Add mutect2 force calling using '--alleles' flag #1952

jchchiu · 2025-08-11T09:03:11Z

PR checklist

Regarding issue #1883 I have:

Added two params (mutect2_force_call, mutect2_force_call_tbi) to supply a path to a vcf.gz file and associated index file for force calling.
Added Channel to collect .vcf.gz; added Channel to collect .vcf.gz tbi (if not found will create a tbi file); added params to main workflow pipeline.
Added params to pipeline initialisation (raise an error if mutect2_force_call file format not in .vcf.gz format.
Added TABIX creation for mutect2_force_call if user does not supply it.
Added params to the [somatic/tumor_only]_[ALL/mutect2] subworkflows.
Added params to GATK4 mutect2 module; created new command in script for force calling (mfc_command) and added to Mutect2 execution.
Temporarily edited some tests configs in tools to test for the force call parameter (also tests if TABIX creation works if associated index file not supplied).

Some questions:

Regarding tests:
- For the test data I'm just thinking of creating a VCF with a few random alleles to be used to force call. Is there any other data you prefer?
- I created test_force_call.vcf.gz which works for both tumor only and somatic mode:

##fileformat=VCFv4.2
##contig=<ID=chr21,length=46709983>
#CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO
chr21	46664275	.	A	C	.	.	.
chr21	46664280	.	A	C	.	.	.
chr21	46664285	.	A	G	.	.	.
chr21	46664290	.	T	A	.	.	.

In the Mutect2 docs there is another boolean --genotype-filtered-alleles (or maybe it's now --force-call-filtered-alleles) flag that can be used in conjunction with --alleles. Is it worth it to also implement this?

jchchiu · 2025-08-25T05:25:46Z

Also, while I was editing ./subworkflows/local/samplesheet_to_channel/main.nf, I had a small question about bcftools annotate here.

// Fails when bcftools annotate is used but no files are supplied
if (tools && tools.split(',').contains('bcfann') && !(bcftools_annotations && bcftools_annotations_tbi && bcftools_header_lines)) {
    error("Please specify --bcftools_annotations, --bcftools_annotations_tbi, and --bcftools_header_lines, when using BCFTools annotations")
}

In the ./main.nf NFCORE_SAREK workflow here, the PREPARE_GENOME workflow is called for bcftools_annotation to create a bcftools_annotation_tbi file if it doesn’t exist.
However, in the main workflow here the PIPELINE_INITIALISATION workflow (and thus, ./subworkflows/local/samplesheet_to_channel/main.nf) is called before this to create a samplesheet to be passed into the NFCORE_SAREK workflow.
Thus, if a user has to has to specify a bcftools_annotations_tbi file in the first place anyway for the pipeline to run, this makes the TABIX call for bcftools_annotation redundant.

Just a nitpick, but should either the !bcftools_annotations_tbi requirement, or the code for the TABIX creation of bcftools_annotation be removed?

If this is of interest and doesn't disrupt too much, I can open another PR with one of these updates.

FriederikeHanssen

This looks good already. I added some comments on where changes should be handeled instead.

What we are missing are updates to the output documentation of Mutect2.

FriederikeHanssen · 2025-08-27T06:49:47Z

conf/test/tools_tumoronly.config

    pon_tbi                = "${params.modules_testdata_base_path}/genomics/homo_sapiens/genome/chr21/germlineresources/mills_and_1000G.indels.hg38.vcf.gz.tbi"
    ngscheckmate_bed       = "${params.modules_testdata_base_path}/genomics/homo_sapiens/genome/chr21/germlineresources/SNP_GRCh38_hg38_wChr.bed"
+    mutect2_force_call     = "${projectDir}/testing/test_force_call.vcf.gz"
+//    mutect2_force_call_tbi = "${projectDir}/testing/test_force_call.vcf.gz.tbi"


we should proabbly have one test that checks things work with a supplied tbi file

modules/nf-core/gatk4/mutect2/main.nf

subworkflows/local/samplesheet_to_channel/main.nf

…s flag. User supplies path to vcf.gz file and associated index file. (Ref: Issue nf-core#1883)

…not supply it

…e not in 'vcf.gz' format

…gatk4/mutect2 module

jchchiu · 2025-09-15T06:54:19Z

Following remove validation error; handled by schema, I have:

Removed validation error for samplesheets (handles in schema) and removed temporary tests
Updated the gatk4/mutect module using nf-core and any associated subworkflows
Updated output documentation and README
Added a new test config specific to mutect2 to test the parameter, but unsure if this is the best way to add a test for it (maybe change the test to tumoronly?). As of Add config for readgroups to fq2bam #1939, I've been running with nextflow run . -profile test,tools_somatic_mutect2,mutect,docker --tools mutect2 --outdir results -step variant_calling

Sorry I also messed up the commit history, should have merged instead of rebased. The previous comments are up to:
temp: added param to config tests; testing TABIX creation

docs/output.md

maxulysse · 2025-09-17T06:57:09Z

conf/test/tools_somatic_mutect2.config

Can't we add the params we need to the tests?

Hey, I've tried two ways to do this, could you have a look at which one you prefer?

jchchiu#2: Is what makes most sense to me by adding test scenarios and updating snap; is this what you had in mind?

jchchiu#3: Just updates the somatic and tumor_only configs to include the parameter, but I believe this changes all mutect2 tests to always test with the force_call parameter which is not very good.

2nd is definitvely best for me

main.nf

subworkflows/local/bam_variant_calling_tumor_only_all/main.nf

subworkflows/local/prepare_genome/main.nf

…lling

Test modifcations by changing tests

…lling

nf-core-bot · 2025-11-12T08:14:10Z

Warning

Newer version of the nf-core template is available.

Your pipeline is using an old version of the nf-core template: 3.3.2.
Please update your pipeline to the latest version.

For more documentation on how to update your pipeline, please see the nf-core documentation and Synchronisation documentation.

…lict raised

…nchanged

… files_unchanged" This reverts commit a1881cf.

jchchiu · 2025-11-13T03:01:10Z

@nf-core-bot fix linting

jchchiu · 2025-11-13T05:53:47Z

Heya, I've seen that 'Prepare genome refactor #1979' has been merged so I've updated my PR.

The linting error is from the updated mutect2 module; haven't updated the module because of the extra ', path(gzi)' in the updated module that I'm guessing should be updated in a different PR
I think everything should be good, just wondering whether there are any issues with the new snapshots for mutect2 using newer nf-test and nextflow versions? ( "nf-test": "0.9.3", "nextflow": "25.10.0" vs "nf-test": "0.9.2", "nextflow": "25.04.6")
Also want to confirm that there is no need for a mutect2_force_call input in the SAMPLESHEET_TO_CHANNEL script; if so I'll also do another quick PR after to update Add mutect2 force calling using '--alleles' flag #1952 (comment) (also removing the bcftools_annotations_tbi // Path: bcftools annotations tbi input since it'll no longer be used in the script)

jchchiu requested review from FriederikeHanssen and maxulysse as code owners August 11, 2025 09:03

jchchiu marked this pull request as draft August 11, 2025 10:58

jchchiu marked this pull request as ready for review August 27, 2025 03:24

FriederikeHanssen reviewed Aug 27, 2025

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jchchiu mentioned this pull request Sep 8, 2025

Update gatk4 mutect2 to add --alleles parameter for force calling nf-core/modules#9020

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17 tasks

jchchiu added 12 commits September 15, 2025 15:28

Added params for force-calling alleles in GATK mutect2 using --allele…

074310f

…s flag. User supplies path to vcf.gz file and associated index file. (Ref: Issue nf-core#1883)

change param name from mutect2_force_alleles to mutect2_force_call

6efed5a

added TABIX creation for mutect2_force_call vcf.gz file if user does …

5a08b97

…not supply it

added param to pipeline intitalisation; raise error if force-call fil…

427ff80

…e not in 'vcf.gz' format

fix param call order in tumor only mode

8636c28

temp: added param to config tests; testing TABIX creation

8956c63

remove validation error; handled by schema

7620aa0

update module using nf-core modules update gatk4/mutect2

dbf1f0a

update subworkflows to match with renaming and reordering in updated …

32e14e0

…gatk4/mutect2 module

docs: updated documentation

bd5bd7d

removed temporary tests

ac92553

added new test config specific to mutect2 (?)

f1f88fb

jchchiu force-pushed the add_mutect2_force_calling branch from 6e49ded to f1f88fb Compare September 15, 2025 05:44

fix formatting

41853e3

fix lint

c7dfe7f

maxulysse reviewed Sep 17, 2025

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maxulysse reviewed Sep 17, 2025

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maxulysse reviewed Sep 17, 2025

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jchchiu added 2 commits September 17, 2025 19:04

Merge remote-tracking branch 'upstream/dev' into add_mutect2_force_ca…

3cf28c1

…lling

Merge remote-tracking branch 'upstream/dev' into add_mutect2_force_ca…

cd2f06c

…lling

fix lint; fix naming order

dcb320d

jchchiu force-pushed the add_mutect2_force_calling branch from d1daaf8 to dcb320d Compare September 17, 2025 09:15

jchchiu and others added 10 commits September 17, 2025 19:19

update docs/output

f58e77a

fix ordering of prepare genome

1a45fd9

fix order of prepare genomes versions

6a5c74d

add tests

83fffb1

remove params from tools configs

cd9465e

update snap

aa54834

fix formatting

e2e6c89

update naming of test

a7d6a95

Merge pull request #2 from jchchiu/test-modification-tests

7912ed3

Test modifcations by changing tests

Merge remote-tracking branch 'upstream/dev' into add_mutect2_force_ca…

6ac91b3

…lling

jchchiu added 6 commits November 13, 2025 12:59

refactor: resolve formatting merge with 'prepare genome' without conf…

af463db

…lict raised

test: updated test snapshots

b1caa07

docs: update using nf-core pipelines lint --fix rocrate_readme_sync

63b7006

docs: update ro-crate-metadata.json

ff5be30

chore: update lint using nf-core pipelines lint --dir . --fix files_u…

a1881cf

…nchanged

Revert "chore: update lint using nf-core pipelines lint --dir . --fix…

c8e9b08

… files_unchanged" This reverts commit a1881cf.

nf-core-bot and others added 3 commits November 13, 2025 03:01

[automated] Fix code linting

d9750ba

docs: reorder CHANGELOG and add name to config

77aac76

style: change to alphabetica order

e2a6f29

refactor: remove unneeded mutect2_force_call from SAMPLESHEET_TO_CHANNEL

6f8a9b8

jchchiu requested review from FriederikeHanssen and maxulysse November 13, 2025 06:13

Add mutect2 force calling using '--alleles' flag #1952

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