I’m writing to request your advice about the ATACseq in primary human cells. I am Si Young Lee who is currently working for VRC/NIH. We recently made a pooled library by pooling each 21 library from 7 different samples. I clearly saw the periodicity of the fragment lengths in each library but 3 libraries have different pattern like high peak in high size, #13-15; they are from same sample. Eventually we wan to run a Hiseq but we may run a Miseq first to check a balance of pooled library. I searched several references and all references are really helpful. However, I am not sure if I can use this pooled library to run Miseq because of the different pattern of library in sample #13-15. Do you think this pooled library is good enough to run a Miseq?
Any comments and suggestion will be appreciated.
Thanks
SY
Hi Si, Did you sequence these libraries? These should be perfectly fine to sequence on the MiSeq. I think the profiles that are more right shifted could be due to lower PCR cycles. Previously we optimized the PCR cycle number and right shifted profiles were shifted to the left (smaller fragments) if you increase PCR cycles by 1-2. Also, if you look at the molar concentrations in the bioanalyzer, you will see that the fragment molarity in the very high MW fraction is actually smaller but due to the size scale in the DNA HS, the higher MW fragments look larger.
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