A collection of tools for the analysis of CpG/5mC data from PacBio HiFi reads aligned to a reference genome (e.g., an aligned BAM). To use these tools, the HiFi reads should already contain 5mC base modification tags, generated on-instrument or by using primrose. The aligned BAM should also be sorted and indexed.

• pb-CpG-tools Github

• Module Name: pb-cpg-tools (see the modules page for more information)
• Multithreaded
• Environment variables set
  • CPG_TEST_DATA
  • CPG_PILEUP_MODEL
• Test data files in $CPG_TEST_DATA
• The models distributed with the git repository can be copied from $CPG_PILEUP_MODEL directory

Allocate an interactive session and run the program.
Sample session (user input in bold):

[user@biowulf]$ sinteractive --mem=32G -c8 --gres=lscratch:20
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144 ~]$ module load pb-cpg-tools

[user@cn3144 ~]$ cd /lscratch/$SLURM_JOB_ID

[user@cn3144 ~]$ cp $CPG_TEST_DATA/*bam* .

[user@cn3144 ~]$ aligned_bam_to_cpg_scores \
                --bam HG002.GRCh38.haplotagged.truncated.bam \
                --output-prefix test \
                --model $CPG_PILEUP_MODEL/pileup_calling_model.v1.tflite \
                --threads $SLURM_CPUS_PER_TASK

[2023-07-24][14:31:38][aligned_bam_to_cpg_scores][INFO] Starting aligned_bam_to_cpg_scores
[2023-07-24][14:31:38][aligned_bam_to_cpg_scores][INFO] cmdline: aligned_bam_to_cpg_scores --bam HG002.GRCh38.haplotagged.truncated.bam --output-prefix test --model /usr/local/apps/pb-cpg-tools/2.3.1/models/pileup_calling_model.v1.tflite --threads 8
[2023-07-24][14:31:38][aligned_bam_to_cpg_scores][INFO] Running on 8 threads
[2023-07-24][14:31:38][aligned_bam_to_cpg_scores][INFO] Processing alignment file 'HG002.GRCh38.haplotagged.truncated.bam'
[2023-07-24][14:32:16][aligned_bam_to_cpg_scores][INFO] Finished processing alignment files.
[2023-07-24][14:32:16][aligned_bam_to_cpg_scores][INFO] Writing hap2 site methylation to bed file: 'test.hap2.bed'
[2023-07-24][14:32:16][aligned_bam_to_cpg_scores][INFO] Writing hap1 site methylation to bed file: 'test.hap1.bed'
[2023-07-24][14:32:16][aligned_bam_to_cpg_scores][INFO] Writing combined site methylation to bed file: 'test.combined.bed'
[2023-07-24][14:32:16][aligned_bam_to_cpg_scores][INFO] Writing combined site methylation to bigwig file: 'test.combined.bw'
[2023-07-24][14:32:16][aligned_bam_to_cpg_scores][INFO] Writing hap2 site methylation to bigwig file: 'test.hap2.bw'
[2023-07-24][14:32:16][aligned_bam_to_cpg_scores][INFO] Writing hap1 site methylation to bigwig file: 'test.hap1.bw'
[2023-07-24][14:32:17][aligned_bam_to_cpg_scores][INFO] aligned_bam_to_cpg_scores completed. Total Runtime: 00:00:39.426

[user@cn3144 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

Create a batch input file (e.g. pb-cpg-tools.sh). For example:

#!/bin/bash
set -e
module load pb-cpg-tools
cd /lscratch/$SLURM_JOB_ID
cp $CPG_TEST_DATA/*bam* .
aligned_bam_to_cpg_scores \
            --bam HG002.GRCh38.haplotagged.truncated.bam \
            --output-prefix test \
            --model $CPG_PILEUP_MODEL/pileup_calling_model.v1.tflite \
            --threads $SLURM_CPUS_PER_TASK

Submit this job using the Slurm sbatch command.

sbatch --mem=32g --cpus-per-task=8 --gres=lscratch:20  pb-cpg-tools.sh

Create a swarmfile (e.g. pb-cpg-tools.swarm). For example:

aligned_bam_to_cpg_scores --bam input1.bam --output-prefix out1 --model v1.tflite --threads $SLURM_CPUS_PER_TASK
aligned_bam_to_cpg_scores --bam input2.bam --output-prefix out2 --model v1.tflite --threads $SLURM_CPUS_PER_TASK
aligned_bam_to_cpg_scores --bam input3.bam --output-prefix out3 --model v1.tflite --threads $SLURM_CPUS_PER_TASK
aligned_bam_to_cpg_scores --bam input4.bam --output-prefix out4 --model v1.tflite --threads $SLURM_CPUS_PER_TASK

Submit this job using the swarm command.

swarm -f pb-cpg-tools.swarm [-g #] [-t #] [--gres=lscratch:#] --module pb-cpg-tools