Hello! I have a question of FFPE samples for NGS.
Are the fresh/frozen samples superior than FFPE samples for NGS? What I know now is that DNA from FFPE samples are more fragmented than common DNAs. Are there some other characters could distinguish these types of tissue in terms of NGS(here I mean RNA-Seq and Targeted DNA sequencing)?
One intertesting fact for me is usually the DNA 260/280 absorbance value 1.8-2.0 is considered as "pure", and for "pure" RNA the value is like 2.0-2.2. But for some commercial kits, DNA 260/280 value of 1.7-1.9 is enough, and for RNA 260/280 value is OK if between 1.9-2.1.
I'm wondering if the paraffin will necessarily decrease the quality of DNA/RNA? And generally, will a slighter higher than normal 260/230 value, i.g.2.3-2.4, will affect the reliability of RNA-Seq and Targeted DNA sequencing?
You guys are always my bioinformatic hero. Short and clear answers are preferred firstly. Then bomb me with links or reference. Thank you. Have a nice day.
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Qingyang Xiao
1 answer
This is not quite a bioinformatics question and more about wet laboratory protocols, I feel. Nevertheless:
The formalin fixation process results in crosslinking of the DNA molecule. Over time, the molecule also becomes heavily fragmented. In this sense, it can be viewed as losing its quality. Through gel electrophoresis, the DNA will look 'blurred' and you will not see large fragments. Most will certainly be <1000bp. The A260/280 reading will depend on how you extract your DNA from the slides. I have previously worked with FFPE and fresh-frozen tumour - the difference in 'quality' between these is astounding (in favour of fresh frozen).
All of this said, it is possible to extrapolate useful information from DNA that has undergone FFPE through NGS: Please refer to the following for useful information:
- Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.
- Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue.
- Reliable Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue Using Single Molecule Tags.
- Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues
I believe that Qiagen also have kits designed specifically for FFPE and NGS.
Kevin
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Here's a recent comparison of formalin-fixed, paraffin-embedded (FFPE) tissues against Fresh-frozen (FF) tissue for the 100,000 genomes project, that may also be useful Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project