Usage

There are 2 main steps in the analysis workflow:

1. Generating read count coverage information using readCounter from the HMMcopy suite.
2. Copy number analysis and prediction of tumor fraction using ichorCNA R package.

The analysis workflow has also been written into a Snakemake Workflow

Generate Read Count File

To create a WIG file from a ULP-WGS BAM, use HMMcopy's readCounter. This example would create a WIG file with 1Mb bins across all chromosomes and only include reads with a mapping quality greater than 20.

Note: The BAM file must be indexed and the index file must have the extension .bam.bai. The index file should be located in the same directory as the BAM file.

/path/to/HMMcopy/bin/readCounter --window 1000000 --quality 20 \
--chromosome "1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,X,Y" \
/path/to/tumor.bam > /path/to/tumor.wig

Run ichorCNA

The easiest way to manually run ichorCNA is to use runIchorCNA.R provided in the ichorCNA/scripts/ directory. Here is an example of how to launch the R script from the command line:

Rscript /path/to/ichorCNA/scripts/runIchorCNA.R --id tumor_sample \
  --WIG /path/to/tumor.wig --ploidy "c(2,3)" --normal "c(0.5,0.6,0.7,0.8,0.9)" --maxCN 5 \
  --gcWig /path/to/ichorCNA/inst/extdata/gc_hg19_1000kb.wig \
  --mapWig /path/to/ichorCNA/inst/extdata/map_hg19_1000kb.wig \
  --centromere /path/to/ichorCNA/inst/extdata/GRCh37.p13_centromere_UCSC-gapTable.txt \
  --normalPanel /path/to/ichorCNA/inst/extdata/HD_ULP_PoN_1Mb_median_normAutosome_mapScoreFiltered_median.rds \
  --includeHOMD False --chrs "c(1:22, \"X\")" --chrTrain "c(1:22)" \
  --estimateNormal True --estimatePloidy True --estimateScPrevalence True \
  --scStates "c(1,3)" --txnE 0.9999 --txnStrength 10000 --outDir ./

Invoking the --help flag will print out the list of options to use. Here, we will briefly describe some of the key arguments to consider.

Rscript runIchorCNA.R --help
Usage: runIchorCNA.R [options]

Options:
--WIG=WIG
	Path to tumor WIG file. Required.

--NORMWIG=NORMWIG
	Path to normal WIG file. Default: [NULL]

--gcWig=GCWIG
	Path to GC-content WIG file; Required

--mapWig=MAPWIG
	Path to mappability score WIG file. Default: [NULL]

--normalPanel=NORMALPANEL
	Median corrected depth from panel of normals. Default: [NULL]

--exons.bed=EXONS.BED
	Path to bed file containing exon regions. Default: [NULL]

--id=ID
	Patient ID. Default: [test]

--centromere=CENTROMERE
	File containing Centromere locations; if not provided then will use hg19 version from ichorCNA package. Default: [NULL]

--rmCentromereFlankLength=RMCENTROMEREFLANKLENGTH
	Length of region flanking centromere to remove. Default: [1e+05]

--normal=NORMAL
	Initial normal contamination; can be more than one value if additional normal initializations are desired. Default: [0.5]

--scStates=SCSTATES
	Subclonal states to consider. Default: [NULL]

--coverage=COVERAGE
	PICARD sequencing coverage. Default: [NULL]

--lambda=LAMBDA
	Initial Student's t precision; must contain 4 values (e.g. c(1500,1500,1500,1500)); if not provided then will automatically use based on variance of data. Default: [NULL]

--lambdaScaleHyperParam=LAMBDASCALEHYPERPARAM
	Hyperparameter (scale) for Gamma prior on Student's-t precision. Default: [3]

--ploidy=PLOIDY
	Initial tumour ploidy; can be more than one value if additional ploidy initializations are desired. Default: [2]

--maxCN=MAXCN
	Total clonal CN states. Default: [7]

--estimateNormal=ESTIMATENORMAL
	Estimate normal. Default: [TRUE]

--estimateScPrevalence=ESTIMATESCPREVALENCE
	Estimate subclonal prevalence. Default: [TRUE]

--estimatePloidy=ESTIMATEPLOIDY
	Estimate tumour ploidy. Default: [TRUE]

--maxFracCNASubclone=MAXFRACCNASUBCLONE
	Exclude solutions with fraction of subclonal events greater than this value. Default: [0.7]

--maxFracGenomeSubclone=MAXFRACGENOMESUBCLONE
	Exclude solutions with subclonal genome fraction greater than this value. Default: [0.5]

--minSegmentBins=MINSEGMENTBINS
	Minimum number of bins for largest segment threshold required to estimate tumor fraction; if below this threshold, then will be assigned zero tumor fraction.

--altFracThreshold=ALTFRACTHRESHOLD
	Minimum proportion of bins altered required to estimate tumor fraction; if below this threshold, then will be assigned zero tumor fraction. Default: [0.05]

--chrNormalize=CHRNORMALIZE
	Specify chromosomes to normalize GC/mappability biases. Default: [c(1:22)]

--chrTrain=CHRTRAIN
	Specify chromosomes to estimate params. Default: [c(1:22)]

--chrs=CHRS
	Specify chromosomes to analyze. Default: [c(1:22,"X")]

--normalizeMaleX=NORMALIZEMALEX
	If male, then normalize chrX by median. Default: [TRUE]

--fracReadsInChrYForMale=FRACREADSINCHRYFORMALE
	Threshold for fraction of reads in chrY to assign as male. Default: [0.001]

--includeHOMD=INCLUDEHOMD
	If FALSE, then exclude HOMD state. Useful when using large bins (e.g. 1Mb). Default: [FALSE]

--txnE=TXNE
	Self-transition probability. Increase to decrease number of segments. Default: [0.9999999]

--txnStrength=TXNSTRENGTH
	Transition pseudo-counts. Exponent should be the same as the number of decimal places of --txnE. Default: [1e+07]

--plotFileType=PLOTFILETYPE
	File format for output plots. Default: [pdf]

--plotYLim=PLOTYLIM
	ylim to use for chromosome plots. Default: [c(-2,2)]

--outDir=OUTDIR
	Output Directory. Default: [./]

--libdir=LIBDIR
	Script library path. Usually exclude this argument unless custom modifications have been made to the ichorCNA R package code and the user would like to source those R files. Default: [NULL]

Tuning ichorCNA Parameters