FastQC:"Per Base Sequence Quality"

Question

[l.ushanoff]

Hi, I am trying to figure out what the "Per Base Sequence Quality" actually implies. I understand the higher the score on Y axis, the better quality. I don't quite get what the yellow box (25-90 %) and whiskers represent-what does a specific bar with specific whiskers say... because some of it is in the green area and some in orange, or red.

Thank you ! Very new to NGS here.

[WouterDeCoster]

Are you familiar with boxplots?

[Joe]

It’s showing you the range of qualities, per base, across all your your reads in that file.

It’s normal to see the sequence quality drop toward the ends. The colours backgrounds are just a rough guide to good/medium/poor (green/orange/blue).

Answer 1

[Ido Tamir]

These are boxplots where the box normally covers the interquartile range i.e. 25%-75%. The wiskers are convention and in fastqc according to the docs 1,9.th decile. Then you have mean and median indicated. The background coloring is of course arbitrary where they say a value below 20 is bad "red" a value below 28 dangerous "orange" and everything above 30 good "green". What you are interested in is if it fits your application. E.g. for SNP calling you care a lot about quality for RNA-Seq or ChIP-Seq where modern aligners can deal with a lot of mismatches, its not really relevant, unless it gets really bad for multiple cycles in many reads.

https://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/2%20Per%20Base%20Sequence%20Quality.html

For the actual meaning of the quality value https://en.wikipedia.org/wiki/FASTQ_format#Quality 40 = 1 in 10000, 30 = 1 in 1000, 20 = 1 in 100 bases called wrong.