I'm currently working to troubleshoot my ChIPseq workflow, and one step where I think I may be having some issues is amplification of my ChIP DNA to create my sequencing library:
We use the Illumina TruSeq ChIP Library Prep Kit, and PippinPrep for gel-purification of the adapter-ligated fragments before PCR.
My concern is the amount of product I end up with after amplification; I would like to minimize amplification bias and avoid over-amplifying my samples, however we have not been able to generate a sufficient amount of DNA (from 10ng of starting material) after amplification, even when running 18 cycles of PCR. I just began making my own library instead of leaving that step to our seq. core. I tried starting the library prep protocol with 100ng of DNA instead of 10ng, and was able to obtain 50uL of ~10nM DNA after 14 cycles of PCR. The Bioanalyzer peak is narrow and the correct size, with no detectable adapter dimers.
While I now have a library for sequencing, I have a gut feeling that I shouldn't need to start with 10 times the amount of DNA spec'd in the protocol to end up with enough DNA in my library after 14 PCR cycles, especially when I read that many people are getting good libraries with only 10-12 cycles (presumably starting with 10ng of ChIP DNA). Also, I won't have the luxury of 100ng starting material for many of my ChIPs.
Could my ligation efficiency be low, causing only a low proportion of my starting material to be amplified? Do these numbers sound reasonable, or is my library prep efficiency low?
We use the Illumina TruSeq ChIP Library Prep Kit, and PippinPrep for gel-purification of the adapter-ligated fragments before PCR.
My concern is the amount of product I end up with after amplification; I would like to minimize amplification bias and avoid over-amplifying my samples, however we have not been able to generate a sufficient amount of DNA (from 10ng of starting material) after amplification, even when running 18 cycles of PCR. I just began making my own library instead of leaving that step to our seq. core. I tried starting the library prep protocol with 100ng of DNA instead of 10ng, and was able to obtain 50uL of ~10nM DNA after 14 cycles of PCR. The Bioanalyzer peak is narrow and the correct size, with no detectable adapter dimers.
While I now have a library for sequencing, I have a gut feeling that I shouldn't need to start with 10 times the amount of DNA spec'd in the protocol to end up with enough DNA in my library after 14 PCR cycles, especially when I read that many people are getting good libraries with only 10-12 cycles (presumably starting with 10ng of ChIP DNA). Also, I won't have the luxury of 100ng starting material for many of my ChIPs.
Could my ligation efficiency be low, causing only a low proportion of my starting material to be amplified? Do these numbers sound reasonable, or is my library prep efficiency low?
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