Hi Everyone,
I have sequenced ChIP samples (Input and IP) - 1X75 bp, 10M depth. DNA shearing was done using sonication (Bioruptor). Once I map reads to the reference genome (budding yeast) and look at the coverage of reads for Input sample, they appear very uneven with peaks and troughs.
I am attaching an image with comparison of Input coverage from two different experiments. The top one looks fine and has even coverage across the chromosome as expected for an Input sample. The bottom one has apparent uneven coverage and messes up the analysis when used to normalize IP sample.
Anyone has any suggestions on what could cause the uneven coverage?
Thanks!
What is the scale on this (kb, Mb, bp?)
Its 300 kb.
...and is this normalized? If not one cannot say anything about it. Uneven coverage in input is normal. Not every part of the genome amplifies and gets sequenced equally well, that is exactly why input is done rather than assuming even coverage and doing stats on that assumption.