I have a question regarding "Per Base Sequence Content" plot for "fastqc":

In the fastqc documentation, it is written: "In a random library you would expect that there would be little to no difference between the different bases of a sequence run, so the lines in this plot should run parallel with each other."

But I don't understand why different bases in a read should follow the same pattern of allele frequency ("A/T/C/G"). I mean they are different positions in the genome and it is normal that each position has different allele.

I would appreciate it if someone could help me, please.


The positions you see in the x-axis of this plot are positions on your reads, not on the reference genome. Since each read comes from a random position on the genome, the frequency of A/T/C/G on each base position should reflect the base composition of the entire genome. Therefore, the percentage of each base should be approximately constant across the read.

Hope that helps!

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