recalibrate-BAM

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 pipeline-align-DNA and performs indel realignment and BQSR. 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: 23.04.2

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 (Template YAMLs).

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

• YAML input

python submit_nextflow_pipeline.py \
       --nextflow_script /path/to/main.nf \
       --nextflow_config /path/to/call-recalibrate-bam.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 into sub-intervals for parallelization

Split the reference genome into intervals for parallel processing. If params.parallelize_by_chromosome is set then the genome will be split by chromosome, otherwise it will be split into up to params.scatter_count intervals.

2. Realign indels

Generate indel realignment targets and realign indels per interval.

3. Generate BQSR (Base Quality Score Recalibration)

Assess how sequencing errors correlate with four covariates (assigned quality score, read group, machine cycle producing this base, and current and immediately upstream base) and output base quality score recalibration table.

4. Apply BQSR per split interval in parallel

Apply the base quality score recalibration to each interval and reheader output as necessary.

5. Merge interval-level BAMs

Merge BAMs from each interval to generate a whole sample BAM.

5a. Deduplicate BAM

If params.parallelize_by_chromosome is not set, run a deduplication process to remove reads duplicated due to overlap on interval splitting sites.

6. Index BAM file

Generate a BAI index file for fast random access of the whole sample BAM.

7. Get pileup summaries

Tabulate pileup metrics for inferring contamination. Summarize counts of reads that support reference, alternate and other alleles for given sites.

8. Calculate contamination

Calculates the fraction of reads coming from cross-sample contamination, given results from Step 7. For paired samples, generates an additional output table containing segmentation of the tumor by minor allele fraction.

9. DepthOfCoverage

If params.is_DOC_run is set, generate coverage summary information for the whole sample BAM from step 5, partitioned by sample, read group, and library.

10. Generate sha512 checksum

Generate sha512 checksum for final BAM and BAI files.

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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 tumour BAM
recalibration_table	path	(Optional) Absolute path to recalibration table

Input without pre-existing recalibration table(s):

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

Input with existing recalibration table(s):

---
patient_id: "patient_id"
input:
  BAM:
    normal:
      - "/absolute/path/to/BAM"
      - "/absolute/path/to/BAM"
    tumor:
      - "/absolute/path/to/BAM"
      - "/absolute/path/to/BAM"
  recalibration_table:
    - "/absolute/path/to/recalibration/table1"
    - "/absolute/path/to/recalibration/table2"

For normal-only or tumour-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
aligner	Yes	string	Original aligner used to align input BAMs; formatted as <aligner>-<aligner-version>
cache_intermediate_pipeline_steps	No	boolean	Set to true to enable process caching from Nextflow; defaults to false
is_emit_original_quals	Yes	boolean	Set to true to emit original quality scores; false to omit
is_DOC_run	Yes	boolean	Set to true to run GATK DepthOfCoverage (very time-consuming for large BAMs); false otherwise
parallelize_by_chromosome	Yes	boolean	Whether the parallelize by chromosome or by scattering intervals
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)
gatk_ir_compression	No	integer	Compression level for BAMs output by IndelRealigner. Default: 0. Range: 0-9
reference_fasta	Yes	path	Absolute path to reference genome fasta file, e.g., /hot/ref/reference/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/ref/tool-specific-input/GATK/GRCh38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
bundle_known_indels_vcf_gz	Yes	path	Absolute path to known indels file, e.g., /hot/ref/tool-specific-input/GATK/GRCh38/Homo_sapiens_assembly38.known_indels.vcf.gz
bundle_v0_dbsnp138_vcf_gz	Yes	path	Absolute path to dbsnp file, e.g., /hot/ref/tool-specific-input/GATK/GRCh38/resources_broad_hg38_v0_Homo_sapiens_assembly38.dbsnp138.vcf.gz
bundle_contest_hapmap_3p3_vcf_gz	Yes	path	Absolute path to HapMap 3.3 biallelic sites file, e.g., /hot/ref/tool-specific-input/GATK/GRCh38/Biallelic/hapmap_3.3.hg38.BIALLELIC.PASS.2021-09-01.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.
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.

The below parameters have default values defined in default.config and generally do not need to be set by the user.

Optional Parameter	Type	Description
metapipeline_delete_input_bams	boolean	Set to true to delete the input BAM files once the initial processing step is complete. WARNING: This option should NOT be used for individual runs of recalibate-BAM; it's intended for metapipeline-DNA to optimize disk space usage by removing files that are no longer needed from the workDir.
metapipeline_final_output_dir	string	Absolute path for the final output directory of metapipeline-DNA that's expected to contain the output BAM from align-DNA. WARNING: This option should not be used for individual runs of recalibrate-BAM; it's intended for metapipeline-DNA to optimize disk space usage.
metapipeline_states_to_delete	list	List of states for which to delete input BAMs. WARNING: This option should not be used for individual runs of recalibrate-BAM; it's intended for metapipeline-DNA to optimize disk space usage.
cache_intermediate_pipeline_steps	boolean	Enable process caching from Nextflow.
ucla_cds	boolean	Overwrite default memory and CPU values by cluster-specific configs.
docker_container_registry	string	Registry containing tool Docker images.
docker_image_gatk, gatk_version	string	Docker image name and version for GATK.
docker_image_pipeval, pipeval_version	string	Docker image name and version for PipeVal.
docker_image_gatk3, gatk3_version	string	Docker image name and version for GATK3.
docker_image_picard, picard_version	string	Docker image name and version for Picard.
docker_image_samtools, samtools_version	string	Docker image name and version for SAMtools.
reference_fasta_fai, reference_fasta_dict	path	Index and dictionary files for the required input. Default: Matching .fai and .dict files in the same directory.
bundle_v0_dbsnp138_vcf_gz_tbi	path	Index file for the required input. Default: Matching .tbi file in the same directory.
bundle_known_indels_vcf_gz_tbi	path	Index file for the required input. Default: Matching .tbi file in the same directory.
bundle_contest_hapmap_3p3_vcf_gz_tbi	path	Index file for the required input. Default: Matching .tbi file in the same directory.
bundle_mills_and_1000g_gold_standard_indels_vcf_gz_tbi	path	Index file for the required input. Default: Matching .tbi file in the same directory.

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_ApplyBQSR_GATK and triple memory for run_validate_PipeVal and run_IndelRealigner_GATK:

base_resource_update {
    memory = [
        ['run_ApplyBQSR_GATK', 2],
        [['run_validate_PipeVal', 'run_IndelRealigner_GATK'], 3]
    ]
}

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

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

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Outputs

Output	Description
<aligner>_<GATK>_<dataset_id>_<sample_id>.bam	Post-processes BAM
<aligner>_<GATK>_<dataset_id>_<sample_id>.bam.sha512	Post-processes BAM SHA512 checksum
<aligner>_<GATK>_<dataset_id>_<sample_id>.bam.bai	Post-processes BAM index
<aligner>_<GATK>_<dataset_id>_<sample_id>.bam.bai.sha512	Post-processes BAM index 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 recalibrate-BAM Discussions
• recalibrate-BAM pull requests are also open for discussion

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Contributors

Update link to repo-specific URL for GitHub Insights Contributors page.

Please see list of Contributors at GitHub.

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References

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License

Authors: Yash Patel ([email protected]), Shu Tao ([email protected]), Stefan Eng ([email protected])

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

Recalibrate-BAM takes BAM files and utilizes GATK to perform indel realignment and BQSR.

Copyright (C) 2023-2024 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.