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I hope somebody will be able to help me.
I am using a quite new assay GeneRead DNAseq Targeted Panels V2 from Qiagen for targeted enrichment of FFPE samples before NGS. I purchased a predesigned assay that covers a specific gene. Because I use FFPE DNA I chose all amplicons have to be within 150 bp range.
Basically the protocol includes following steps:
1. DNA purification from the paraffin slides
2. QC of extracted DNA
3. Targeted Enrichment PCR
4. Purification and pooling of the PCR products
5. Library construction
6. NGS
6. postNGS analysis
For targeted enrichment PCR the Qiagen recommends the GeneRead PCR mix followed by purification of PCR products using AMPure XP beads and this is where I have issues. As you can see on attached picture of my 6% polyacrylamide gel I can't really get rid off the larger fragments, i.e. > 150 bp.
The gel is a bit complicated as I tried to test a few things simultaneously. I used FFPE DNA that passed QC control (Qiagen's QuantiMIZE assay) and DNA extracted from RCC cell line cells and fragmented by sonication (dabbed Fresh). Then besides Qiagen's GeneRead PCR mix I also used in-house PCR mix where I could control for MgCl2 concentration to increase specificity of the reaction.
And I used two different concentration of beads to see whether I can improve the cleaning. x0.9/x1.6 is recommended by Qiagen's protocol and I used a higher concentration of x1.4/x1.6.
Issue #1
Seems like higher concentration gave me a bit cleaner results in the higher size range, though by reading how the beads work one could expect that lower concentration of beads should help to eliminate larger fragments. Can someone suggest what is going here or how I might improve the cleaning?
Issue #2
I can't understand what sits in each well of the gel where I run FFPE samples? Could it be some cross linked DNA with some proteins that just couldn't migrate through the gel?
Issue#3
What also doesn't make too much sense is that it seems like Qiagen's PCR mix works with FFPE DNA but not with Fresh one while our in-house mix doesn't amplify FFPE DNA at all.
If someone can help me withe any suggestion what is going on here I will really appreciate it!
I am using a quite new assay GeneRead DNAseq Targeted Panels V2 from Qiagen for targeted enrichment of FFPE samples before NGS. I purchased a predesigned assay that covers a specific gene. Because I use FFPE DNA I chose all amplicons have to be within 150 bp range.
Basically the protocol includes following steps:
1. DNA purification from the paraffin slides
2. QC of extracted DNA
3. Targeted Enrichment PCR
4. Purification and pooling of the PCR products
5. Library construction
6. NGS
6. postNGS analysis
For targeted enrichment PCR the Qiagen recommends the GeneRead PCR mix followed by purification of PCR products using AMPure XP beads and this is where I have issues. As you can see on attached picture of my 6% polyacrylamide gel I can't really get rid off the larger fragments, i.e. > 150 bp.
The gel is a bit complicated as I tried to test a few things simultaneously. I used FFPE DNA that passed QC control (Qiagen's QuantiMIZE assay) and DNA extracted from RCC cell line cells and fragmented by sonication (dabbed Fresh). Then besides Qiagen's GeneRead PCR mix I also used in-house PCR mix where I could control for MgCl2 concentration to increase specificity of the reaction.
And I used two different concentration of beads to see whether I can improve the cleaning. x0.9/x1.6 is recommended by Qiagen's protocol and I used a higher concentration of x1.4/x1.6.
Issue #1
Seems like higher concentration gave me a bit cleaner results in the higher size range, though by reading how the beads work one could expect that lower concentration of beads should help to eliminate larger fragments. Can someone suggest what is going here or how I might improve the cleaning?
Issue #2
I can't understand what sits in each well of the gel where I run FFPE samples? Could it be some cross linked DNA with some proteins that just couldn't migrate through the gel?
Issue#3
What also doesn't make too much sense is that it seems like Qiagen's PCR mix works with FFPE DNA but not with Fresh one while our in-house mix doesn't amplify FFPE DNA at all.
If someone can help me withe any suggestion what is going on here I will really appreciate it!