Draft: Add feature for sample demultiplexing followed by immune profiling #365

herpov · 2024-08-16T07:12:04Z

Description of changes

The current nf-core/scrnaseq version (v1.7.0) does not handle the use case where data is both sample multiplexed and requires immune profiling. The current version can however handle either situation separately.

The cellranger software provided by 10x does not handle this situation either, but this tutorial guides the user to follow 3 steps:

Run cellranger multi to demultiplex the samples
Convert the output .bam files to .fq files
Run cellranger multi with immune profiling for each sample using the generated .fq files

This PR serves to enable that for the workflow. This has required an additional tool (nf-core/bamtofastq10x), some new code, and a bit of rearrangement of existing code.

Rearrangement of code

Since cellranger multi is to be run multiple times during a nextflow run, I moved the code for preparing the reference genome from subworkflows/local/align_cellrangermulti.nf and generated a new subworkflow, cellrangermulti_ref.nf.

Added tool

I added the tool nf-core/bamtofastq10x.

New code

I added the subworkflow align_cellrangermulti_vdj.nf based on align_cellrangermulti.nf which contains the above described steps: 1,2, and 3. For step 1 and 3 the nf-core cellranger multi module is invoked as in align_cellrangermulti.nf.

The main changes lie in channel operations in scrnaseq.nf and align_cellrangermulti_vdj.nf.

PR checklist

…kflows

…ng. additionally changing input channels

…by immune profiling

… cellranger multi or cellranger multi+vdj

…r to bamtofastq process

conf/modules.config

herpov · 2024-08-16T07:28:06Z

modules/nf-core/bamtofastq10x/main.nf

I have made several changes to this script, but idk if I should make a PR for the module itself?

The changes you made seem pretty specific to your use-case, so it doesn't make sense to update the central module.
It is also not allows to change modules in a pipeline (-> linting error).

The preferred way would be to make it somehow work with the nf-core module unchanged. If this is not possible, you can make a copy of the module in the "local" folder and adapt it as needed.

@grst now the changes are minimal to this module. The rest I could fix by manipulating the output channel. Should I make a PR for bamtofastq or should I still just move this to "local" dir?

Should I make a PR for bamtofastq

Yes please, the changes look like everyone will benefit from them.

modules/nf-core/bamtofastq10x/main.nf

herpov · 2024-08-16T07:33:26Z

modules/nf-core/bamtofastq10x/main.nf

+    bamtofastq \\
+        $args \\
+        $bam \\
+        $prefix


Changed the output path from ${prefix}.fastq.gz.

bamtofastq generates a directory containing two folders: one for GEX and one for CMO .fastq files.
The two folders are prefixed with the .bam prefix.
All files are automatically prefixed with bamtofastq.

modules/nf-core/bamtofastq10x/main.nf

conf/modules.config

grst · 2024-08-20T07:21:06Z

modules/nf-core/bamtofastq10x/main.nf

The changes you made seem pretty specific to your use-case, so it doesn't make sense to update the central module.
It is also not allows to change modules in a pipeline (-> linting error).

The preferred way would be to make it somehow work with the nf-core module unchanged. If this is not possible, you can make a copy of the module in the "local" folder and adapt it as needed.

grst · 2024-08-20T07:25:33Z

subworkflows/local/align_cellrangermulti_idx.nf

+include { CELLRANGER_MKVDJREF               } from "../../modules/nf-core/cellranger/mkvdjref/main.nf"
+
+// Define workflow to subset and index a genome region fasta file
+workflow CELLRANGER_MULTI_REF {


Please make sure the workflow name and filename match.

Do you accept the new name of the file?
cellrangermulti_ref.nf

subworkflows/local/align_cellrangermulti_idx.nf

modules/nf-core/bamtofastq10x/main.nf

github-actions · 2024-08-20T07:48:52Z

`nf-core lint` overall result: Passed ✅ ⚠️

Posted for pipeline commit 0132d9b

+| ✅ 205 tests passed       |+
#| ❔   4 tests were ignored |#
!| ❗   3 tests had warnings |!

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pipeline_todos - TODO string in main.nf: Optionally add in-text citation tools to this list.
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files_exist - File is ignored: lib/Utils.groovy
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Run details

nf-core/tools version 2.14.1
Run at 2024-08-20 07:48:34

grst · 2024-08-20T07:49:50Z

@grst is this a 10x thing or is it an issue of how nextflow stages the files? Cellranger does not require all files to have the same prefix, ie fastq_id. I'd like some guidance to how I can debug this.

How nextflow stages the files you can check by investigating the process work directory. I haven't worked much wich cellranger multi, but cellranger is pretty strict about the filenames. They need to follow the {sample_name}_S{i}_L00{j}_{R1,R2}_001.fastq.gz convention or they won't be found.

…ied extract_gex_fq

herpov · 2024-08-27T08:47:34Z

@grst is this a 10x thing or is it an issue of how nextflow stages the files? Cellranger does not require all files to have the same prefix, ie fastq_id. I'd like some guidance to how I can debug this.

How nextflow stages the files you can check by investigating the process work directory. I haven't worked much wich cellranger multi, but cellranger is pretty strict about the filenames. They need to follow the {sample_name}_S{i}_L00{j}_{R1,R2}_001.fastq.gz convention or they won't be found.

I realized the workflow renamed the files according to the GEX sample name, so I had to ensure that the IDs of the VDJ and AB channels were consistent with the demultiplexed GEX IDs.

… updated extract_gex_fq()

herpov · 2024-09-04T07:54:56Z

I have not tested the pipeline with frna data. Further, I had to exclude the sample containing probe barcodes from my test set, ie: 4PLEX_HUMAN from assets/cellranger_barcodes_samplesheet.csv. I have not further investigated the reason for failure.
I have been testing the workflow with the linked metadata.

When I tried including another dataset which had been hashed with the same cmo as one of the others I ran into this error:

When running the pipeline separately on the dataset which failed, I had no issues. I have not spent more time trying to work around it, because I don't expect it to be an issue in our use case and, probably, it is a rare event - but thought you'd like to know.

samplesheet.csv
fb_reference.csv
cmo.csv
barcodes_samplesheet.csv

…es which require immune profiling

…tiplexed samples

nf-core-bot · 2024-11-01T14:16:24Z

Warning

Newer version of the nf-core template is available.

Your pipeline is using an old version of the nf-core template: 2.14.1.
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.

…nstead of inside cellranger_multi.py

Helle Rus Povlsen added 11 commits August 16, 2024 09:08

cp cellrangermulti to cellrangermulti_vdj in SCRNASEQ workflow

5812f72

cp align_cellrangermulti to align_cellrangermulti_vdj in local subwor…

a7a5cd3

…kflows

separate processes that generate reference files from cellranger multi

06e9932

installed nf-core/module bamtofastq10x

9bafa8d

add null/ to the gitignore list

2e32c2e

add description of demultiplexing combined with immuneprofiling

6072a4c

add bamtofastq10x module with amendments

27a8c16

move reference creation outside the cellranger multi to avoid rerunni…

a1d0170

…ng. additionally changing input channels

add subworkflow specific for handling sample demultiplexing followed …

170ec07

…by immune profiling

implement cellranger multi ref and vdj. branch channels to either run…

3b71ff8

… cellranger multi or cellranger multi+vdj

update publishDir for the two cellranger multi outputs. add publishDi…

0132d9b

…r to bamtofastq process

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Helle Rus Povlsen added 2 commits August 21, 2024 08:52

add func to expand feature channels to match demultiplexed gex. modif…

1434e45

…ied extract_gex_fq

remove renaming of files

18b9eae

Helle Rus Povlsen added 4 commits September 3, 2024 16:59

removed arg for BAMTOFASTQ and updated CELLRANGER_MULTI with regex

ecf453f

changed faux channels to value channels to be consumed infinitely and…

8359254

… updated extract_gex_fq()

remove unused code

c4356b0

renamed file

ced5ddc

update output path for fastq

8c6e1c0

Helle Rus Povlsen added 3 commits September 4, 2024 10:13

update output dir for emptydrops analysis

e22f8fb

update filename for generating reference files for cellranger multi

eff6158

remove frna option for immune-profiling

998f967

herpov mentioned this pull request Oct 2, 2024

[FEATURE] Update BAMTOFASTQ10X for multiplexed samples nf-core/modules#6725

Closed

Helle Rus Povlsen added 4 commits October 4, 2024 15:09

updated bamtofastq10x module

cef8759

update changelog and output docs

3f28b0b

add separate test for cellranger multi when we have multiplexed sampl…

503122b

…es which require immune profiling

update element to do cross product on when expanding channels for mul…

4735642

…tiplexed samples

Helle Rus Povlsen added 6 commits November 4, 2024 13:36

update the sorting mechanism for fq files in cellranger_multi.py

ae94059

added printout of fastqs list

5d0a0b6

move expanded sorting mechanism of fq files to channel manipulation i…

de26ecb

…nstead of inside cellranger_multi.py

update sorting mechanism of bam2fq outputs - fstq_dr is not an integer

fbddb27

update the regex search term for sorting bamtofastq output files

b6a7795

add specific vdj feature types for B, T and gamma/delta cells

050d8a8

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Draft: Add feature for sample demultiplexing followed by immune profiling #365

Draft: Add feature for sample demultiplexing followed by immune profiling #365

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Draft: Add feature for sample demultiplexing followed by immune profiling #365

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Draft: Add feature for sample demultiplexing followed by immune profiling #365

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herpov commented Aug 16, 2024 • edited Loading

Description of changes

Rearrangement of code

Added tool

New code

PR checklist

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github-actions bot commented Aug 20, 2024

nf-core lint overall result: Passed ✅ ⚠️

❗ Test warnings:

❔ Tests ignored:

✅ Tests passed:

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grst commented Aug 20, 2024

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herpov commented Aug 16, 2024 •

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`nf-core lint` overall result: Passed ✅ ⚠️