call-gSNP

1. Overview
2. How To Run
3. Flow Diagram
4. Pipeline Steps
5. Inputs
6. Outputs
7. Discussions
8. Contributors
9. References

Overview

This pipeline takes BAMs and corresponding indices from recalibrate-BAM and runs through GATK4 best practice to call germline short variant (SNP and INDEL). It can be run with any combination of normal and tumor samples (normal only, tumor only, normal-tumor paired, multiple normal and tumor samples).

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How To Run

Requirements: Currently supported Nextflow versions: v23.04.2

The pipeline is currently configured to run on a SINGLE NODE mode with normal only, tumor only, normal-tumor paired, or multiple normal and tumor samples.

1. Update the params section of the .config file (Example config).

2. Update the YAML.

3. Download the submission script (submit_nextflow_pipeline.py) from here, and submit your pipeline below.

Note: Because this pipeline uses an image stored in the GitHub Container Registry, you must follow the steps listed in the Docker Introduction on Confluence to set up a PAT for your GitHub account and log into the registry on the cluster before running this pipeline.

• YAML input

python submit_nextflow_pipeline.py \
       --nextflow_script /path/to/main.nf \
       --nextflow_config /path/to/call-gSNP.config \
       --nextflow_yaml /path/to/sample.yaml \
       --pipeline_run_name job_name \
       --partition_type <type> \
       --email email_address

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Flow Diagram

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Pipeline Steps

1. Split genome or target intervals into sub-intervals for parallelization

Use the input target intervals or the whole genome intervals and split them into sub-intervals for parallel processing.

2. HaplotypeCaller

Generate VCF for each split interval using HaplotypeCaller. Generate GVCF for SNPs and INDELs.

3. Merge raw VCFs and GVCFs

Merge raw variants from each interval.

4. VQSR - SNPs

Generate VQSR (Variant Quality Score Recalibration) model for SNPs.

5. VQSR - INDELs

Generate VQSR model for INDELs.

6. VQSR - Apply SNP model

Take the whole sample raw VCF from Step 3 as input, and apply the model in Step 4 to generate variants in which only SNPs are recalibrated.

7. VQSR Apply INDEL model

Take the output from Step 6 as input, and apply the model in Step 5 to recalibrate only INDELs.

Steps 4 through 7 model the technical profile of variants in a training set and uses that to filter out probable artifacts from the raw VCF. After these four steps, a recalibrated VCF is generated.

8. Filter gSNP – Filter out ambiguous variants

Use customized Perl script to filter out ambiguous variants.

9. Generate sha512 checksum

Generate sha512 checksum for VCFs and GVCFs.

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Inputs

Input YAML

Field	Type	Description
patient_id	string	Patient ID (will be standardized according to data storage structure in the near future)
normal_BAM	path	Set to absolute path to normal BAM
tumor_BAM	path	Set to absolute path to tumor BAM

---
patient_id: "patient_id"
input:
  BAM:
    normal:
      - "/absolute/path/to/BAM"
      - "/absolute/path/to/BAM"
    tumor:
      - "/absolute/path/to/BAM"
      - "/absolute/path/to/BAM"

For normal-only or tumor-only samples, exclude the fields for the other state.

Config

Input Parameter	Required	Type	Description
dataset_id	Yes	string	Dataset ID
blcds_registered_dataset	Yes	boolean	Set to true when using BLCDS folder structure; use false for now
output_dir	Yes	string	Need to set if blcds_registered_dataset = false
save_intermediate_files	Yes	boolean	Set to false to disable publishing of intermediate files; true otherwise; disabling option will delete intermediate files to allow for processing of large BAMs
cache_intermediate_pipeline_steps	No	boolean	Set to true to enable process caching from Nextflow; defaults to false
scatter_count	Yes	integer	Number of intervals to divide into for parallelization
intervals	Yes	path	Use all .list in inputs for WGS; Set to absolute path to targeted exome interval file (with .interval_list, .list, .intervals, or .bed suffix)
reference_fasta	Yes	path	Absolute path to reference genome fasta file, e.g., /hot/resource/reference-genome/GRCh38-BI-20160721/Homo_sapiens_assembly38.fasta
bundle_mills_and_1000g_gold_standard_indels_vcf_gz	Yes	path	Absolute path to Mills & 1000G Gold Standard Indels file, e.g., /hot/resource/tool-specific-input/GATK/GRCh38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
bundle_v0_dbsnp138_vcf_gz	Yes	path	Absolute path to dbsnp file, e.g., /hot/resource/tool-specific-input/GATK/GRCh38/resources_broad_hg38_v0_Homo_sapiens_assembly38.dbsnp138.vcf.gz
bundle_hapmap_3p3_vcf_gz	Yes	path	Absolute path to HapMap 3.3 file, e.g., /hot/resource/tool-specific-input/GATK/GRCh38/hapmap_3.3.hg38.vcf.gz
bundle_omni_1000g_2p5_vcf_gz	Yes	path	Absolute path to 1000 genomes OMNI 2.5 file, e.g., /hot/resource/tool-specific-input/GATK/GRCh38/1000G_omni2.5.hg38.vcf.gz
bundle_phase1_1000g_snps_high_conf_vcf_gz	Yes	path	Absolute path to 1000 genomes phase 1 high-confidence file, e.g., /hot/resource/tool-specific-input/GATK/GRCh38/1000G_phase1.snps.high_confidence.hg38.vcf.gz
work_dir	optional	path	Path of working directory for Nextflow. When included in the sample config file, Nextflow intermediate files and logs will be saved to this directory. With ucla_cds, the default is /scratch and should only be changed for testing/development. Changing this directory to /hot or /tmp can lead to high server latency and potential disk space limitations, respectively.
docker_container_registry	optional	string	Registry containing tool Docker images. Default: ghcr.io/uclahs-cds
base_resource_update	optional	namespace	Namespace of parameters to update base resource allocations in the pipeline. Usage and structure are detailed in template.config and below.

Base resource allocation updaters

To update the base resource (cpus or memory) allocations for processes, use the following structure and add the necessary parts. The default allocations can be found in the node-specific config files

base_resource_update {
    memory = [
        [['process_name', 'process_name2'], <multiplier for resource>],
        [['process_name3', 'process_name4'], <different multiplier for resource>]
    ]
    cpus = [
        [['process_name', 'process_name2'], <multiplier for resource>],
        [['process_name3', 'process_name4'], <different multiplier for resource>]
    ]
}

Note Resource updates will be applied in the order they're provided so if a process is included twice in the memory list, it will be updated twice in the order it's given.

Examples:

• To double memory of all processes:

base_resource_update {
    memory = [
        [[], 2]
    ]
}

• To double memory for run_ApplyVQSR_GATK and triple memory for run_validate_PipeVal and run_HaplotypeCallerVCF_GATK:

base_resource_update {
    memory = [
        ['run_ApplyVQSR_GATK', 2],
        [['run_validate_PipeVal', 'run_HaplotypeCallerVCF_GATK'], 3]
    ]
}

• To double CPUs and memory for run_ApplyVQSR_GATK and double memory for run_validate_PipeVal:

base_resource_update {
    cpus = [
        ['run_ApplyVQSR_GATK', 2]
    ]
    memory = [
        [['run_ApplyVQSR_GATK', 'run_validate_PipeVal'], 2]
    ]
}

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Outputs

Output	Description
<GATK>_<dataset_id>_<sample_id>.g.vcf.gz	Per-sample GVCF
<GATK>_<dataset_id>_<sample_id>.g.vcf.gz.sha512	Per-sample GVCF checksum
<GATK>_<dataset_id>_<sample_id>.g.vcf.gz.tbi	Per-sample GVCF index
<GATK>_<dataset_id>_<sample_id>.g.vcf.gz.tbi.sha512	Per-sample GVCF index checksum
<GATK>_<dataset_id>_<patient_id>.vcf	Raw variant calls
<GATK>_<dataset_id>_<patient_id>.vcf.idx	Raw variant calls index
<GATK>_<dataset_id>_<patient_id>_VQSR-SNP-AND_INDEL.vcf.gz	SNP and INDEL recalibrated variants
<GATK>_<dataset_id>_<patient_id>_VQSR-SNP-AND_INDEL.vcf.gz.sha512	SNP and INDEL recalibrated variants checksum
<GATK>_<dataset_id>_<patient_id>_VQSR-SNP-AND_INDEL.vcf.gz.tbi	SNP and INDEL recalibrated variants index
<GATK>_<dataset_id>_<patient_id>_VQSR-SNP-AND_INDEL.vcf.gz.tbi.sha512	SNP and INDEL recalibrated variants index checksum
<GATK>_<dataset_id>_<patient_id>_snv.vcf.gz	Filtered SNVs with non-germline and ambiguous variants removed
<GATK>_<dataset_id>_<patient_id>_snv.vcf.gz.tbi	Filtered germline SNVs index
<GATK>_<dataset_id>_<patient_id>_snv.vcf.gz.sha512	Filtered germline SNVs sha512 checksum
<GATK>_<dataset_id>_<patient_id>_indel.vcf.gz	Filtered INDELs with non-germline and ambiguous variants removed
<GATK>_<dataset_id>_<patient_id>_indel.vcf.gz.tbi	Filtered germline INDELs index
<GATK>_<dataset_id>_<patient_id>_indel.vcf.gz.sha512	Filtered germline INDELs sha512 checksum
report.html, timeline.html and trace.txt	Nextflow report, timeline and trace files
*.command.*	Process specific logging files created by nextflow

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Discussions

• Issue tracker to report errors and enhancement ideas.
• Discussions can take place in call-gSNP Discussions
• call-gSNP pull requests are also open for discussion

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Contributors

Please see list of Contributors at GitHub.

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References

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License

Authors: Yash Patel (YashPatel@mednet.ucla.edu), Shu Tao (shutao@mednet.ucla.edu), Stefan Eng (stefaneng@mednet.ucla.edu)

Call-gSNP is licensed under the GNU General Public License version 2. See the file LICENSE for the terms of the GNU GPL license.

Call-gSNP takes BAM files and utilizes GATK to call short germline variants (SNP and INDEL).

Copyright (C) 2021-2023 University of California Los Angeles ("Boutros Lab") All rights reserved.

This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or (at your option) any later version.

This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.