Snakemake workflow for TitanCNA_SV_WGS_tumorOnly

Modules to load (for users of Hutch)

• ml snakemake/5.19.2-foss-2019b-Python-3.7.4
• ml R/3.6.2-foss-2019b-fh1
• ml Python/3.7.4-foss-2019b-fh1
• ml BCFtools/1.9-GCC-8.3.0
• ml Pysam/0.15.4-GCC-8.3.0-Python-3.7.4
• ml PyYAML/5.1.2-GCCcore-8.3.0-Python-3.7.4

Software packages or libraries (for users outside of Hutch)

Please see https://github.com/gavinha/TitanCNA/blob/edb1fd7bbdd273f8432c6167a0182d152f02dc7b/scripts/snakemake/README.md#software-packages-or-libraries

Requires to install ichorCNA from our GavinHaLab github https://github.com/GavinHaLab/ichorCNA

Requires to install TitanCNA https://github.com/gavinha/TitanCNA

Set-up

config/samples.yaml

Please specify the samples to be analyzed in config/samples.yaml, following the format explained therein.

config/config.yaml

There are a number of parameters to adjust in config/config.yaml. Filepaths to where your TitanCNA and ichorCNA repository as well as the filepath to tools (samTools, bcfTools, svaba) and readCounterScript.

Running the snakemake workflows on slurm cluster

snakemake -s TitanCNA.snakefile --latency-wait 60 --restart-times 3 --keep-going --cluster-config config/cluster_slurm.yaml --cluster "sbatch -p {cluster.partition} --mem={cluster.mem} -t {cluster.time} -c {cluster.ncpus} -n {cluster.ntasks} -o {cluster.output}" -j 30

snakemake -s svaba.snakefile --latency-wait 60 --cluster-config config/cluster_slurm.yaml --cluster "sbatch -p {cluster.partition} --mem={cluster.mem} -t {cluster.time} -c {cluster.ncpus} -n {cluster.ntasks} -o {cluster.output}" -j 30

snakemake -s combineSvabaTitan.snakefile --latency-wait 60 --keep-going --restart-times 3 --cluster-config config/cluster_slurm.yaml --cluster "sbatch -p {cluster.partition} --mem={cluster.mem} -t {cluster.time} -c {cluster.ncpus} -n {cluster.ntasks} -o {cluster.output}" -j 30

Whole Exome Sequencing Analysis

The tumor-only pipeline can be applied to whole exome sequencing (WES) data. This pipeline is applicable for 2 scenarios:

1. There is only a single normal sample that was processed and sequenced identically as the tumor samples of interest.
2. There are a set of normal samples that was processed and sequenced identically as the tumor samples. The normal samples may or may not be patient-matched to the tumor samples.

There are 3 main steps to set up this analysis.

1. Create a Panel Of Normals (PoN)

Make sure to use the updated version of the R script https://github.com/GavinHaLab/ichorCNA/blob/master/scripts/createPanelOfNormals.R

1. Create WIG Files

Create a WIG file for each sample in your PoN.

(Example) with 50kb bin size

/path/to/readCounter --window 50000 --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/normal.bam > /path/to/normal.wig

2. Generate PoN

Use the createPanelOfNormals.R script provided in the scripts directory of ichorCNA to generate your PoN.

As input, this script takes a file that has the path to each WIG file you'd like to use in your panel (one per line, no header).

(Example)

Rscript createPanelOfNormals.R 
     --filelist /path/to/wig_files.txt \
     --gcWig /path/to/gc.wig --mapWig /path/to/map.wig 
     --repTimeWig /path/to/repTiming.wig \
     --centromere /path/to/centromeres_file.txt \
     --exons.bed /path/to/WES_target.bed \
     --libdir /path/to/ichorCNA \
     --outfile my_new_pon

--filelist - file containing a list of the paths to all the normals in the panel to analyze

--gcWig - GC Wig file for reference genome (e.g. ichorCNA/inst/extdata/gc_hg38_50kb.wig)

--mapWig - Mappabiliy Wig file for reference genome (e.g. ichorCNA/inst/extdata/map_hg38_50kb.wig)

--repTimeWig - Rep Time Wig file for reference genome (e.g. ichorCNA/inst/extdata/Koren_repTiming_hg38_50kb.wig)

--centromere - File containing Centromere locations (e.g. GRCh38.GCA_000001405.2_centromere_acen.txt)

--exons.bed - Specify the exon target bed file

Must use gc/map/repTime wig file corresponding to same binSize matching to window size above (/path/to/readCounter --window).

2. Set config.yaml parameters

• Specify the exon target bed file

ichorCNA_exons: WES_target.bed

https://github.com/GavinHaLab/ichorCNA/blob/85c4339d7ced280d8e2113055f832911ea81cd08/scripts/snakemake/config/config.yaml#L20

• Specify the newly created PoN file

ichorCNA_normalPanel: my_new_pon.rds

https://github.com/GavinHaLab/ichorCNA/blob/85c4339d7ced280d8e2113055f832911ea81cd08/scripts/snakemake/config/config.yaml#L13

3. Use normalpanel parameter from ichorCNA.snakefile

• Uncomment following line

  TitanCNA_SV_WGS_tumorOnly/ichorCNA.snakefile

  Line 50 in 0032aae

  #normalpanel=config["ichorCNA_normalPanel"], #skip normal panel, not available for 10kb

• Make sure to add --normalPanel {params.normalpanel} to here

  TitanCNA_SV_WGS_tumorOnly/ichorCNA.snakefile

  Line 76 in 0032aae

  "Rscript {params.rscript} --id {params.id} --libdir {params.libdir} --WIG {input.tum} --gcWig {params.gcwig} --mapWig {params.mapwig} --repTimeWig {params.repTimeWig} --sex {params.sex} --ploidy \"{params.ploidy}\" --normal \"{params.normal}\" --maxCN {params.maxCN} --includeHOMD {params.includeHOMD} --chrs \"{params.chrs}\" --chrTrain \"{params.chrTrain}\" --genomeStyle {params.genomeStyle} --genomeBuild {params.genomeBuild} --estimateNormal {params.estimateNormal} --estimatePloidy {params.estimatePloidy} --estimateScPrevalence {params.estimateClonality} --scStates \"{params.scStates}\" --centromere {params.centromere} --exons.bed {params.exons} --txnE {params.txnE} --txnStrength {params.txnStrength} --minMapScore {params.minMapScore} --fracReadsInChrYForMale {params.fracReadsChrYMale} --maxFracGenomeSubclone {params.maxFracGenomeSubclone} --maxFracCNASubclone {params.maxFracCNASubclone} --plotFileType {params.plotFileType} --plotYLim \"{params.plotYlim}\" --outDir {params.outDir} > {log} 2> {log}"