Pipeline to verify BAM/CRAM files from WES/WGS

Requirements

1. Download and compile VerifyBamId2.
2. Verify that you are running Python 3 and the following packages are installed: argparse, pandas, matplotlib, fpdf.
3. Verify that you have samtools installed.

Running

1. Clone this repository to the directory where you will run the pipeline:

   git clone https://github.com/CERC-Genomic-Medicine/verifybam_pipeline.git
   

2. Modify nextflow.config configuration file.

   • params.bams -- path to your BAM/CRAM file(s). You can use glob expressions to selecect multiple files.
   • params.targets_bed -- path to the WES target BED file. Used only when analyzing WES BAM/CRAM.
   • params.fasta -- path to the genome reference FASTA file.
   • params.genome_build -- set to "b37" or "b38".
   • params.verifyBamId_dir -- path to the VerifyBamID directory.
   • params.reported_sex -- path to the file with the reported sex. It is a tab-delimied file with two columns named: "NAME", "SEX". The "NAME" column stores the sample name/id. The sample name/id must match the BAM/CRAM filename without suffixes. For example, if the BAM file name is "Sample123.run2.phase1.bam", then the sample will be referred to as "Sample123". The "SEX" column stores self-reported sex values encoded as: F - female, M - male, NA - missing.
   • executor.$slurm.queueSize -- maximal number of SLURM jobs to submit at once.

3. Run pipeline.

   WGS:

   module load nextflow
   module load samtools
   nextflow run Verify.nf -w ~/scratch/work_directory
   

   WES:

   module load nextflow
   module load samtools
   nextflow run Verify_wes.nf -w ~/scratch/work_directory
   

   Important: when working on Compute Canada HPC, set working directory to ~/scratch/<new directory name>. This will speed up IO and also save space on your project partition. After the execution, if there were no errors and you are happy with the results, you can remove this working directory.

Results

Results will saved in the results directory:

• results/report.pdf -- PDF file with all plots.
• results/summary.txt -- Space-delimited table with all the results, which was used to generate PDF.
• results/plots/ -- All plots in JPEG format from PDF.
• results/verifyBamId_logs/ -- All log files from the verifyBamId tool.
• results/dp/ -- Files with averagte DP values by chromosome for each sample. These files were used to generate summary.txt.

Below you can find description of the summary.txt columns:

Column	Description
NAME	Sample name, which is equal to the corresponding BAM/CRAM name without a suffix.
HGDP_SNPS_OVERLAP	number of SNPs used by VerifyBamId for estimating contamination using the HGDP reference panel. This columns is not present when analyzing WES data.
HGDP_SNPS	number of SNPs within the HGDP reference panel. This columns is not present when analyzing WES data.
HGDP_AVG_DP	average read depth at SNPs used by VerifyBamId for estimating contamination using the HGDP reference panel. This columns is not present when analyzing WES data.
HGDP_FREEMIX	VerifyBamId estimated contamination using the HGDP reference panel. This columns is not present when analyzing WES data.
HGDP_PC1_CONTAMINATING	Contaminating sample's PC1 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
HGDP_PC1_INTENDED	Intended sample's PC1 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
HGDP_PC2_CONTAMINATING	Contaminating sample's PC2 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
HGDP_PC2_INTENDED	Intended sample's PC2 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
HGDP_PC3_CONTAMINATING	Contaminating sample's PC3 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
HGDP_PC3_INTENDED	Intended sample's PC3 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
HGDP_PC4_CONTAMINATING	Contaminating sample's PC4 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
HGDP_PC4_INTENDED	Intended sample's PC4 projected to the HGDP PCA space. This columns is not present when analyzing WES data.
1000G_SNPS_OVERLAP	number of SNPs used by VerifyBamId for estimating contamination using the 1000G reference panel.
1000G_SNPS	number of SNPs within the 1000G reference panel.
1000G_AVG_DP	average read depth at SNPs used by VerifyBamId for estimating contamination using the 1000G reference panel.
1000G_FREEMIX	VerifyBamId estimated contamination using the 1000G reference panel.
1000G_PC1_CONTAMINATING	Contaminating sample's PC1 projected to the 1000G PCA space.
1000G_PC1_INTENDED	Intended sample's PC1 projected to the 1000G PCA space.
1000G_PC2_CONTAMINATING	Contaminating sample's PC2 projected to the 1000G PCA space.
1000G_PC2_INTENDED	Intended sample's PC2 projected to the 1000G PCA space.
1000G_PC3_CONTAMINATING	Contaminating sample's PC3 projected to the 1000G PCA space.
1000G_PC3_INTENDED	Intended sample's PC3 projected to the 1000G PCA space.
1000G_PC4_CONTAMINATING	Contaminating sample's PC4 projected to the 1000G PCA space.
1000G_PC4_INTENDED	Intended sample's PC4 projected to the 1000G PCA space.
ALL_CHROMS	Total number of chromosomes.
ALL_N_BASES	Total number of bases across all chromosomes.
ALL_PRCNT_COVERED	Percent of covered bases across all chromosomes.
ALL_PRCNT_DP1	Percent of bases covered at DP>=1 across all chromosomes.
ALL_PRCNT_DP2	Percent of bases covered at DP>=2 across all chromosomes.
ALL_PRCNT_DP3	Percent of bases covered at DP>=3 across all chromosomes.
ALL_PRCNT_DP4	Percent of bases covered at DP>=4 across all chromosomes.
ALL_PRCNT_DP5	Percent of bases covered at DP>=5 across all chromosomes.
ALL_PRCNT_DP10	Percent of bases covered at DP>=10 across all chromosomes.
ALL_PRCNT_DP20	Percent of bases covered at DP>=20 across all chromosomes.
ALL_PRCNT_DP30	Percent of bases covered at DP>=30 across all chromosomes.
ALL_PRCNT_DP40	Percent of bases covered at DP>=40 across all chromosomes.
ALL_PRCNT_DP50	Percent of bases covered at DP>=50 across all chromosomes.
ALL_PRCNT_DP60	Percent of bases covered at DP>=60 across all chromosomes.
ALL_PRCNT_DP70	Percent of bases covered at DP>=70 across all chromosomes.
ALL_PRCNT_DP80	Percent of bases covered at DP>=80 across all chromosomes.
ALL_PRCNT_DP90	Percent of bases covered at DP>=90 across all chromosomes.
ALL_PRCNT_DP100	Percent of bases covered at DP>=100 across all chromosomes.
ALL_PRCNT_DP110	Percent of bases covered at DP>=110 across all chromosomes.
ALL_AVG_DP	Average depth across all chromosomes.
AUTO_CHROMS	Total number of autosomal chromosomes.
AUTO_N_BASES	Total number of bases across autosomal chromosomes.
AUTO_PRCNT_COVERED	Percent of covered bases across autosomal chromosomes.
AUTO_PRCNT_DP1	Percent of bases covered at DP>=1 across autosomal chromosomes.
AUTO_PRCNT_DP2	Percent of bases covered at DP>=2 across autosomal chromosomes.
AUTO_PRCNT_DP3	Percent of bases covered at DP>=3 across autosomal chromosomes.
AUTO_PRCNT_DP4	Percent of bases covered at DP>=4 across autosomal chromosomes.
AUTO_PRCNT_DP5	Percent of bases covered at DP>=5 across autosomal chromosomes.
AUTO_PRCNT_DP10	Percent of bases covered at DP>=10 across autosomal chromosomes.
AUTO_PRCNT_DP20	Percent of bases covered at DP>=20 across autosomal chromosomes.
AUTO_PRCNT_DP30	Percent of bases covered at DP>=30 across autosomal chromosomes.
AUTO_PRCNT_DP40	Percent of bases covered at DP>=40 across autosomal chromosomes.
AUTO_PRCNT_DP50	Percent of bases covered at DP>=50 across autosomal chromosomes.
AUTO_PRCNT_DP60	Percent of bases covered at DP>=60 across autosomal chromosomes.
AUTO_PRCNT_DP70	Percent of bases covered at DP>=70 across autosomal chromosomes.
AUTO_PRCNT_DP80	Percent of bases covered at DP>=80 across autosomal chromosomes.
AUTO_PRCNT_DP90	Percent of bases covered at DP>=90 across autosomal chromosomes.
AUTO_PRCNT_DP100	Percent of bases covered at DP>=100 across autosomal chromosomes.
AUTO_PRCNT_DP110	Percent of bases covered at DP>=110 across autosomal chromosomes.
AUTO_AVG_DP	Average depth across autosomal chromosomes.
X	1 - X chromose is present, 0 - otherwise.
X_AVG_DP	average read depth for X chromosome.
X_NORM_AVG_DP	average read depth for X chromosome normalized by the average read depth across autosomal chromosomes.
Y	1 - Y chromosome is present, 0 - othewise.
Y_AVG_DP	average read depth for Y chromosome.
Y_NORM_AVG_DP	average read depth for Y chromosome normalized by the average read depth across autosomal chromosomes.