A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

Abstract

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.

Keywords: Bisulfite sequencing; DNA extraction; DNA methylation; FFPE tissue; Genome-wide; RRBS.

Fig. 1

Diagram summarizing the workflow of experiments and key points for genome-wide DNA methylation analysis using FFPE samples

Fig. 2

H&E stained 5 μm cut sections from formalin fixed paraffin embedded (FFPE) melanoma. Eight analysed FFPE samples (a–h) are shown. Scales bars are shown

Fig. 3

Bioanalyser images demonstrating quality of two FFPE RRBS libraries. Each of the RRBS libraries (FFPE1 and FFPE2) was run on an Agilent 2100 Bioanalyzer using the high sensitivity DNA kit. The electropherogram displays a plot of fragment size (bp) versus fluorescence intensity. Peaks at 35 bp and 10,380 bp represent lower and upper markers. The 160–340 bp peaks represent the RRBS library

Fig. 4

MLH1 PCR to test efficiency of bisulfite conversion on FFPE derived DNA. a) MLH1 PCR of FFPE-derived bisulfite-treated DNA. Lane 1: 1Kb + ladder, Lane 2: 200 ng input DNA, Lane 3: 500 ng input DNA, Lane 4: Zymo methylated control DNA, Lane 5: unconverted genomic DNA, Lane 6: PCR negative (water). 2% agarose, run for 25 mins at 100 V. b) MLH1 PCR of RRBS libraries prepared from different amounts of FFPE-derived DNA. FFPE DNA was digested with MspI enzyme, A-tailed, end repaired and ligated to Illumina adaptors, and bisulfite converted. Then PCR was performed with MLH1 primers. Lane 1: 1Kb + ladder, Lane 2: 50 ng input DNA, Lane 3: 100 ng input DNA, Lane 4: 500 ng input DNA, Lane 5: Zymo methylated control DNA, Lane 6: PCR negative (water). 2% agarose, run for 25 mins at 100 V