---
title: "PureCN best practices"
author:
      name: Markus Riester
      affiliation: Novartis Institutes for BioMedical Research 
output: 
  BiocStyle::html_document:
    toc_float: true
  BiocStyle::pdf_document: default  
package: PureCN
abstract: |
    This tutorial provides a quick overview of the command line tools shipping
    with _PureCN_. These tools implement highly recommended best practices. For
    the R package and more detailed information, see the main vignette. 
vignette: |
  %\VignetteIndexEntry{Best practices, quick start and command line usage}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
---


```{r load-purecn, echo=FALSE, message=FALSE}
library(PureCN)
library(BiocStyle)
```

# Prerequisites

## Update from previous stable versions

`r Biocpkg("PureCN")` is backward compatible with input generated by
versions 1.16 and 1.18. For versions 1.8 to 1.14, please re-run `NormalDB.R`
(see also below): 

```
$ Rscript $PURECN/NormalDB.R --outdir $OUT_REF \
    --coveragefiles example_normal.list \
    --genome hg19 --normal_panel $NORMAL_PANEL --assay agilent_v6
```

For upgrades from version 1.6, we highly recommend starting from scratch
following this tutorial.


## Installation

For the command line scripts described in this tutorial, we will need to 
install `r Biocpkg("PureCN")` with suggested dependencies:

```
if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("PureCN", dependencies = TRUE)
```

Alternatively, manually install the packages required by the command line
scripts:

```
BiocManager::install(c("PureCN", "optparse", 
    "TxDb.Hsapiens.UCSC.hg19.knownGene", "org.Hs.eg.db"))
```

(Replace `hg19` with your genome version).

To use the alternative and in many cases recommended `r CRANpkg("PSCBS")`
segmentation:

```
# default PSCBS without support of interval weights
BiocManager::install("PSCBS")

# patched PSCBS with support of interval weights
BiocManager::install("lima1/PSCBS", ref="add_dnacopy_weighting")
```

To call mutational signatures, install the GitHub version of the
`r CRANpkg("deconstructSigs")` package:

```
BiocManager::install("raerose01/deconstructSigs")
```

For the experimental support of importing variant calls from 
GATK4 GenomicsDB, follow the installations instructions from
[GenomicsDB-R](https://github.com/nalinigans/GenomicsDB-R).


# Prepare environment and assay-specific reference files


- Start R and enter the following to get the path to the command line
  scripts:

```{r path}
system.file("extdata", package="PureCN")
```

- Exit R and store this path in an environment variable, for example in 
  BASH:

```
$ export PURECN="/path/to/PureCN/extdata"
$ Rscript $PURECN/PureCN.R --help
Usage: /path/to/PureCN/inst/extdata/PureCN.R [options] ...
```

- Generate an interval file from a BED file containing baits coordinates (not
  necessarily required with third-party segmentations, see in the corresponding
  Section \@ref(run-purecn-with-third-party-segmentation)):

```
# specify path where PureCN should store reference files
$ export OUT_REF="reference_files"
$ Rscript $PURECN/IntervalFile.R --infile baits_hg19.bed \ 
    --fasta hg19.fa --outfile $OUT_REF/baits_hg19_intervals.txt \
    --offtarget --genome hg19 \
    --export $OUT_REF/baits_optimized_hg19.bed \
    --mappability wgEncodeCrgMapabilityAlign100mer.bigWig \
    --reptiming wgEncodeUwRepliSeqK562WaveSignalRep1.bigWig
```
Internally, this script uses `r Biocpkg("rtracklayer")` to parse the
`infile`. Make sure that the file format matches the file extension.
See the `r Biocpkg("rtracklayer")` documentation for problems loading the file.
Check that the genome version of the baits file matches the reference.  Do not
include chrM baits in case the capture kit includes some.

The `--offtarget` flag will include off-target reads. Including them is
recommended except for Amplicon data.

The `--genome` version is needed to annotate exons with gene symbols.  Use
hg19/hg38 for human genomes, not b37/b38. You might get a warning that an
annotation package is missing.  For hg19, install
`r Biocpkg("TxDb.Hsapiens.UCSC.hg19.knownGene")` in R.

The `--export` argument is optional. If provided, this script will store the
modified intervals as BED file for example (again every 
`r Biocpkg("rtracklayer")` format is supported). This is useful when the coverages
are calculated with third-party tools like GATK.

The `--mappability` argument should provide a `r Biocpkg("rtracklayer")`
parsable file with a mappability score in the first meta data column. If
provided, off-target regions will be restricted to regions specified in this
file.  On-target regions with low mappability will be excluded. For hg19,
download the file from the UCSC website. Choose the kmer size that best fits
your average mapped read length. For hg38, download recommended 76-kmer or
100-kmer mappability files through the courtesy of the Waldron lab from: 

   - [GCA_000001405.15_GRCh38_no_alt_analysis\_set_76.bw](https://s3.amazonaws.com/purecn/GCA_000001405.15_GRCh38_no_alt_analysis\_set_76.bw)
   - [GCA_000001405.15_GRCh38_no_alt_analysis_set_100.bw](https://s3.amazonaws.com/purecn/GCA_000001405.15_GRCh38_no_alt_analysis_set_100.bw)

See the FAQ section of the main vignette for instruction how to generate such a
file for other references.

Similarly, the `--reptiming` argument takes a replication timing score in the
same format.  If provided, GC-normalized and log-transformed coverage is tested
for a linear relationship with this score and normalized accordingly.  This is
optional and provides only a minor benefit for coverage normalization, but can
identify samples with high proliferation. Requires `--offtarget` to be useful. 


# Create VCF files

`r Biocpkg("PureCN")` does not ship with a variant caller. Use a third-party
tool to generate a VCF for each sample. 

Important recommendations:

- Use _MuTect 1.1.7_ if possible; _Mutect 2_ from _GATK 4.1.7+_ is now out of
  alpha.

- VCFs from most other tumor-only callers such as _VarScan2_ and
  _FreeBayes_ are supported, but only very limited artifact filtering will be
  performed for these callers.  Make sure to provide filtered VCFs. See the FAQ
  section in the main vignette for common problems and questions related to
  input data.

- Since germline SNPs are needed to infer allele-specific copy numbers, the
  provided VCF needs to contain both somatic and germline variants.  Make sure
  that upstream filtering does not remove high quality SNPs, in particular due
  to presence in germline databases.

- Run the variant caller with a 50-75 base pair interval padding to increase the
  number of heterozygous SNPs


# Run PureCN with internal segmentation

The following describes `r Biocpkg("PureCN")` runs with internal copy number
normalization and segmentation. 

What you will need:

- The interval file generated above

- BAM files of tumor samples. 

- BAM files of normal samples (see main vignette for recommendations). These
  normal samples are not required to be patient-matched to the tumor samples,
  but they need to be processed-matched (same assay run through the same
  alignment pipeline, ideally sequenced in the same lab)

- VCF files generated above for all tumor and normal BAM files


## Coverage

For each sample, tumor and normal, calculate GC-normalized coverages:

```
# Calculate and GC-normalize coverage from a BAM file 
$ Rscript $PURECN/Coverage.R --outdir $OUT/$SAMPLEID \ 
    --bam ${SAMPLEID}.bam \
    --intervals $OUT_REF/baits_hg19_intervals.txt

# GC-normalize coverage from a GATK DepthOfCoverage file
Rscript $PURECN/Coverage.R --outdir $OUT/$SAMPLEID \
    --coverage ${SAMPLEID}.coverage.sample_interval_summary \ 
    --intervals $OUT_REF/baits_hg19_intervals.txt
```

Similar to GATK, this script also takes a text file containing a list of BAM or
coverage file names (one per line). The file extension must be `.list`:

```
# Calculate and GC-normalize coverage from a list of BAM files 
$ Rscript $PURECN/Coverage.R --outdir $OUT \ 
    --bam normals.list \
    --intervals $OUT_REF/baits_hg19_intervals.txt \
    --cores 4
```

Important recommendations:

- Only provide `--keepduplicates` or `--removemapq0` if you know what you are
doing and always use the same command line arguments for tumor and the normals


## NormalDB

To build a normal database for coverage normalization, copy the paths to all
GC-normalized normal coverage files in a single text file, line-by-line:

```
ls -a normal*loess.txt.gz | cat > example_normal.list

# From already GC-normalized files
$ Rscript $PURECN/NormalDB.R --outdir $OUT_REF \
    --coveragefiles example_normal.list \
    --genome hg19 --assay agilent_v6

# When normal panel VCF is available (highly recommended for
# unmatched samples)
$ Rscript $PURECN/NormalDB.R --outdir $OUT_REF \
    --coveragefiles example_normal.list \
    --normal_panel $NORMAL_PANEL \
    --genome hg19 \
    --assay agilent_v6

# For a Mutect2/GATK4 normal panel GenomicsDB (experimental)
$ Rscript $PURECN/NormalDB.R --outdir $OUT_REF \
    --coveragefiles example_normal.list \
    --normal_panel $GENOMICSDB-WORKSPACE-PATH/pon_db \
    --genome hg19 \
    --assay agilent_v6
```

Important recommendations:

- Consider generating different databases when differences are significant, e.g.
  for samples with different read lengths or insert size distributions

- In particular, do not mix normal data obtained with different capture kits
  (e.g. _Agilent SureSelect v4_ and _v6_)

- Provide a normal panel VCF here to precompute mapping bias for faster
  runtimes. The only requirement for the VCF is an `AD` format field containing
  the number of reference and alt reads for all samples. See the example file
  `$PURECN/normalpanel.vcf.gz`.

- For ideal results, examine the `interval_weights.png` file to find good
  off-target bin widths. You will need to re-run `IntervalFile.R` with the
  `--offtargetwidth` parameter and re-calculate the coverages.

- The `--assay` argument is optional and is only used to add the provided assay
  name to all output files

- A warning pointing to the likely use of a wrong baits file means that more
  than 5% of targets have close to 0 coverage in all normal samples. A BED file
  with the low coverage targets will be generated in `--outdir`.  If for any
  reason there is no access to the correct file, it is recommended to re-run the
  `IntervalFile.R` command and provide this BED file with `--exclude`.


## PureCN

Now that the assay-specific files are created and all coverages calculated, we
run `PureCN.R` to normalize, segment and determine purity and ploidy:

```
mkdir $OUT/$SAMPLEID

# Without a matched normal (minimal test run)
$ Rscript $PURECN/PureCN.R --out $OUT/$SAMPLEID \
    --tumor $OUT/$SAMPLEID/${SAMPLEID}_coverage_loess.txt.gz \
    --sampleid $SAMPLEID \
    --vcf ${SAMPLEID}_mutect.vcf \
    --normaldb $OUT_REF/normalDB_hg19.rds \
    --intervals $OUT_REF/baits_hg19_intervals.txt \
    --genome hg19 

# Production pipeline run
$ Rscript $PURECN/PureCN.R --out $OUT/$SAMPLEID \
    --tumor $OUT/$SAMPLEID/${SAMPLEID}_coverage_loess.txt.gz \
    --sampleid $SAMPLEID \
    --vcf ${SAMPLEID}_mutect.vcf \
    --statsfile ${SAMPLEID}_mutect_stats.txt \
    --normaldb $OUT_REF/normalDB_hg19.rds \
    --mappingbiasfile $OUT_REF/mapping_bias_hg19.rds \
    --intervals $OUT_REF/baits_hg19_intervals.txt \
    --snpblacklist hg19_simpleRepeats.bed \
    --genome hg19 \
    --force --postoptimize --seed 123

# With a matched normal (test run; for production pipelines we recommend the
# unmatched workflow described above)
$ Rscript $PURECN/PureCN.R --out $OUT/$SAMPLEID \
    --tumor $OUT/$SAMPLEID/${SAMPLEID}_coverage_loess.txt.gz \
    --normal $OUT/$SAMPLEID/${SAMPLEID_NORMAL}_coverage_loess.txt.gz \
    --sampleid $SAMPLEID \
    --vcf ${SAMPLEID}_mutect.vcf \
    --normaldb $OUT_REF/normalDB_hg19.rds \
    --intervals $OUT_REF/baits_hg19_intervals.txt \
    --genome hg19

# Recreate output after manual curation of ${SAMPLEID}.csv
$ Rscript $PURECN/PureCN.R --rds $OUT/$SAMPLEID/${SAMPLEID}.rds
```

Important recommendations:

- Even if matched normals are available, it is often better to use the normal
  database for coverage normalization. **When a matched normal coverage is
  provided with `--normal` then the pool of normal coverage normalization and
  denoising steps are skipped!**

- Always provide the normal coverage database to ignore low quality regions in
  the segmentation and to increase the sensitivity for homozygous deletions in
  high purity samples.

- The normal panel VCF file is useful for mapping bias correction and especially
  recommended without matched normals. See the FAQ of the main vignette how to
  generate this file. It is not essential for test runs.  

- The _MuTect 1.1.7_ stats file (the main output file besides the VCF) should be
  provided for better artifact filtering. If the VCF was generated by a pipeline
  that performs good artifact filtering, this file is not needed.

- The `--postoptimize` flag defines that purity should be optimized using both
  variant allelic fractions and copy number instead of copy number only.  This
  results in a significant runtime increase for whole-exome data.

- If `--out` is a directory, it will use the sample id as file prefix for all
  output files. Otherwise `r Biocpkg("PureCN")` will use `--out` as prefix.   
    
- The `--parallel` flag will enable the parallel fitting of local optima. See 
  `r Biocpkg("BiocParallel")` for details. This script will use the default
  backend.

- `--funsegmentation PSCBS` will become the new default in 1.22.  Support for
  interval weights currently requires a patch (see Section
  \@ref(installation)).

- `--model betabin` will become the new default in 1.22 with larger panel of
  normals (probably more than 10-15 normal samples). We encourage users to try
  this model and appreciate feedback.

- Defaults are well calibrated and should produce close to ideal results for
  most samples. A few common cases where changing defaults makes sense:

    - High purity and high quality: For cancer types with a high expected
      purity, such as ovarian cancer, AND when quality is expected to be very
      good (high coverage, young samples), `--maxcopynumber 8`

    - Small panels with high coverage: `--padding 100` (or higher), requires
      running the variant caller with this padding or without interval file. Use
      the same settings for the panel of normals VCF so that SNPs in the
      flanking regions have reliable mapping bias estimates. The
      `--maxhomozygousloss` parameter might also need some adjustment for very
      small panels with large gaps around captured deletions.

    - Cell lines: Safely skip the search for low purity solutions in cell lines:
      `--maxcopynumber 8`, `--minpurity 0.9`, `--maxpurity 0.99`.  Add
      `--modelhomozygous` to find regions of LOH in samples without normal
      contamination (do not provide this flag when matched normal data are
      available in the VCF).

    - cfDNA: `--minpurity 0.1`, `--minaf 0.01` (or lower) and `--error 0.0005`
      (or lower, when there is UMI-based error correction). Note that the
      estimated purity can be very wrong when the true purity is below 5-7%;
      these samples are usually flagged as non-aberrant.

    - Amplicon data: `--model betabin` (Amplicon data is not officially
      supported)

    - All assays: `--maxsegments` should set to a value so that with few
      exceptions only poor quality samples exceed this cutoff. For cancer types
      with high heterogenity, it is also recommended to increase
      `--maxnonclonal` to 0.3-0.4 (this will increase the runtime significantly
      for whole-exome data).

# Run PureCN with third-party segmentation

What you will need:

- Output of third-party tools (see details below)

- VCF files for all tumor samples and some normal files (see main vignette for
  questions related to required normal samples) 


## General usage

If you already have a segmentation from third-party tools (for example _CNVkit_,
_GATK4_, _EXCAVATOR2_). For a minimal test run:

```
Rscript $PURECN/PureCN.R --out $OUT/$SAMPLEID  \
    --sampleid $SAMPLEID \
    --segfile $OUT/$SAMPLEID/${SAMPLEID}.cnvkit.seg \
    --vcf ${SAMPLEID}_mutect.vcf \
    --intervals $OUT_REF/baits_hg19_intervals.txt \
    --genome hg19 
```

See the main vignette for more details and file formats.


## Recommended _CNVkit_ usage

For a production pipeline run we provide again more information about the assay
and genome. Here an _CNVkit_ example: 

```
# Recommended: Provide a normal panel VCF to remove mapping biases, pre-compute
# position-specific bias for much faster runtimes with large panels
# This needs to be done only once for each assay
Rscript $PURECN/NormalDB.R --outdir $OUT_REF --normal_panel $NORMAL_PANEL \
    --assay agilent_v6 --genome hg19 --force

# Export the segmentation in DNAcopy format
cnvkit.py export seg $OUT/$SAMPLEID/${SAMPLEID}_cnvkit.cns --enumerate-chroms \
    -o $OUT/$SAMPLEID/${SAMPLEID}_cnvkit.seg

# Run PureCN by providing the *.cnr and *.seg files 
Rscript $PURECN/PureCN.R --out $OUT/$SAMPLEID  \
    --sampleid $SAMPLEID \
    --tumor $OUT/$SAMPLEID/${SAMPLEID}_cnvkit.cnr \
    --segfile $OUT/$SAMPLEID/${SAMPLEID}_cnvkit.seg \
    --mappingbiasfile $OUT_REF/mapping_bias_agilent_v6_hg19.rds \
    --vcf ${SAMPLEID}_mutect.vcf \
    --statsfile ${SAMPLEID}_mutect_stats.txt \
    --snpblacklist hg19_simpleRepeats.bed \
    --genome hg19 \
    --funsegmentation Hclust \
    --force --postoptimize --seed 123
```

Important recommendations:

- The `--funsegmentation` argument controls if the data should to be
  re-segmented using germline BAFs (default). Set this value to `none` if the
  provided segmentation should be used as is.  The recommended `Hclust` will
  only cluster provided segments.

- Since _CNVkit_ provides all necessary information in the `*.cnr` output files,
  the `--intervals` argument is not required.

- In test runs, especially when the input VCF contains matched normal
  information, `--mappingbiasfile` can be skipped

- _CNVkit_ runs without normal reference samples are not recommended

## Recommended _GATK4_ usage

```
# Recommended: Provide a normal panel GenomicsDB to remove mapping
# biases, pre-compute position-specific bias for much faster runtimes
# with large panels. This needs to be done only once for each assay. 
Rscript $PURECN/NormalDB.R --outdir $OUT_REF \
    --normal_panel $GENOMICSDB-WORKSPACE-PATH/pon_db \
    --assay agilent_v6 --genome hg19 --force

Rscript $PURECN/PureCN.R --out $OUT/$SAMPLEID  \
    --sampleid $SAMPLEID \
    --tumor $OUT/$SAMPLEID/${SAMPLEID}.hdf5 \
    --logratiofile $OUT/$SAMPLEID/${SAMPLEID}.denoisedCR.tsv \
    --segfile $OUT/$SAMPLEID/${SAMPLEID}.modelFinal.seg \
    --mappingbiasfile $OUT_REF/mapping_bias_agilent_v6_hg19.rds \
    --vcf ${SAMPLEID}_mutect2_filtered.vcf \
    --snpblacklist hg19_simpleRepeats.bed \
    --genome hg19 \
    --funsegmentation Hclust \
    --force --postoptimize --seed 123
```

# Biomarkers

`Dx.R` provides copy number and mutation metrics commonly used as biomarkers,
most importantly tumor mutational burden (TMB), chromosomal instability (CIN)
and mutational signatures.

```
# Provide a BED file with callable regions, for examples obtained by
# GATK CallableLoci. Useful to calculate mutations per megabase and
# to exclude low quality regions.
grep CALLABLE ${SAMPLEID}_callable_status.bed > \ 
    ${SAMPLEID}_callable_status_filtered.bed

# Only count mutations in callable regions, also subtract what was
# ignored in PureCN.R via --snpblacklist, like simple repeats, from the
# mutation per megabase calculation
# Also search for the COSMIC mutation signatures
# (http://cancer.sanger.ac.uk/cosmic/signatures)
Rscript $PureCN/Dx.R --out $OUT/$SAMPLEID/$SAMPLEID \
    --rds $OUT/SAMPLEID/${SAMPLEID}.rds \
    --callable ${SAMPLEID}_callable_status_filtered.bed \
    --exclude hg19_simpleRepeats.bed \
    --signatures 

# Restrict mutation burden calculation to coding sequences
Rscript $PureCN/FilterCallableLoci.R --genome hg19 \
    --infile ${SAMPLEID}_callable_status_filtered.bed \
    --outfile ${SAMPLEID}_callable_status_filtered_cds.bed \
    --exclude '^HLA'
 
Rscript $PureCN/Dx.R --out $OUT/$SAMPLEID/${SAMPLEID}_cds \
    --rds $OUT/SAMPLEID/${SAMPLEID}.rds \
    --callable ${SAMPLEID}_callable_status_filtered_cds.bed \
    --exclude hg19_simpleRepeats.bed
```

Important recommendations:

- Run _GATK CallableLoci_ with `--minDepth N` where N is roughly 20% of the mean
  target coverage of all samples.


# Reference

Argument name          | Corresponding PureCN argument | PureCN function 
-----------------------|-------------------------------|----------------
`--fasta`              | `reference.file` | `preprocessIntervals` 
`--infile`             | `interval.file` | `preprocessIntervals` 
`--offtarget`          |     `off.target`    | `preprocessIntervals` 
`--targetwidth`        | `average.target.width` | `preprocessIntervals` 
`--mintargetwidth`     | `min.target.width` | `preprocessIntervals` 
`--smalltargets`       | `small.targets` | `preprocessIntervals` 
`--offtargetwidth`     | `average.off.target.width` | `preprocessIntervals` 
`--offtargetseqlevels` | `off.target.seqlevels` | `preprocessIntervals` 
`--mappability`        | `mappability`   | `preprocessIntervals` 
`--minmappability`     | `min.mappability`   | `preprocessIntervals` 
`--reptiming`          | `reptiming`   | `preprocessIntervals` 
`--reptimingwidth`     | `average.reptiming.width` | `preprocessIntervals`  
`--genome`             | `txdb`, `org` | `annotateTargets` 
`--outfile`            | | 
`--export`             | | `rtracklayer::export`  
`--version -v`         | | 
`--force -f`           | | 
`--help -h`            | | 

: (\#tab:intervalfile) IntervalFile


Argument name      | Corresponding PureCN argument | PureCN function 
-------------------|-------------------------------|----------------
`--bam`            | `bam.file` | `calculateBamCoverageByInterval`
`--bai`            | `index.file` | `calculateBamCoverageByInterval` 
`--coverage`       | `coverage.file` | `correctCoverageBias`
`--intervals`      | `interval.file`  | `correctCoverageBias` 
`--method`         | `method`  | `correctCoverageBias`
`--keepduplicates` | `keep.duplicates`  | `calculateBamCoverageByInterval`
`--removemapq0`    | `mapqFilter`  | `ScanBamParam`
`--outdir`         | | 
`--cores`          | | Number of CPUs to use when multiple BAMs are provided 
`--parallel`       | | Use default `r Biocpkg("BiocParallel")` backend when multiple BAMs are provided 
`--seed`           | | 
`--version -v`     | | 
`--force -f`       | | 
`--help -h`        | | 

: (\#tab:coverage) Coverage


Argument name          | Corresponding PureCN argument | PureCN function 
-----------------------|-------------------------------|----------------
`--coveragefiles`   | `normal.coverage.files` | `createNormalDatabase`
`--normal_panel`    | `normal.panel.vcf.file` | `calculateMappingBiasVcf` 
`--assay -a`          | Optional assay name | Used in output file names. 
`--genome -g`        | Optional genome version | Used in output file names. 
`--outdir -o`         | | 
`--version -v`        | | 
`--force -f`          | | 
`--help -h`           | | 

: (\#tab:normaldb) NormalDB

Argument name          | Corresponding PureCN argument | PureCN function 
-----------------------|-------------------------------|----------------
`--sampleid -i`      | `sampleid`  | `runAbsoluteCN`  
`--normal`           | `normal.coverage.file`   | `runAbsoluteCN`  
`--tumor`            | `tumor.coverage.file`     | `runAbsoluteCN` 
`--vcf`              | `vcf.file`          | `runAbsoluteCN`   
`--rds`              | `file.rds`          | `readCurationFile`   
`--mappingbiasfile`  | `mapping.bias.file` | `setMappingBiasVcf` 
`--normaldb`         | `normalDB` (serialized with `saveRDS`) | `calculateTangentNormal`, `filterTargets` 
`--segfile`          | `seg.file`           | `runAbsoluteCN`  
`--logratiofile`     | `log.ratio`          | `runAbsoluteCN`  
`--additionaltumors` | `tumor.coverage.files` | `processMultipleSamples`  
`--sex`              | `sex`                | `runAbsoluteCN`  
`--genome`           | `genome`             | `runAbsoluteCN`  
`--intervals`        | `interval.file`      | `runAbsoluteCN`   
`--statsfile`        | `stats.file` | `filterVcfMuTect`        
`--minaf`            | `af.range` | `filterVcfBasic` 
`--snpblacklist`     | `snp.blacklist` | `filterVcfBasic`     
`--error`            | `error` | `runAbsoluteCN`     
`--dbinfoflag`       | `DB.info.flag` | `runAbsoluteCN`     
`--popafinfofield`   | `POPAF.info.field` | `runAbsoluteCN`     
`--mincosmiccnt`     | `min.cosmic.cnt` | `setPriorVcf`     
`--funsegmentation`  | `fun.segmentation` | `runAbsoluteCN`  
`--alpha`            | `alpha` | `segmentationCBS` 
`--undosd`           | `undo.SD` | `segmentationCBS` 
`--maxsegments`      | `max.segments` | `runAbsoluteCN` 
`--minpurity`        | `test.purity` | `runAbsoluteCN` 
`--maxpurity`        | `test.purity` | `runAbsoluteCN` 
`--minploidy`        | `min.ploidy` | `runAbsoluteCN` 
`--maxploidy`        | `max.ploidy` | `runAbsoluteCN` 
`--maxcopynumber`   | `test.num.copy` | `runAbsoluteCN` 
`--postoptimize`     | `post.optimize` | `runAbsoluteCN`     
`--bootstrapn`       | `n` | `bootstrapResults` 
`--modelhomozygous`  | `model.homozygous` | `runAbsoluteCN`  
`--model`            | `model` | `runAbsoluteCN`  
`--logratiocalibration` | `log.ratio.calibration` | `runAbsoluteCN`  
`--maxnonclonal`     | `max.non.clonal` | `runAbsoluteCN`  
`--maxhomozygousloss` | `max.homozygous.loss` | `runAbsoluteCN`  
`--outvcf`           | `return.vcf` | `predictSomatic` 
`--out -o`           | |                            
`--parallel`         | `BPPARAM` | `runAbsoluteCN` 
`--cores`            | `BPPARAM` | `runAbsoluteCN` 
`--seed`             | | 
`--version -v`       | | 
`--force -f`         | | 
`--help -h`          | | 

: (\#tab:purecn) PureCN

Argument name   | Corresponding PureCN argument | PureCN function 
----------------|-------------------------------|----------------
`--rds`         | `file.rds` | `readCurationFile`   
`--callable`    | `callable` | `callMutationBurden` 
`--exclude`     | `exclude`  | `callMutationBurden` 
`--maxpriorsomatic` | `max.prior.somatic`  | `callMutationBurden` 
`--signatures` | | `deconstructSigs::whichSignatures` 
`--signature_databases` | | `deconstructSigs::whichSignatures` 
`--out`         | | 
`--version -v`  | | 
`--force -f`    | | 
`--help -h`     | | 

: (\#tab:dx) Dx