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# Quality Control of Illumina Data - PowerPoint PPT Presentation

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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## PowerPoint Slideshow about 'Quality Control of Illumina Data' - kat

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### Quality Control of Illumina Data

Mick Watson

Director of ARK-Genomics

The Roslin Institute

• 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

• 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")

• 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")

• Q30 – 1 in 1000 chance base is incorrect

• Q20 – 1 in 100 chance base is incorrect

• 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

• p(probability base is wrong) : 0.01

• Q (-10 * log10(p)) : 30

• Encode as character : ?

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

• 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

• 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

• Per sequence N content

• May identify cycles that are unreliable

• Over-represented sequences

• May identify Illumina adapters and primers