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Old 11-14-2018, 04:35 AM   #4
kmcarr
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Location: USA, Midwest

Join Date: May 2008
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Quote:
Originally Posted by frymor View Post
Hi,

we have a 36-samples ChIP-Seq experiment with a very strange GC-content behavior (s. images)
The samples are from yeast. This strange behavior comes in the IP samples, some of them shows (the first image is from such a file) in the fastqc report an over-representative accumulation of certain sequences, such as
Code:
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
TGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG	
TTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
AGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
But some of them don't ( the second image is from a file with no(!) over-rep. sequences at all.
Other than that the report looks quite good with no abnormalities (except the GC content).

I was thinking that this curve might be due to the over-rep. polyG sequences, but what about the samples where there is no apparent accumulation of such sequences. Is there a possible biological reason for this thing?

The problem occurs only in the IP samples. Is it possible that lack of biological material for the sequencer for this samples causes the machine to put a G (or identify a G ) intread of the real nt?
we are using a nextseq500 with a high kit from Illumina


thanks
Assa
frymor,

You correctly identified the cause in your last statement above. The Illumina NextSeq500 (and NovaSeq) use 2 Color Chemistry for tagging and identifying bases. Using this system G's are defined by the absence of a fluorescent signal. This means that anything which can cause a cluster to stop producing a signal with result in a polyG output. A much better and more thorough explanation can be found in this QC Fail post.
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