# FASTQC overrepresented sequences after trimming

I have a set of RNA-seq samples from different experiments (Single and Paired End, depending on the experiment). I ran FASTQC in all the samples and found overrepresented adapter sequences:

I removed the adaptors (TruSeq adaptors) using Cutadapt (in addition, I removed low quality and N bases from the 3' end of the reads). After that, I ran again FASTQC and the output is the following (representative example) :

Does anyone know what is happening? Now I have an overrepresented sequence for which no sequence is provided. What does this mean?

• Looks like you trimmed your sequences to nothing. Do you have empty fastq entries in the file? – Bioathlete Aug 4 '17 at 16:39
• Yes, thanks for your comment, this was the problem. – plat Aug 7 '17 at 11:06

As @AaronBerlin mentioned, you didn't remove reads that were completely trimmed. Next time use the --minimum-length option and set it to something reasonable, like 20. Alternatively, use "Trim Galore!", which is a wrapper around cutadapt that has more reasonable defaults.