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Quality Control of Illumina Data. Mick Watson Director of ARK-Genomics The Roslin Institute. Quality scores. Quality scores. The sequencer outputs base calls at each position of a read It also outputs a quality value at each position

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Quality control of illumina data

Quality Control of Illumina Data

Mick Watson

Director of ARK-Genomics

The Roslin Institute



Quality scores1
Quality scores

  • The sequencer outputs base calls at each position of a read

  • It also outputs a quality value at each position

    • This relates to the probability that that base call is incorrect

  • The most common Quality value is the Sanger Q score, or Phred score

    • Qsanger -10 * log10(p)

    • Where p is the probability that the call is incorrect

    • If p = 0.05, there is a 5% chance, or 1 in 20 chance, it is incorrect

    • If p = 0.01, there is a 1% chance, or 1 in 100 chance, it is incorrect

    • If p = 0.001, there is a 0.1% chance, or 1 in 1000 chance, it is incorrect

  • Using the equation:

    • p=0.05, Qsanger= 13

    • p=0.01, Qsanger= 20

    • p=0.001, Qsanger= 30


For the geeks
For the geeks….

  • In R, you can investigate this:

    sangerq<- function(x) {return(-10 * log10(x))}

    sangerq(0.05)

    sangerq(0.01)

    sangerq(0.001)

    plot(seq(0,1,by=0.00001),sangerq(seq(0,1,by=0.00001)), type="l")



For the geeks1
For the geeks….

  • And the other way round….

    qtop<- function(x) {return(10^(x/-10))}

    qtop(30)

    qtop(20)

    qtop(13)

    plot(seq(40,1,by=-1), qtop(seq(40,1,by=-1)), type="l")


The important stuff
The important stuff

  • Q30 – 1 in 1000 chance base is incorrect

  • Q20 – 1 in 100 chance base is incorrect



Quality encoding1
Quality Encoding

  • Bioinformaticians do not like to make your life easy!

  • Q scores of 20, 30 etc take two digits

  • Bioinformaticians would prefer they only took 1

  • In computers, letters have a corresponding ASCII code:

  • Therefore, to save space, we convert the Q score (two digits) to a single letter using this scheme


The process in full
The process in full

  • p(probability base is wrong) : 0.01

  • Q (-10 * log10(p)) : 30

  • Add 33 : 63

  • Encode as character : ?


For the geeks2
For the geeks….

code2Q <- function(x) { return(utf8ToInt(x)-33) }

code2Q(".")

code2Q("5")

code2Q("?")

code2P <- function(x) { return(10^((utf8ToInt(x)-33)/-10)) }

code2P(".")

code2P("5")

code2P("?")


Qc of illumina data
QC of Illumina data


Fastqc
FastQC

  • FastQC is a free piece of software

  • Written by Babraham Bioinformatics group

  • http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

  • Available on Linux, Windows etc

  • Command-line or GUI


Read the documentation

Follow the course notes


Per sequence quality
Per sequence quality

  • One of the most important plots from FastQC

  • Plots a box at each position

  • The box shows the distribution of quality values at that position across all reads





Other useful plots
Other useful plots

  • Per sequence N content

    • May identify cycles that are unreliable

  • Over-represented sequences

    • May identify Illumina adapters and primers


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