Genome-widecopy-numbercalling(CNAsnotCNVs!)

Dr GeoffMacintyre

Structuralvariation(SVs)

Deletion

Duplication

Inversion

Translocation

ABC

C

ABABC

BAC

Copy-numbervariations

Balancedrearrangements

Causes• Replicationerrors• Retrotransposition• Repairerrors• Recombinationerrors

Whyiscopy-numberimportant?

Ciriello et al (2015). Nature Genetics

Thedata

Genome-wideSNPallelefrequencies

SomemeasureoftheamountofDNAforagivenlocus(e.g.sequencingdepth)

ArraybaseddetectionofSNPs(hybridisation)

SNPcallingusingaffymetrix arrays

NucleicAcidsRes.2006;34(14):e1002006doi: 10.1093/nar/gkl475

Furtherreadingongenotyping

• Birdseed:http://www.nature.com/ng/journal/v40/n10/full/ng.237.html• CRLMM:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329223/• HaplotypeCaller andUnifiedGenotyper:http://www.nature.com/ng/journal/v43/n5/full/ng.806.html• Varscan:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2734323/• GWASprimer:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181332/

Basicworkflow

Quantifysignal• depth/intensity• B-allele

Segmentgenome• HiddenMarkovmodel• Smoothingmethods

Callcopy-numberchanges• Threshold• Cluster• Probabilisticmodels

Thedata:logR

Sequencing:log2(depth)

Array:R(θ)subject= normalised intensityof

probesfromsample

R(θ)expected= normalised intensityofprobesfromcontrol

logR =log2(R(θ)observed/R(θ)expected)

Quantifysignal

Segmentgenome

Callcopy-number

logR ofHCC1143cell-lineusingaffy SNP6

logR/depthnormalisation

• Different proportions of GC in each region can produce a bias in the read depth (wave artifact)• We can fit a loess model and remove the effect.

35 40 45 50 55 60

0.5

1.0

1.5

2.0

LS041 Sample (CNAnorm package)

GC content

Cop

y N

umbe

r rat

io

Quantifysignal

Segmentgenome

Callcopy-number

Thedata:B-allelefrequency

θA= intensityofprobeforalleleA

BAF=θA/(θA +θB)

AA

AB

BB

AA

BB

AAB

ABB

Quantifysignal

Segmentgenome

Callcopy-number

BAFbanding

• 1 band:– Backgroundnoise(0copies).

• 2 bands: – {A,B},{AA,BB},or{AAA,BBB},…Copynumbers(0,j).

• 3 bands:– {AA,AB,BB}or{AAAA,AABB,BBBB},...Copynumbers(i,j=i)

• 4 bands: – {AAA,ABB,AAB,BBB}or{AAAA,ABBB,AAAB,BBBB}or{AAAAA,ABBBB,AAAAB,BBBBB},…Copynumbers(i,j)/i <j

Quantifysignal

Segmentgenome

Callcopy-number

BAFofHCC1143cell-lineusingaffy SNP6

Segmentation:CircularbinarysegmentationOlshen etal.,2004.• Itcanbeusedwitharrayandsequencingdata• Findschangepointsusingat-testunderapermutationmodel.• BioconductorpackageDNAcopy.

https://academic.oup.com/biostatistics/article/5/4/557/275197/Circular-binary-segmentation-for-the-analysis-of?searchresult=1

Quantifysignal

Segmentgenome

Callcopy-number

Segmentation:Hiddenmarkov modelsQuantifysignal

Segmentgenome

Callcopy-number

Copy-numbercalling:thresholdbased

Individual thresholds based on the variability of each sample:

Quantifysignal

Segmentgenome

Callcopy-number

Copy-numbercalling:clusterbased

van de Wiel et al., 2007 (CGHCall Bioconductor package).

• The segmented means come from a mixture of six normal

populations.

• The model is fit by EM algorithm.

• Classification reduced to 3 or 4 states. (Usually loss, gain, normal)

http://bioconductor.org/packages/release/bioc/html/CGHcall.html

Quantifysignal

Segmentgenome

Callcopy-number

Relativecopy-numberprofile(ovariancancer)

Method:QDNAseq

Scheinin I et al., 2014 (QDNAseqBioconductor package).

• Divides genome into bins of equal size.

• Normalisation based on blacklisted

regions, GC content,....

• Segmentation with DNAcopy.

• Optional calling with CGHcall.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248318/

Exercise1

Problems:purityandheterogeneity

Whataretheeffectsonrelativecopy-number?

Absolutecopy-numberprofile(ovariancancer)

Method:Allele-SpecificCopynumberAnalysisofTumours (ASCAT)

constanttumour fraction

ploidy

B-allelecopy-number

A-allelecopy-number

Furtherreadingoncopy-number

• MethodsforCNdetection(arraydata):https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697494/

• ToolsforCNdetection(sequencedata):http://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-14-S11-S1• PennCNV,apackageforCNVcalling:http://penncnv.openbioinformatics.org/en/latest/• LargescaleanalysisofCNAsincancer:http://www.nature.com/ng/journal/v45/n10/full/ng.2760.html

Exercise2