I am struggling to get the next generation sequencing panel done for a few tumor cases: They are low-cellularity tumors. We only have scant FFPE tissue. The cases kept getting rejected by a sequencing facility due to quality or quantity issue. The facility runs the Illumina platform. They need a minimum of 50 ng DNA for panel sequencing (200 genes), which I think is a lot. Will Ion Torrent Ampliseq solve the issue?
Any suggestions between Illumina or Ion Torrent platform for low-cellularity tumors?
Dear Cheng-Ying Ho,
The big problem is not the equipment you use. I think the problem is the use of FFPE tissue samples scant. You have to test the best extraction and cleaning option for it.
Hello Cheng-Ying,
It sounds that the problem is the library prep method and not the platform. There are examples out there of sequencing on an Illumina platform with as low as 1ng of DNA, so your quantity is not a problem. If you have really poor quality DNA (i.e. highly degraded) there is not much that can be done. Have you tried using a KAPA kit? They are made for FFPE samples and are becoming quite popular.
https://www.kapabiosystems.com/product-applications/platforms/illumina/library-preparation-for-illumina/
You can also try the NEBNext Ultra kit.
https://www.neb.com/products/e7370-nebnext-ultra-dna-library-prep-kit-for-illumina
Or a TruSeq Nano kit.
http://www.illumina.com/products/truseq-nano-dna-library-prep-kit.html
Both have been around for a while and are commonly used.
Not all facilities like to mess with low quantity DNA, no matter the platform, so you may have to make the libraries yourself (really not all that hard to do with one of these kits and can save you money if you are doing a large number of samples) or you can shop around for different core facilities (I am sure someone out there would be willing to mess with this but for an increase cost due to inconsistent results and increase work hours that accompany low quantity DNA library prep). You can also try messy with different extraction methods.
Also I should note that most core facilities request 50ng but may only use a faction of that. Thus, if you talk to someone at the facility they may be able to work with you. For example we use the University of Wisconsin at Madison for our sequencing and they request 50ng, which we usually can provide no problem, but for one project we only had 10ng and they had no problem with it. However, I should point out this was already prepared libraries that we made and not raw DNA. Low starting quantity of DNA is a little harder to deal with, but there are core facilities that will have no problem with this, for a fee of course.
Hope this helps.
Best Regards
Cherry,
Are you asking, because of the requirements of a commercial laboratory/send out or establishing your own testing? I have some thoughts, send me an e-mail through research gate with some specifics regarding the cases and I will get back to you within a couple of days.
Thanks for the answers. The facility does everything from DNA extraction to data analysis. I don't know what extraction method they use but when we extract DNA ourselves, the yield seems higher. Perhaps we should try submitting extracted DNA instead of tissue blocks or slides.
I agree with Nirmal to use Ampliseq from Ion Torrent, 20-40 ng DNA is needed. Poeple working wit Illumina platforms mostly work with capture techniques with probes for the target enrichment, Sureselect from Agilent or other, for this you need 100 ng of starting material . However a Ampliseq panel for 200 genes will need a investment of about 20.000 Euro, Sureselect panels are even more expensive .
Hello Cheng-Ying,
Do you have any details on what the exact problem is. For example, is it a problem with the starting quantity being to low, and if so how low is it? Or is it a problem with the quality of the starting DNA, and if so what is the issue (i.e. highly degraded)? I think everyone can give you some suggestions if we know better know the problem.
Best Regards
Our DNA sources are variable. Samples are from different institutions. There are two main issues: low DNA quantity in some cases (in our hands we get 90-100 ng at concentration of 3-5 ng/ul but we can only give half to the sequencing facility just in case something goes wrong) and possible degradation in others (low coverage, told by the facility).
Input DNA requirements for ampliseq panels depend on the number of primer pools in the design. So the bigger the design, the more primer pools, the more DNA needed. My current panel is designed for 6 genes (exons only) and consists of 2 primer pools. My input DNA is 10ng per pool, so 20ng per sample.
The facility you are using is most likely asking for a bit more on top of what is required on the chance that your quantitation equipment may be calibrated differently to theirs. But I don't think 50ng per sample for your size panel is that much at all.
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