Contents

1 Introduction

The conumee package provides tools for performing copy-number variation (CNV) analysis using Illumina 450k or EPIC DNA methylation arrays. Although the primary purpose of these arrays is the detection of genome-wide DNA methylation levels [1], it can additionally be used to extract useful information about copy-number alterations, e.g. in clinical cancer samples. The approach was initially described in Sturm et al., 2012 [2]. Other tools following a similar strategy include ChAMP [3] and CopyNumber450k (now deprecated).

CNV analysis is performed using a two-step approach. First, the combined intensity values of both ‘methylated’ and ‘unmethylated’ channel of each CpG are normalized using a set of normal controls (i.e. with a flat genome not showing any copy-number alterations). This step is required for correcting for probe and sample bias (e.g. caused by GC-content, type I/II differences or technical variability). Secondly, neighboring probes are combined in a hybrid approach, resulting in bins of a minimum size and a minimum number of probes. This step is required to reduce remaining technical variability and enable meaningful segmentation results.

The conumee package has been written for seamless integration with frequently-used Bioconductor packages. It is recommended that methylation array data is loaded using the minfi package [4]. Segmentation is performed using the circular binary segmentation (CBS) algorithm implemented in the DNAcopy package [5]. Processing time is typically less than a minute per sample.

Finally, the conumee package also provides a set of plotting methods to create publication-grade CNV plots of the whole genome, selected chromosomes or previously defined regions of interest. Writing of text-based output files for visualization in other tools (e.g. the IGV browser) or downstream processing (e.g. using GISTIC) is also supported.

2 Load data

The recommended input format are Mset objects generated from raw IDAT files using the minfi package. Depending on the analysis workflow, these objects might be already available. A good example 450k data-set for testing the conumee package can be downloaded from TCGA (971.6 MB):

https://tcga-data.nci.nih.gov/tcgafiles/ftp_auth/distro_ftpusers/anonymous/tumor/brca/cgcc/jhu-usc.edu/humanmethylation450/methylation/jhu-usc.edu_BRCA.HumanMethylation450.Level_1.8.8.0.tar.gz

UPDATE October 2016: The TCGA Data Portal is no longer operational. TCGA DNA methylation data can now be downloaded from the NCI GDC Legacy Archive: https://gdc-portal.nci.nih.gov/legacy-archive

This data-set comprises of 42 primary breast cancer samples, 2 breast cancer cell lines and 16 control samples. Make sure to unpack the archive. For the purpose of this vignette, only two illustrative examples are downloaded using the read.450k.url method.

library("minfi")
library("conumee")
#RGsetTCGA <- read.450k.exp(base = "~/conumee_analysis/jhu-usc.edu_BRCA.HumanMethylation450.Level_1.8.8.0")  # adjust path
RGsetTCGA <- read.450k.url()  # use default parameters for vignette examples
MsetTCGA <- preprocessIllumina(RGsetTCGA)
MsetTCGA
## class: MethylSet 
## dim: 485512 2 
## metadata(0):
## assays(2): Meth Unmeth
## rownames(485512): cg00050873 cg00212031 ... ch.22.47579720R
##   ch.22.48274842R
## rowData names(0):
## colnames(2): 6042324037_R05C02 6042324037_R06C01
## colData names(0):
## Annotation
##   array: IlluminaHumanMethylation450k
##   annotation: ilmn12.hg19
## Preprocessing
##   Method: Illumina, bg.correct = TRUE, normalize = controls, reference = 1
##   minfi version: 1.22.0
##   Manifest version: 0.4.0

Alternatively, you can use the minfiData example data-set, comprising of 3 cancer samples and 3 normal controls.

library("minfiData")
data(MsetEx)
MsetEx
## class: MethylSet 
## dim: 485512 6 
## metadata(0):
## assays(2): Meth Unmeth
## rownames(485512): cg00050873 cg00212031 ... ch.22.47579720R
##   ch.22.48274842R
## rowData names(0):
## colnames(6): 5723646052_R02C02 5723646052_R04C01 ...
##   5723646053_R05C02 5723646053_R06C02
## colData names(13): Sample_Name Sample_Well ... Basename filenames
## Annotation
##   array: IlluminaHumanMethylation450k
##   annotation: ilmn12.hg19
## Preprocessing
##   Method: Raw (no normalization or bg correction)
##   minfi version: 1.21.2
##   Manifest version: 0.4.0

The CopyNumber450k package provides a large data-set of 52 control samples which can be used for normalization.

UPDATE April 2017: The CopyNumber450k and CopyNumber450kData packages are now depreacted. We will use the 3 normal controls from the minfiData package for now. Using a larger set of control samples would give better results.

# library("CopyNumber450kData")
# data(RGcontrolSetEx)
# MsetControls <- preprocessIllumina(RGcontrolSetEx)

If raw IDAT files are unavailable, data can also be supplied by importing text-based input files, e.g. as generated by GenomeStudio or downloaded from the GEO repository. More details are given later.

3 Create annotation object

To begin with the CNV analysis, an annotation object, generated by the CNV.create_anno function, is required. This object holds information which only needs to be generated once, irrespective of the number of samples that are processed.

Arguments bin_minprobes and bin_minsize define the minimum number of probes per bin and the minimum bin size (default values that were optimized for 450k data are 15 and 50000, respectively). Argument exclude_regions defines regions to be excluded (e.g. polymorphic regions, an example is given in data(exclude_regions)). Please see ?CNV.create_anno for more details.

Argument detail_regions defines regions to be examined in detail (e.g. dedicated detail plots or text output, see below). For example, detail regions can contain known oncogenes or tumor suppressor genes. These regions should either be supplied as a path to a BED file or as a GRanges object (an example is given in data(detail_regions)). The start and stop coordinates indicate the regions in which probes are analyzed in detail. The plotting range of detail plots are defined in a second set of start and stop coordinates in the GRanges object values (or the 7th and 8th column in the BED file).

data(exclude_regions)
data(detail_regions)
head(detail_regions, n = 2)
## GRanges object with 2 ranges and 5 metadata columns:
##       seqnames               ranges strand |        name
##          <Rle>            <IRanges>  <Rle> | <character>
##   [1]    chr11 [69453874, 69469242]      + |       CCND1
##   [2]    chr19 [30300902, 30315215]      + |       CCNE1
##                      thick     score probes_gene probes_promoter
##                  <IRanges> <integer>   <integer>       <integer>
##   [1] [68961558, 69961557]        70          38              32
##   [2] [29808059, 30808058]        16          10               6
##   -------
##   seqinfo: 11 sequences from an unspecified genome; no seqlengths

anno <- CNV.create_anno(array_type = "450k", exclude_regions = exclude_regions, detail_regions = detail_regions)
## using genome annotations from UCSC
## getting 450k annotations
##  - 470870 probes used
## importing regions to exclude from analysis
## importing regions for detailed analysis
## creating bins
##  - 53891 bins created
## merging bins
##  - 15820 bins remaining

anno
## CNV annotation object
##    created  : Mon Apr 24 20:17:53 2017
##   @genome   : 22 chromosomes
##   @gap      : 313 regions
##   @probes   : 470870 probes
##   @exclude  : 3 regions (overlapping 570 probes)
##   @detail   : 20 regions (overlapping 768 probes)
##   @bins     : 15820 bins (min/avg/max size: 50/168.5/5000kb, probes: 15/29.7/478)

4 Combine intensity values

Intensity values of the ‘methylated’ and ‘unmethylated’ channel are combined using the CNV.load function. Input can be either an Mset object (recommended, see above), or a data.frame or matrix object containing intensities generated by GenomeStudio or downloaded from GEO (imported using e.g. read.table, should work without reformatting for most tables, please refer to ?CNV.load for more details).

data(tcgaBRCA.sentrix2name)  # TCGA sample IDs are supplied with the conumee package
sampleNames(MsetTCGA) <- tcgaBRCA.sentrix2name[sampleNames(MsetTCGA)]
tcga.data <- CNV.load(MsetTCGA)
tcga.controls <- grep("-11A-", names(tcga.data))
names(tcga.data)
## [1] "TCGA-AR-A1AU-01A-11D-A12R-05" "TCGA-AR-A1AY-01A-21D-A12R-05"
tcga.data
## CNV data object
##    created   : 
##   @intensity : available (2 samples, 485512 probes)

minfi.data <- CNV.load(MsetEx)
minfi.controls <- pData(MsetEx)$status == "normal"

# controls.data <- CNV.load(MsetControls)

5 Perform CNV analysis

The main CNV analysis is separated into four parts:

x <- CNV.fit(minfi.data["GroupB_1"], minfi.data[minfi.controls], anno)
# y <- CNV.fit(tcga.data["TCGA-AR-A1AU-01A-11D-A12R-05"], controls.data, anno)  # CopyNumber450kData package is deprecated
# z <- CNV.fit(tcga.data["TCGA-AR-A1AY-01A-21D-A12R-05"], controls.data, anno)
y <- CNV.fit(tcga.data["TCGA-AR-A1AU-01A-11D-A12R-05"], minfi.data[minfi.controls], anno)  # minfiData control samples
z <- CNV.fit(tcga.data["TCGA-AR-A1AY-01A-21D-A12R-05"], minfi.data[minfi.controls], anno)

x <- CNV.bin(x)
x <- CNV.detail(x)
x <- CNV.segment(x)

y <- CNV.segment(CNV.detail(CNV.bin(y)))
z <- CNV.segment(CNV.detail(CNV.bin(z)))

x
## CNV analysis object
##    created   : Mon Apr 24 20:18:30 2017
##   @name      : GroupB_1
##   @anno      : 22 chromosomes, 470870 probes, 15820 bins
##   @fit       : available (noise: 0.237)
##   @bin       : available (shift: -0.018)
##   @detail    : available (20 regions)
##   @seg       : available (48 segments)

6 Output plots and text files

The conumee package supports two types of plots:

Text output is generated using the CNV.write method. Parameter what specifies if “probes”, “bins”, “detail” or “segments” should be returned. If parameter file is specified, the output is written into a file, otherwise a data.frame is returned. See ?CNV.write for more details.

#pdf("~/conumee_analysis/CNVplots.pdf", height = 9, width = 18)
CNV.genomeplot(x)

CNV.genomeplot(y)
CNV.genomeplot(y, chr = "chr6")

CNV.genomeplot(z)
CNV.genomeplot(z, chr = "chr10")
CNV.detailplot(z, name = "PTEN")
CNV.detailplot_wrap(z)
#dev.off()

head(CNV.write(y, what = "segments"), n = 5)
##                             ID chrom loc.start   loc.end num.mark    bstat
## 1 TCGA-AR-A1AU-01A-11D-A12R-05  chr1    635684  88100000      726 29.32946
## 2 TCGA-AR-A1AU-01A-11D-A12R-05  chr1  88675000 118475000      163 32.18477
## 3 TCGA-AR-A1AU-01A-11D-A12R-05  chr1 119000000 249195311      704       NA
## 4 TCGA-AR-A1AU-01A-11D-A12R-05 chr10    105000 135462374      840       NA
## 5 TCGA-AR-A1AU-01A-11D-A12R-05 chr11    130000  50516927      326 22.74454
##            pval seg.mean seg.median
## 1 2.906752e-186    0.009      0.015
## 2 1.991610e-224   -0.264     -0.254
## 3            NA    0.097      0.098
## 4            NA   -0.023     -0.021
## 5 5.343554e-112    0.141      0.136

#CNV.write(y, what = "segments", file = "~/conumee_analysis/TCGA-AR-A1AU.CNVsegments.seg")  # adjust path
#CNV.write(y, what = "bins", file = "~/conumee_analysis/TCGA-AR-A1AU.CNVbins.igv")
#CNV.write(y, what = "detail", file = "~/conumee_analysis/TCGA-AR-A1AU.CNVdetail.txt")
#CNV.write(y, what = "probes", file = "~/conumee_analysis/TCGA-AR-A1AU.CNVprobes.igv")