Here is the background of my questions: During the CUT & TAG library preparation, we found the library concentration of some samples is low, and can not match the required concentration of the sequence factory. so we give some extra PCR cycles to low concentration(even different cycles between the replicates of the sample), the PCR cycles are ranged from 15 to 18 depending on their DNA concentration. After this, the sequence factory performed Cut & TAG seq. The duplicate rates got from MultiQC report range from 47% to 76%.
Here are the questions:
- Give different PCR cycles(extra PCR cycles for low concentration samples) instead of giving the same cycle for all samples to match the sequence factory requires, does this make sense?
- Will those extra PCR circles cover the biological difference when performing Differential Binding Analysis between samples?
- Will different cycles between replicates pull down common peak numbers that appear in replicates?
- According to the protocol from Steven Henikoff's lab, they said "extra PCR cycles reduce the complexity of the library and may result in an unacceptable level of PCR duplicates", are our data's duplicate rates(47% to 76%) acceptable?
Thanks for any advice!