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Old 02-18-2014, 09:16 AM   #1
JTCoastal
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Default FFPE DNA for PGM

Hi Everyone,

I am new to sequencing and would welcome any and all comments regarding FFPE DNA yield on the PGM with the AmpliSeq Cancer Hot Spot Panel.

First, let me tell what we are doing:

We currently use the Qiagen DNA Tissue Kit on the Qiagen EZ1 instrument with two 10micron sections for our extraction. This has worked great with our current method (pyrosequencing with the PyroMark24) but we are not getting consistent, if any, results on the PGM.

We are quantitating our DNA with the Qubit fluorometer before making our libraries of 10ng of DNA. We know this will include fragmented and degraded DNA because of their source (FFPE).

We are then making libraries (including 8 barcoded patient samples, Sample Id, and the Cancer Hot Spot Panel), amplifying, doing template prep on the One Touch2, then enriching on the One Touch ES, then loading a 318 chip on the PGM.

Our results vary with some samples getting 500X reads and some with little or no reads. We are told that it all goes back to our starting DNA.

So finally, my question.... Are there ANY labs out there using only FFPE samples and getting consistent results on the PGM with the Cancer Hot Spot Panel? And if so, will you please share what you are doing to make it work? (extra amplification cycles, etc...)

Thank you for taking the time to read this lengthy post.
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Old 02-18-2014, 02:28 PM   #2
mikeg
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Hi JT,

I am pretty much doing the same thing as you, but I am getting very nice data. A couple of things to integrate:
1. RNase P kit to quantify your amplifiable DNA before PCR. This will help!!!
2. Library Quantitation kit (qPCR of libraries)
2. Ion Quality control Kit- To measure the templating of your spheres. I try to stick to about 25-30% templated ISP's. My input after normalizing to 100pM is 4.75ul/sample, pool, then add 4.75ul to my emPCR reaction.

Last 8 sample run, I got about 5.5x106 reads. Probably with the samples with no reads, you are not putting enough amplifiable material. I would also take out the sample ID. Not unless your doing pairing with normal tissue, it really does not help. Just tells you the sex of the sample.

Quantification is extremely important! Get those qPCR kits!
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Old 02-19-2014, 11:37 AM   #3
JTCoastal
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Hi Mike,

Thank you so much for your reply!

We do not have a qPCR machine. That is why we were using the Qubit for our initial DNA stock concentration and for the library concentration. We have been doing all of the QC steps along the way (using the library Quant Kit with Qubit and the ISP QC Kit). We usually get around 20% templated ISPs (though I feel like washing 3 times and leaving behind the "imaginary pellet", leaves room for error).

In most cases our initial DNA conc. is so low we put the maximum amount 11uL in from the beginning and hope it will amplify(though, this could increase to 12uL if we stop using Sample Id, which we were only using as a QC step, so we will try).

We are thinking the problem is how we were are doing the DNA extraction and are trying some new methods/kits.

Do you do 20 or 22 cycles for your initial amplification?

Thanks again for your reply.
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Old 02-19-2014, 12:00 PM   #4
mikeg
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Hi JT,

I still would not trust the qubit numbers. The important thing here is "amplifiability", and the qubit does not tell you that. I always measure my FFPE DNA with the Qubit first, before I do RNaseP qPCR. The concentrations always seem to never match, so this is why I stress doing qPCR. As far as the QC kit, don't worry so much about losing some of your ISP's. Actually you want to wash well, because you might get false readings if you don't! You are measuring a percentage and not so much the ISP amount. If you take half of that prep and read it, you should get the same percentage.

How much volume are you eluting your samples in? I usually elute in 100ul, and the input for PCR is rarely above 7ul. For initial PCR, I am following the protocol and using 20 cycles for the 207 FFPE Hotspot amplicons.

The kit we use is specific for FFPE. I see you are using just a DNA kit. Our kit is Qiagen, FFPE, and it is a manual extraction.

I really think your problem is quantification. If you don't get the first part right, the subsequent steps will suffer.
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Old 02-19-2014, 12:17 PM   #5
JTCoastal
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Mike,

We elute them to 100uL also but we are using the EZ1 Robot automated extraction and we just recently heard that the manual method will give better yields. We ordered the FFPE manual one today so hopefully this will increase our DNA yield. Which qPCR instrument do you use? Looks like we may have to buy another instrument.

We only bumped it up to 22 cycles after our Life Tech person suggested it.

Thanks again!
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Old 02-19-2014, 12:49 PM   #6
mikeg
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Right now, I am having to use the Roche Light cycler Z-480. If you have to buy an instrument, I would say go with one of Life Tech's. Maybe the 7500?

Good luck!
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Old 05-27-2014, 09:41 PM   #7
Tejaswini
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I am using RNAseP kit which is quite helpful. I use Quagen FFPE kit. According to RNAse P Kit, my amplifiable DNA is generally 10 times less than the Qubit quantification. In other words, my DNA input for library preperation is around 100 ng total as per Qubit values. This is mainly because of the compromised DNA quality of the FFPE tissue.
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