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.
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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