# PureCN best practices

# Prerequisites

## Update from previous stable versions

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

    $ Rscript $PURECN/NormalDB.R --out-dir $OUT_REF \
        --coverage-files example_normal_coverages.list \
        --genome hg19 --normal-panel $NORMAL_PANEL --assay agilent_v6

When using `--model betabin` in `PureCN.R`, we recommend for all previous versions re-creating the mapping bias database by re-running `NormalDB.R`:

    # only re-creating the mapping bias file
    $ Rscript $PURECN/NormalDB.R --out-dir $OUT_REF \
        --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 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", "R.utils",
        "TxDb.Hsapiens.UCSC.hg19.knownGene", "org.Hs.eg.db"))

(Replace `hg19` with your genome version).

To use the alternative and in many cases recommended 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 deconstructSigs package:

    BiocManager::install("raerose01/deconstructSigs")

For the experimental support of importing variant calls from GATK4 GenomicsDB, follow the installations instructions from GenomicsDB-R.

The GATK4 segmentation requires the `gatk` binary in path. Versions 4.1.7.0 and newer are supported.

# Prepare environment and assay-specific reference files

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

    system.file("extdata", package = "PureCN")

    ## [1] "/tmp/RtmpESe3pR/Rinst186a6b27a58c/PureCN/extdata"

- 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 --in-file baits_hg19.bed \
        --fasta hg19.fa --out-file $OUT_REF/baits_hg19_intervals.txt \
        --off-target --genome hg19 \
        --export $OUT_REF/baits_optimized_hg19.bed \
        --mappability wgEncodeCrgMapabilityAlign100mer.bigWig \
        --reptiming wgEncodeUwRepliSeqK562WaveSignalRep1.bigWig

Internally, this script uses rtracklayer to parse the `--in-file`. Make sure that the file format matches the file extension. See the 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.

We do not recommend padding the baits file manually unless the coverages are very low (<30X) where the increased counts from the padded regions might decrease sampling variance slightly. Note that we do however strongly recommend running the variant caller with a padding of at least 50bp to increase the number of informative SNPs, see below in the VCF section. Double check that the genome version of the `--in-file` is correct - many assays are still designed using older references and might need to be lifted over to the pipeline reference. If possible, do NOT use a BED file that contains the targeted exons, instead use the coordinates of the baits. These are optimized for GC-content and mappability and will produce cleaner coverage profiles.

The `--off-target` flag will include off-target reads. Including them is recommended except for Amplicon data. For whole-exome data, the benefit is usually also limited unless the assay is inefficient with a high fraction of off-target reads (>10-15%).

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 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 rtracklayer format is supported). This is useful when the coverages are calculated with third-party tools like GATK.

The `--mappability` argument should provide a 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
- 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 `--off-target` to be useful.

# Create VCF files

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 and VCFs generated following the best practices somatic workflow should work (earlier Mutect 2 versions are not supported and will not work).

- 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. Mutect 1.1.7 automatically calls SNPs, but Mutect 2 does not. Make sure to run Mutect 2 with `--genotype-germline-sites true --genotype-pon-sites true`. You will not get usuable output without those flags. Since Mutect 2 from GATK 4.2.0+, average base quality scores can be very low and variants will be too aggressively removed by PureCN. You will need to set `--min-base-quality 20` in PureCN.R to keep them.

- Run the variant caller with a 50-75 base pair interval padding to increase the number of heterozygous SNPs (for example `--interval_padding` and `--interval-padding` in Mutect 1.1.7 and Mutect 2, respectively). For very high coverages beyond 1000X, it is safe to increase this value up to 200bp.

# Run PureCN with internal segmentation

The following describes 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 --out-dir $OUT/$SAMPLEID \
        --bam ${SAMPLEID}.bam \
        --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 --out-dir $OUT/normals \
        --bam normals.list \
        --intervals $OUT_REF/baits_hg19_intervals.txt \
        --cores 4

Important recommendations:

- Only provide `--keep-duplicates` or `--remove-mapq0` if you know what you are doing and always use the same command line arguments for tumor and the normals

- It can be safe to skip the GC-normalization with `--skip-gc-norm` when tumor and normal samples are expected to exhibit similar biases and a sufficient number of normal samples is available. A good example would be plasma sequencing. In contrast, old FFPE samples normalized against blood controls will more likely benefit from GC-normalization.

- A potential negative impact of GC-normalization is much more likely in very small targeted panels (< 0.5Mb) and worth benchmarking.

- When supported third-party tools are used to calculate coverage (currently CNVkit, GATK3 and GATK4), it is possible to GC-normalize those coverages with a matching interval file:

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

## 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 $OUT/normals/*_loess.txt.gz | cat > example_normal_coverages.list
    # In case no GC-normalization is performed:
    # ls -a $OUT/normals/*_coverage.txt.gz | cat > example_normal_coverages.list

    $ Rscript $PURECN/NormalDB.R --out-dir $OUT_REF \
        --coverage-files example_normal_coverages.list \
        --genome hg19 --assay agilent_v6

    # When normal panel VCF is available (highly recommended for
    # unmatched samples)
    $ Rscript $PURECN/NormalDB.R --out-dir $OUT_REF \
        --coverage-files example_normal_coverages.list \
        --normal-panel $NORMAL_PANEL \
        --genome hg19 \
        --assay agilent_v6

    # For a Mutect2/GATK4 normal panel GenomicsDB (beta)
    $ Rscript $PURECN/NormalDB.R --out-dir $OUT_REF \
        --coverage-files example_normal_coverages.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 `--average-off-target-width` parameter and re-calculate the coverages. `NormalDB.R` will also give a suggestion for a good minimum width. We do not recommend going lower than this estimate; setting `--average-off-target-width` to value larger than this value can decrease noise at the cost of loss in resolution. Setting it to 1.2-1.5x the minimum recommendation (that should be ideally < 250kb) is a good starting point.

- 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 `--out-dir`. 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 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 \
        --stats-file ${SAMPLEID}_mutect_stats.txt \
        --fun-segmentation PSCBS \
        --normaldb $OUT_REF/normalDB_hg19.rds \
        --mapping-bias-file $OUT_REF/mapping_bias_hg19.rds \
        --intervals $OUT_REF/baits_hg19_intervals.txt \
        --snp-blacklist hg19_simpleRepeats.bed \
        --genome hg19 \
        --model betabin \
        --force --post-optimize --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.

- Double check that in `--tumor` and `--normaldb`, GC-normalization is either used in both (`*_loess.txt.gz`) or skipped in both (`*_coverage.txt.gz`).

- 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. Do NOT provide this file for Mutect 2.

- The `--post-optimize` 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 PureCN will use `--out` as prefix.

- The `--parallel` flag will enable the parallel fitting of local optima. See BiocParallel for details. This script will use the default backend. `--cores` is a short cut to use the specified number of CPUs instead of the default backend. Only specify one of the two arguments. Note that memory usage can increase linearly with number of cores and insufficient memory can result in random crashes.

- `--fun-segmentation PSCBS` is the new recommendation in 1.22. Support for interval weights currently requires a patch (see Section @ref(installation)). See below for some more details on the best choice of the method.

- `--model betabin` is the new recommendation in 1.22 with larger panel of normals (more than 10-15 normal samples).

- 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), `--max-copy-number 8`. (PureCN reports copy numbers greater than this value, but will stop fitting SNP allelic fractions to the exact allele-specific copy number because this will get impossible very quickly with high copy numbers - and computationally expensive.)

  - Small panels with high coverage: `--interval-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 `--max-homozygous-loss` 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: `--max-copy-number 8`, `--min-purity 0.9`, `--max-purity 0.99`. Add `--model-homozygous` 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: `--min-purity 0.1`, `--min-af 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.

  - All assays: `--max-segments` 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 `--max-non-clonal` to 0.3-0.4 (this will increase the runtime significantly for whole-exome data).

  - The choice of the segmentation function can also make a significant difference and unfortunately there is not yet a universal method that works best in all scenarios.

    - PSCBS: A good and safe starting point, especially with off-target regions that might exhibit different noise profiles compared to on-target.

    - GATK4: Most recent addition. Not yet well tested in PureCN, but theoretically best choice with a larger number of SNPs per intervals, for example assays with copy number backbones. We appreciate feedback.

    - CBS: Simple, fast and well tested. Does not fully support SNP information, so only recommended for settings with a very small SNPs/intervals ratio, for example small targeted panels (<1Mb) with healthy off-target coverage (<150kb resolution and similar log ratio noise compared to on-target).

    - copynumber: For cases with multiple time points or biopsies. This is
      automatically chosen with `--additional-tumors` and currently not supported in a single-sample analysis.

    - Hclust/none: For third-party segmentations. `Hclust` clusters segments in an attempt to calibrate log-ratios across chromosomes, `none` largely keeps everything as provided.

- A few recommendations for checks whether the PureCN setup is correct:

  - The “Mean standard deviation of log-ratios” reported in the log file should be fairly low for high quality data. Older FFPE data can be around 0.4, but high coverage, relatively recent samples should approach the 0.15 minimum. If off-target is consistently noisier than on-target, it is probably worth increasing the off-target bin size and start from scratch (or in case of whole-exome sequencing, ignore off-target reads since they do not provide much additional information when bins are large and/or noisy).

  - Related to that, a warning is thrown when less than 10% of all intervals passing filters are off-target intervals. Whole-exome sequencing is usually around that value. If the log-ratio standard deviation is similar or even lower than the one for on-target, it is worth keeping off-target regions. Otherwise off-target might add more noise than signal. Off-target information is automatically ignored when the passing rate falls below 5% of all intervals.

  - The fraction of targets with SNPs should be between 10 and 15 percent. If it is significantly lower, make sure that the variant caller was used with 50-100bp interval padding or no interval file at all. Also check that the interval file was generated using the baits coordinates, not the targets (the baits BED file should have a more even size distribution, e.g. 120bp and multiples of it). If many variants are removed by the default 25 base quality feature, you might be using Mutect 2 and need to re-run PureCN.R with `--min-base-quality 20`.

  - “Initial testing for significant sample cross-contamination” in the log file should not have many false positives, i.e. should be “unlikely” for most samples, not “maybe”. Insufficient artifact removal can result in too many false SNPs calls with low allelic fractions, confusing the contamination caller.

  - Read all warnings.

# Run PureCN with third-party segmentation

Our internal PureCN normalization combined with the PSCBS or GATK4 segmentation should produce highly competitive results and we encourage users to try it and compare it to their existing pipelines. However, we realize that often it is not an option to change tools in production pipelines and we therefore made it relatively easy to use PureCN with third-party tools. We provide examples for CNVkit and GATK4 and it should be straightforward to adapt those for other tools.

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 \
        --seg-file $OUT/$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 --out-dir $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 \
        --seg-file $OUT/$SAMPLEID/${SAMPLEID}_cnvkit.seg \
        --mapping-bias-file $OUT_REF/mapping_bias_agilent_v6_hg19.rds \
        --vcf ${SAMPLEID}_mutect.vcf \
        --stats-file ${SAMPLEID}_mutect_stats.txt \
        --snp-blacklist hg19_simpleRepeats.bed \
        --genome hg19 \
        --fun-segmentation Hclust \
        --force --post-optimize --seed 123

Important recommendations:

- The `--fun-segmentation` 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, `--mapping-bias-file` can be skipped

- CNVkit runs without normal reference samples are not recommended

- The `--stats-file` is only supported for Mutect 1.1.7. Mutect 2 provides the filter flags directly in the VCF.

## 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 --out-dir $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 \
        --log-ratio-file $OUT/$SAMPLEID/${SAMPLEID}.denoisedCR.tsv \
        --seg-file $OUT/$SAMPLEID/${SAMPLEID}.modelFinal.seg \
        --mapping-bias-file $OUT_REF/mapping_bias_agilent_v6_hg19.rds \
        --vcf ${SAMPLEID}_mutect2_filtered.vcf \
        --snp-blacklist hg19_simpleRepeats.bed \
        --genome hg19 \
        --fun-segmentation Hclust \
        --force --post-optimize --seed 123

Important recommendations:

- The `--fun-segmentation` can be set to none in most cases. This will keep the segmentation largely as provided. `Hclust` clusters segments to avoid over-segmentation and to calibrate log-ratios across chromosomes. This will thus alter the GATK4 segmentation, which might not be desired.

- Beta support for providing CollectAllelicCounts output instead of Mutect is available. Use `--vcf ${SAMPLEID}.allelicCounts.tsv` to automatically import the SNP counts and convert them into a supported VCF. Note that this will not use any somatic SNV and indel information available in Mutect VCFs and thus will also not provide any clonality annotation.

# 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 --snp-blacklist, 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 \
        --in-file ${SAMPLEID}_callable_status_filtered.bed \
        --out-file ${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.

- If `--callable` is missing, all intervals passing filters are assumed to be callable.

# Reference

(#tab:intervalfile) IntervalFile
Argument name | Corresponding PureCN argument | PureCN function
--- | --- | ---
`--fasta` | `reference.file` | `preprocessIntervals`
`--in-file` | `interval.file` | `preprocessIntervals`
`--off-target` | `off.target` | `preprocessIntervals`
`--average-target-width` | `average.target.width` | `preprocessIntervals`
`--min-target-width` | `min.target.width` | `preprocessIntervals`
`--small-targets` | `small.targets` | `preprocessIntervals`
`--average-off-target-width` | `average.off.target.width` | `preprocessIntervals`
`--off-target-seqlevels` | `off.target.seqlevels` | `preprocessIntervals`
`--mappability` | `mappability` | `preprocessIntervals`
`--min-mappability` | `min.mappability` | `preprocessIntervals`
`--reptiming` | `reptiming` | `preprocessIntervals`
`--average-reptiming-width` | `average.reptiming.width` | `preprocessIntervals`
`--genome` | `txdb`, `org` | `annotateTargets`
`--out-file` |  |
`--export` |  | `rtracklayer::export`
`--version -v` |  |
`--force -f` |  |
`--help -h` |  |

(#tab:coverage) Coverage
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`
`--keep-duplicates` | `keep.duplicates` | `calculateBamCoverageByInterval`
`--chunks` | `chunks` | `calculateBamCoverageByInterval`
`--remove-mapq0` | `mapqFilter` | `ScanBamParam`
`--skip-gc-norm` |  | `correctCoverageBias`
`--out-dir` |  |
`--cores` |  | Number of CPUs to use when multiple BAMs are provided
`--parallel` |  | Use default BiocParallel backend when multiple BAMs are provided
`--seed` |  |
`--version -v` |  |
`--force -f` |  |
`--help -h` |  |

(#tab:normaldb) NormalDB
Argument name | Corresponding PureCN argument | PureCN function
--- | --- | ---
`--coverage-files` | `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.
`--genomicsdb-af-field` | For GenomicsDB import, allelic fraction field | `calculateMappingBiasGatk4`
`--min-normals-position-specific-fit` | `min.normals.position.specific.fit` | `calculateMappingBiasVcf`, `calculateMappingBiasGatk4`
`--out-dir -o` |  |
`--version -v` |  |
`--force -f` |  |
`--help -h` |  |

(#tab:purecn) PureCN
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`
`--mapping-bias-file` | `mapping.bias.file` | `setMappingBiasVcf`
`--normaldb` | `normalDB` (serialized with `saveRDS`) | `calculateTangentNormal`, `filterTargets`
`--seg-file` | `seg.file` | `runAbsoluteCN`
`--log-ratio-file` | `log.ratio` | `runAbsoluteCN`
`--additional-tumors` | `tumor.coverage.files` | `processMultipleSamples`
`--sex` | `sex` | `runAbsoluteCN`
`--genome` | `genome` | `runAbsoluteCN`
`--intervals` | `interval.file` | `runAbsoluteCN`
`--stats-file` | `stats.file` | `filterVcfMuTect`
`--min-af` | `af.range` | `filterVcfBasic`
`--snp-blacklist` | `snp.blacklist` | `filterVcfBasic`
`--error` | `error` | `runAbsoluteCN`
`--db-info-flag` | `DB.info.flag` | `runAbsoluteCN`
`--popaf-info-field` | `POPAF.info.field` | `runAbsoluteCN`
`--cosmic-cnt-info-field` | `Cosmic.CNT.info.field` | `runAbsoluteCN`
`--min-cosmic-cnt` | `min.cosmic.cnt` | `setPriorVcf`
`--interval-padding` | `interval.padding` | `filterVcfBasic`
`--min-total-counts` | `min.total.counts` | `filterIntervals`
`--min-fraction-offtarget` | `min.fraction.offtarget` | `filterIntervals`
`--fun-segmentation` | `fun.segmentation` | `runAbsoluteCN`
`--alpha` | `alpha` | `segmentationCBS`
`--undo-sd` | `undo.SD` | `segmentationCBS`
`--changepoints-penalty` | `changepoints.penalty` | `segmentationGATK4`
`--additional-cmd-args` | `additional.cmd.args` | `segmentationGATK4`
`--max-segments` | `max.segments` | `runAbsoluteCN`
`--min-logr-sdev` | `min.logr.sdev` | `runAbsoluteCN`
`--min-purity` | `test.purity` | `runAbsoluteCN`
`--max-purity` | `test.purity` | `runAbsoluteCN`
`--min-ploidy` | `min.ploidy` | `runAbsoluteCN`
`--max-ploidy` | `max.ploidy` | `runAbsoluteCN`
`--max-copy-number` | `test.num.copy` | `runAbsoluteCN`
`--post-optimize` | `post.optimize` | `runAbsoluteCN`
`--bootstrap-n` | `n` | `bootstrapResults`
`--speedup-heuristics` | `speedup.heuristics` | `runAbsoluteCN`
`--model-homozygous` | `model.homozygous` | `runAbsoluteCN`
`--model` | `model` | `runAbsoluteCN`
`--log-ratio-calibration` | `log.ratio.calibration` | `runAbsoluteCN`
`--max-non-clonal` | `max.non.clonal` | `runAbsoluteCN`
`--max-homozygous-loss` | `max.homozygous.loss` | `runAbsoluteCN`
`--out-vcf` | `return.vcf` | `predictSomatic`
`--out -o` |  |
`--parallel` | `BPPARAM` | `runAbsoluteCN`
`--cores` | `BPPARAM` | `runAbsoluteCN`
`--seed` |  |
`--version -v` |  |
`--force -f` |  |
`--help -h` |  |

(#tab:dx) Dx
Argument name | Corresponding PureCN argument | PureCN function
--- | --- | ---
`--rds` | `file.rds` | `readCurationFile`
`--callable` | `callable` | `callMutationBurden`
`--exclude` | `exclude` | `callMutationBurden`
`--max-prior-somatic` | `max.prior.somatic` | `callMutationBurden`
`--signatures` |  | `deconstructSigs::whichSignatures`
`--signature-databases` |  | `deconstructSigs::whichSignatures`
`--out` |  |
`--version -v` |  |
`--force -f` |  |
`--help -h` |  |