Vignette: ACNE: Allele-specific copy numbers using non-negative matrix factorization

Author: Maria Ortiz (cleanup by Henrik Bengtsson)
Created on: 2009-11-18
Last updated: 2014-12-21

Figure: Allele-specific copy numbers (C_A,C_B) using ACNE (left), AS-dChip (center) and ASCRMA v2 (right) in a normal region where we know there should be three clouds located around (2,0), (1,1) and (0,2). Data is from an Mapping250K_Nsp data set.

This document describes how to estimate allele specific copy numbers (ASCNs) in aroma.affymetrix using ACNE (Ortiz-Estevez, Bengtsson, and Rubio, 2010). ACNE is a summarization method that provides ASCNs based on signals normalized in a similar way to as CRMA v2 (Bengtsson, Wirapati, and Speed, 2009).

Eight (8) Affymetrix GenomeWideSNP_6 arrays deposited in NCBI-GEO under accession numbers GSE14996 (GSM374529 -- 36) will be used to illustrate the necessary steps in aroma.affymetrix in order to apply ACNE summarization method.

Note: This analysis requires the ACNE package in addition to aroma.affymetrix.

Setup

Annotation data

annotationData/
  chipTypes/
    GenomeWideSNP_6/
      GenomeWideSNP_6,Full.cdf
        GenomeWideSNP_6,Full,na26,HB20080821.ugp
        GenomeWideSNP_6,Full,na26,HB20080722.ufl
        GenomeWideSNP_6,Full,HB20080710.acs

Note that *.Full.cdf have to be renamed to *,Full.cdf (w/ a comma).

Raw data

rawData/
  GSE14996,testSet/
    GenomeWideSNP_6/
       GSM374529.CEL, GSM374530.CEL, GSM374531.CEL, GSM374532.CEL,
       GSM374533.CEL, GSM374534.CEL, GSM374535.CEL, GSM374536.CEL

Analysis

Setup

library("aroma.affymetrix")
library("ACNE")
verbose <- Arguments$getVerbose(-10, timestamp=TRUE)

dataSet <- "GSE14996,testSet"
chipType <- "GenomeWideSNP_6"

Annotation data

cdf <- AffymetrixCdfFile$byChipType(chipType, tags="Full")
print(cdf)

which gives:

AffymetrixCdfFile:  
Path: annotationData/chipTypes/GenomeWideSNP_6  
Filename: GenomeWideSNP_6,Full.cdf  
Filesize: 470.44MB  
Chip type: GenomeWideSNP_6,Full  
RAM: 0.00MB  
File format: v4 (binary; XDA)  
Dimension: 2572x2680  
Number of cells: 6892960  
Number of units: 1881415  
Cells per unit: 3.66  
Number of QC units: 4

and

gi <- getGenomeInformation(cdf)
print(gi)

which gives:

UgpGenomeInformation:  
Name: GenomeWideSNP_6  
Tags: Full,na26,HB20080821  
Full name: GenomeWideSNP_6,Full,na26,HB20080821  
Pathname:
annotationData/chipTypes/GenomeWideSNP_6/GenomeWideSNP_6,Full,na26,HB20080821.ugp  
File size: 8.97 MB (9407937 bytes)  
RAM: 0.00 MB  
Chip type: GenomeWideSNP_6,Full

and

print(si)

which gives:

UflSnpInformation:  
Name: GenomeWideSNP_6  
Tags: Full,na26,HB20080722  
Full name: GenomeWideSNP_6,Full,na26,HB20080722  
Pathname:
annotationData/chipTypes/GenomeWideSNP_6/GenomeWideSNP_6,Full,na26,HB20080722.ufl  
File size: 7.18 MB (7526454 bytes)  
RAM: 0.00 MB  
Chip type: GenomeWideSNP_6,Full  
Number of enzymes: 2

Then

acs <- AromaCellSequenceFile$byChipType(getChipType(cdf, fullname=FALSE))
print(acs)

which outputs:

AromaCellSequenceFile:  
Name: GenomeWideSNP_6  
Tags: Full,HB20080710  
Full name: GenomeWideSNP_6,Full,HB20080710  
Pathname:
annotationData/chipTypes/GenomeWideSNP_6/GenomeWideSNP_6,Full,HB20080710.acs  
File size: 170.92 MB (179217531 bytes)  
RAM: 0.00 MB  
Number of data rows: 6892960  
File format: v1  
Dimensions: 6892960x26  
Column classes: raw, raw, raw, raw, raw, raw, raw, raw, raw, raw, raw,
raw, raw, raw, raw, raw, raw, raw, raw, raw, raw, raw, raw, raw, raw,
raw  
Number of bytes per column: 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1  
Footer: \<createdOn\>20080710 22:47:02
PDT\</createdOn\>\<platform\>Affymetrix\</platform\>\<chipType\>GenomeWideSNP_6\</chipType\>\<srcFile\>\<filename\>GenomeWideSNP_6.probe_tab\</filename\>\<filesize\>341479928\</filesize\>\<checksum\>2037c033c09fd8f7c06bd042a77aef15\</checksum\>\</srcFile\>\<srcFile2\>\<filename\>GenomeWideSNP_6.CN_probe_tab\</filename\>\<filesize\>96968290\</filesize\>\<checksum\>3dc2d3178f5eafdbea9c8b6eca88a89c\</checksum\>\</srcFile2\>  
Chip type: GenomeWideSNP_6  
Platform: Affymetrix

Raw data

cs <- AffymetrixCelSet$byName(dataSet, cdf=cdf)
print(cs)

AffymetrixCelSet:  
Name: GSE14996  
Tags: testSet  
Path: rawData/GSE14996,testSet/GenomeWideSNP_6  
Platform: Affymetrix  
Chip type: GenomeWideSNP_6,Full  
Number of arrays: 8  
Names: GSM374529, GSM374530, ..., GSM374536  
Time period: 2007-09-21 18:56:58 -- 2007-12-31 12:29:36  
Total file size: 526.64MB  
RAM: 0.01MB

Pre-processing

Cross-talk calibration

acc <- AllelicCrosstalkCalibration(cs, model="CRMAv2")
print(acc)

AllelicCrosstalkCalibration:  
Data set: GSE14996  
Input tags: testSet  
User tags: *  
Asterisk ('*') tags: ACC,ra,-XY  
Output tags: testSet,ACC,ra,-XY  
Number of files: 8 (526.64MB)  
Platform: Affymetrix  
Chip type: GenomeWideSNP_6,Full  
Algorithm parameters: (rescaleBy: chr "all", targetAvg: num 2200,
subsetToAvg: chr "-XY", mergeShifts: logi TRUE, B: int 1, flavor: chr
"sfit", algorithmParameters:List of 3, ..\$ alpha: num [1:8] 0.1 0.075
0.05 0.03 0.01 0.0025 0.001 0.0001, ..\$ q: num 2, ..\$ Q: num 98)  
Output path: probeData/GSE14996,testSet,ACC,ra,-XY/GenomeWideSNP_6  
Is done: FALSE  
RAM: 0.00MB

csC <- process(acc, verbose=verbose)
print(csC)

AffymetrixCelSet:  
Name: GSE14996  
Tags: testSet,ACC,ra,-XY  
Path: probeData/GSE14996,testSet,ACC,ra,-XY/GenomeWideSNP_6  
Platform: Affymetrix  
Chip type: GenomeWideSNP_6,Full  
Number of arrays: 8  
Names: GSM374529, GSM374530, ..., GSM374536  
Time period: 2007-09-21 18:56:58 -- 2007-12-31 12:29:36  
Total file size: 526.64MB  
RAM: 0.01MB

Nucleotide-position sequence normalization

bpn <- BasePositionNormalization(csC, target="zero")
print(bpn)

BasePositionNormalization:  
Data set: GSE14996  
Input tags: testSet,ACC,ra,-XY  
User tags: *  
Asterisk ('*') tags: BPN,-XY  
Output tags: testSet,ACC,ra,-XY,BPN,-XY  
Number of files: 8 (526.64MB)  
Platform: Affymetrix  
Chip type: GenomeWideSNP_6,Full  
Algorithm parameters: (unitsToFit: chr "-XY", typesToFit: chr "pm",
unitsToUpdate: NULL, typesToUpdate: chr "pm", shift: num 0, target: chr
"zero", model: chr "smooth.spline", df: int 5)  
Output path:
probeData/GSE14996,testSet,ACC,ra,-XY,BPN,-XY/GenomeWideSNP_6  
Is done: FALSE  
RAM: 0.00MB

csN <- process(bpn, verbose=verbose)
print(csN)

AffymetrixCelSet:  
Name: GSE14996  
Tags: testSet,ACC,ra,-XY,BPN,-XY  
Path: probeData/GSE14996,testSet,ACC,ra,-XY,BPN,-XY/GenomeWideSNP_6  
Platform: Affymetrix  
Chip type: GenomeWideSNP_6,Full  
Number of arrays: 8  
Names: GSM374529, GSM374530, ..., GSM374536  
Time period: 2007-09-21 18:56:58 -- 2007-12-31 12:29:36  
Total file size: 526.64MB  
RAM: 0.01MB

Probe summarization using non-negative-matrix factorization (NMF)

plm <- NmfSnpPlm(csN, mergeStrands=TRUE)
print(plm)

NmfSnpPlm:
Data set: GSE14996
Chip type: GenomeWideSNP_6,Full
Input tags: testSet,ACC,ra,-XY,BPN,-XY
Output tags: testSet,ACC,ra,-XY,BPN,-XY,NMF,v4
Parameters: (probeModel: chr "pm"; shift: num 0; mergeStrands: logiTRUE).
Path: plmData/GSE14996,testSet,ACC,ra,-XY,BPN,-XY,NMF,v4/GenomeWideSNP_6
RAM: 0.00MB

if (length(findUnitsTodo(plm)) > 0) {
   # Fit CN probes quickly (~5-10s/array + some overhead)
  units <- fitCnProbes(plm, verbose=verbose)
  str(units)
  # int [1:945826] 935590 935591 935592 935593 935594 935595 ...

  # Fit remaining units, i.e. SNPs (~5-10min/array)
  units <- fit(plm, verbose=verbose)
  str(units)
}

ces <- getChipEffectSet(plm)
print(ces)

SnpChipEffectSet:  
Name: GSE14996  
Tags: testSet,ACC,ra,-XY,BPN,-XY,NMF,v4  
Path:
plmData/GSE14996,testSet,ACC,ra,-XY,BPN,-XY,NMF,v4/GenomeWideSNP_6  
Platform: Affymetrix  
Chip type: GenomeWideSNP_6,Full,monocell  
Number of arrays: 8  
Names: GSM374529, GSM374530, ..., GSM374536  
Time period: 2009-11-19 10:51:15 -- 2009-11-19 10:51:16  
Total file size: 215.59MB  
RAM: 0.01MB  
Parameters: (probeModel: chr "pm", mergeStrands: logi TRUE)

Results

Extracting allele-specific CNs

Example: ASCNs for Chromosome 2

chromosome <- 2
units <- getUnitsOnChromosome(gi, chromosome=chromosome)
str(units)

## int [1:153663] 26048 26049 26050 26052 26053 26054 26055 26056 26057 26058 ...

pos <- getPositions(gi, units=units)
str(pos)

## int [1:153663] 102496 141464 155674 160576 160616 166395 179818 179972 214086 214192 ...

Example: ASCNs for Sample #1

cf <- ces[[1]]
data <- extractTotalAndFreqB(cf, units=units)
CT <- data[,"total"]

# NmfSnpPlm returns relative ASCNs (CA, CB) already standardized to the pool of all arrays.
C <- CT
cn <- RawCopyNumbers(C, pos, chromosome=chromosome)
print(cn)

RawCopyNumbers:  
Name:  
Chromosome: 2  
Position range: [2785,2.42738e+08]  
Number of loci: 153663  
Mean distance between loci: 1579.67  
Loci fields: x [153663xnumeric], y [153663xnumeric]  
RAM: 1.76MB

beta <- data[,"freqB"]
fracB <- RawAlleleBFractions(beta, pos, chromosome=chromosome)
print(fracB)

RawAlleleBFractions:
Name:
Chromosome: 2
Position range: [2785,2.42738e+08]
Number of loci: 153663
Mean distance between loci: 1579.67
Loci fields: x [153663xnumeric], y [153663xnumeric]
RAM: 1.76MB

Plotting TCN and BAF

xScale <- 1e-6
cn <- extractSubset(cn, which(!is.na(beta))) # to erase the CN probes

subplots(2, ncol=1)
plot(cn, xScale=xScale, ylim = c(0,6), cex = .3)
stext(side=3, pos=0, getName(cn))
stext(side=3, pos=1, sprintf("Chr%d", chromosome))
plot(fracB, xScale=xScale, cex = .3, ylim = c(0,1))

Total Copy Number and Fraction of Allele B

References

[1] H. Bengtsson, P. Wirapati, and T. P. Speed. "A single-array preprocessing method for estimating full-resolution raw copy numbers from all Affymetrix genotyping arrays including GenomeWideSNP 5 & 6". Eng. In: Bioinformatics (Oxford, England) 25.17 (Sep. 2009), pp. 2149-56. ISSN: 1367-4811. DOI: 10.1093/bioinformatics/btp371. PMID: 19535535.

[2] M. Ortiz-Estevez, H. Bengtsson, and A. Rubio. "ACNE: a summarization method to estimate allele-specific copy numbers for Affymetrix SNP arrays". Eng. In: Bioinformatics (Oxford, England) 26.15 (Aug. 2010), pp. 1827-33. ISSN: 1367-4811. DOI: 10.1093/bioinformatics/btq300. PMID: 20529889.