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FASTQC Per sequence GC content failed

Sorry for asking a question related to a topic that has been discussed multiple times in previous threads, but I have not been able to find a clear solution to my case.

As the title indicates, FastQC has indicated multiple fails in the Per Sequence GC Content, in several of my human RNA seq samples.

Below, I show two of the plots I have obtained, where a sharper than expected curve can be observed and, next to it, a moderately small shoulder appears. From what I have read, the appearance of very sharp peaks could be indicative of contamination, but I do not know if my case would be sufficient.

GC Content Plot 1

GC Content Plot 2

Regarding the rest of the results, I also observed a large number of duplicated sequences and overrepresented sequences, which are common in RNAseq data analyzed with Illumina.

Thanks, Gerard.

rnaseq content human gc
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updated by GenoMax GenoMax • written by Gerard • Gerard

1 answer

There is no rule that says that every FastQC parameters needs to pass before one can move on to analyzing the data. Parameters in FastQC are tuned for normal genomic sequencing and thus many tests fail if one is using other types of data. Only contamination here may be of rRNA depending on how you did your experiments.

You should go ahead with the analysis. IF there is an issue with the downstream results then you can spend time back tracking and figuring where the problem may lie.

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written by GenoMax GenoMax

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