FFPE DNA Repair

[djmastermix]

Hi,

I am working on optimizing FFPE extraction for our next generation sequencing applications. I am currently interested in trying to repair the DNA, so that we have more dsDNA available for improved library construction yield and ultimately better quality sequencing.

I am looking into using the PreCR repair mix from NEB. I am also looking into using Restorase from Sigma-Aldrich. I was wondering if anyone has had any experience using these reagents for DNA repair? If not, does anyone have any advice on how to boost yields of dsDNA from FFPE material, either by repairing the DNA or by other methods? Any advice would be much appreciated, as obtaining adequate material from FFPE tissue can be tricky. Thanks!

[niceday]

Out of interest what protocol are you using to extract from ffpe? And do you use zylene?

Thanks.

[AST]

Hi,
did you have success with the 2 restoring cocktails you mentioned? I was thinking along the same lines and it would be helpful to see if it works on FFPE material or not.

In my case, FFPE DNA could be extracted, and quantified with dsDNA Qubit, the fragmentation range is 100-4000bp. However, after shearing and also during library prep, the amounts gradually decrease. Although there is a shift after adapter ligation (however to an smaller degree than with normal DNA), the PCR does not seem to work (well).

Thanks.

[polymerase1986]

have you already tested the PreCR mix?Was it beneficial?

[dexterslab]

sorry to bump an old thread. is there any new insight on this?

I am having difficulties preparing libraries from FFPE DNA (I can't get amplification after adapter ligation). test-PCRs on the same DNAs are unsuccessful for amplicons >200 bp, which makes me think that DNA modifications might inhibit the amplification steps in the library prepa

[BerlindaV]

Hi, you are indeed likely experience problems due to DNA modifications from the formalin treatment. Also in standard PCR, it is difficult to amplify fragmetns of >200 bp from FFPE material. Was buffered formalin used for fixation? The use of unbuffered formalin makes this problem much worse.