next-generation sequencing libraries for DNA methylation analysis from FFPE tissues.
- To overcome the challenges associated with FFPE sample quality for epigenetic studies.
Main Methods:
- A comprehensive workflow involving cell counting from FFPE blocks and DNA extraction from FFPE slides.
- Implementation of a polymerase chain reaction (PCR)-based test to evaluate bisulfite conversion efficiency.
- Preparation of reduced representation bisulfite sequencing libraries followed by massively parallel sequencing.
Main Results:
- An optimized method yielding high-quality DNA from FFPE tissues.
- An efficient bisulfite conversion and library preparation protocol utilizing as little as 50 ng of FFPE DNA.
- Successful integration of a PCR-based assay to confirm bisulfite conversion efficacy before sequencing.
Conclusions:
- A complete and integrated protocol for genome-scale DNA methylation analysis from FFPE tissues is provided.
- This workflow is expected to significantly advance clinical epigenetic research utilizing FFPE samples.
