nf-core / pixelator

Pipeline to generate Molecular Pixelation data with Pixelator (Pixelgen Technologies AB)

Introduction

nf-core/pixelator is a bioinformatics best-practice analysis pipeline for analysis of Molecular Pixelation assays. It takes a samplesheet as input and will process your data using pixelator to produce final antibody counts.

1. Build amplicon from input reads (pixelator amplicon)
2. Read QC and filtering, correctness of the pixel binding sequence sequences (pixelator preqc | pixelator adapterqc)
3. Assign a marker (barcode) to each read (pixelator demux)
4. Error correction, duplicate removal, compute read counts (pixelator collapse)
5. Compute the components of the graph from the edge list in order to create putative cells (pixelator graph)
6. Call and annotate cells (pixelator annotate)
7. Analyze the cells for polarization and colocalization (pixelator analysis)
8. Generate 3D graph layouts for visualization of cells (pixelator layout)
9. Report generation (pixelator report)

Warning

Since Nextflow 23.07.0-edge, Nextflow no longer mounts the host's home directory when using Apptainer or Singularity. This causes issues in some dependencies. As a workaround, you can revert to the old behavior by setting the environment variable NXF_APPTAINER_HOME_MOUNT or NXF_SINGULARITY_HOME_MOUNT to true in the machine from which you launch the pipeline.

Usage

Note

If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

First, prepare a samplesheet with your input data that looks as follows:

samplesheet.csv:

sample,design,panel,fastq_1,fastq_2
uropod_control,D21,human-sc-immunology-spatial-proteomics,uropod_control_300k_S1_R1_001.fastq.gz,uropod_control_300k_S1_R2_001.fastq.gz

Each row represents a sample and gives the design, a panel file and the input fastq files.

Now, you can run the pipeline using:

nextflow run nf-core/pixelator \
   -profile <docker/singularity/.../institute> \
   --input samplesheet.csv \
   --outdir <OUTDIR>

Warning

Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

For more details and further functionality, please refer to the usage documentation and the parameter documentation.

Pipeline output

To see the results of an example test run with a full size dataset refer to the results tab on the nf-core website pipeline page. For more details about the output files and reports, please refer to the output documentation.

Credits

nf-core/pixelator was originally written for Pixelgen Technologies AB by:

• Florian De Temmerman
• Johan Dahlberg
• Alvaro Martinez Barrio

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

For further information or help, don't hesitate to get in touch on the Slack #pixelator channel (you can join with this invite).

Citations

If you use nf-core/pixelator for your analysis, please cite it using the following doi: 10.5281/zenodo.10015112

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.

You can cite the molecular pixelation technology as follows:

Molecular pixelation: spatial proteomics of single cells by sequencing.

Filip Karlsson, Tomasz Kallas, Divya Thiagarajan, Max Karlsson, Maud Schweitzer, Jose Fernandez Navarro, Louise Leijonancker, Sylvain Geny, Erik Pettersson, Jan Rhomberg-Kauert, Ludvig Larsson, Hanna van Ooijen, Stefan Petkov, Marcela González-Granillo, Jessica Bunz, Johan Dahlberg, Michele Simonetti, Prajakta Sathe, Petter Brodin, Alvaro Martinez Barrio & Simon Fredriksson

Nat Methods. 2024 May 08. doi: 10.1038/s41592-024-02268-9

pixelator's Issues

Create module for collapse command

As of now use the same arguments as the command

Create module for cluster command

As of now use the same arguments as the command

Update nf-core skelton

Do this only if necessary

Create module for demux command

As of now use the same arguments as the command

Create module for report command

As of now use the same arguments as the command

First speed pass

First couple of quick things from looking over the files, without actually reading the real code...

Please delete these files:

• CODEOWNERS
• samplesheet.transformed.csv
  • Is this some kind of testing data? Needs to go into nf-core/test-datasets if so

Minor changes:

• Markdown render error in links in CITATIONS.md (cutadapt, fastp)
• Please update this to https://nf-co.re/pixelator :
  https://github.com/PixelgenTechnologies/nf-core-pixelator/blob/ccaabedecb40d7817f728473d94a0bfb2a8fde73/nextflow.config#L261
• Then remove this:
  https://github.com/PixelgenTechnologies/nf-core-pixelator/blob/ccaabedecb40d7817f728473d94a0bfb2a8fde73/.nf-core.yml#L5-L7

Questions

• What is this file for? Apologies if I missed some docs etc, just glancing at the files right now.

Mac support for `nf-core-pixelator`

Should we support Mac OS?

This is mine, still doesn't work:

% nextflow run PixelgenTechnologies/nf-core-pixelator -r main -profile test,docker --testdata_root ../nf-core-pixelator-datasets
N E X T F L O W  ~  version 22.04.5
NOTE: Your local project version looks outdated - a different revision is available in the remote repository [b80f30889b]
Launching `https://github.com/PixelgenTechnologies/nf-core-pixelator` [stupefied_pike] DSL2 - revision: c2f98772aa [main]

WARN: Found unexpected parameters:
* --publish_dir_mode: copy
* --report_name: report
* --percentile: 0.0
* --compute_coabundance: false
* --compute_colocalization: false
* --compute_polarization: false
* --cluster_min_count: 2
* --condition: optimal
* --big_clusters_recover: false
* --max_size_recover: 10000
* --max_size: null
* --min_size: null
* --use_counts: false
* --collapse_min_count: 1
* --collapse_mismatches: 2
* --neighbours: 60
* --umi2_end: null
* --umi2_start: null
* --umi1_end: null
* --umi1_start: null
* --upi2_end: null
* --upi2_start: null
* --upi1_end: null
* --upi1_start: null
* --algorithm: adjacency
* --demux_min_length: null
* --demux_mismatches: 0.1
* --pbs2: null
* --pbs1: null
* --mismatches: 0.1
* --remove_polyg: false
* --dedup: false
* --avg_qual: 20
* --max_n_bases: 3
* --min_length: null
* --max_length: null
* --trim_tail: 0
* --trim_front: 0
* --testdata_root: ../nf-core-pixelator-datasets
- Ignore this warning: params.schema_ignore_params = "publish_dir_mode,report_name,percentile,compute_coabundance,compute_colocalization,compute_polarization,cluster_min_count,condition,big_clusters_recover,max_size_recover,max_size,min_size,use_counts,collapse_min_count,collapse_mismatches,neighbours,umi2_end,umi2_start,umi1_end,umi1_start,upi2_end,upi2_start,upi1_end,upi1_start,algorithm,demux_min_length,demux_mismatches,pbs2,pbs1,mismatches,remove_polyg,dedup,avg_qual,max_n_bases,min_length,max_length,trim_tail,trim_front,testdata_root"



------------------------------------------------------
                                        ,--./,-.
        ___     __   __   __   ___     /,-._.--~'
  |\ | |__  __ /  ` /  \ |__) |__         }  {
  | \| |       \__, \__/ |  \ |___     \`-._,-`-,
                                        `._,._,'
  nf-core/pixelator v0.1.0dev
------------------------------------------------------
Core Nextflow options
  revision                  : main
  runName                   : stupefied_pike
  containerEngine           : docker
  launchDir                 : /Users/ambarrio/WORK/IDEO/Colibri/Pixelgen_Technologies/src/nf-core-pixelator
  workDir                   : /Users/ambarrio/WORK/IDEO/Colibri/Pixelgen_Technologies/src/nf-core-pixelator/work
  projectDir                : /Users/ambarrio/.nextflow/assets/PixelgenTechnologies/nf-core-pixelator
  userName                  : ambarrio
  profile                   : test,docker
  configFiles               : /Users/ambarrio/.nextflow/assets/PixelgenTechnologies/nf-core-pixelator/nextflow.config, /Users/ambarrio/WORK/IDEO/Colibri/Pixelgen_Technologies/src/nf-core-pixelator/nextflow.config

Input/output options
  input                     : ../nf-core-pixelator-datasets/testdata/micro/test_samplesheet.csv

Reference genome options
  genome                    : R64-1-1
  fasta                     : s3://ngi-igenomes/igenomes/Saccharomyces_cerevisiae/Ensembl/R64-1-1/Sequence/WholeGenomeFasta/genome.fa

Institutional config options
  config_profile_name       : Test profile
  config_profile_description: Minimal test dataset to check pipeline function

Max job request options
  max_cpus                  : 2
  max_memory                : 6.GB
  max_time                  : 6.h

Generic options
  max_multiqc_email_size    : 2.MB

!! Only displaying parameters that differ from the pipeline defaults !!
------------------------------------------------------
If you use nf-core/pixelator for your analysis please cite:

* The nf-core framework
  https://doi.org/10.1038/s41587-020-0439-x

* Software dependencies
  https://github.com/nf-core/pixelator/blob/master/CITATIONS.md
------------------------------------------------------
executor >  local (1)
[2c/e83c80] process > NFCORE_PIXELATOR:PIXELATOR:INPUT_CHECK:SAMPLESHEET_CHECK (test_samplesheet.csv) [100%] 1 of 1, failed: 1 ✘
[-        ] process > NFCORE_PIXELATOR:PIXELATOR:FASTQC                                               -
[-        ] process > NFCORE_PIXELATOR:PIXELATOR:CUSTOM_DUMPSOFTWAREVERSIONS                          -
[-        ] process > NFCORE_PIXELATOR:PIXELATOR:MULTIQC                                              [  0%] 0 of 1
Execution cancelled -- Finishing pending tasks before exit
-[nf-core/pixelator] Pipeline completed with errors-
WARN: There's no process matching config selector: PIXELATOR.*
WARN: There's no process matching config selector: RENAME_READS
WARN: There's no process matching config selector: PIXELATOR_PREQC
WARN: There's no process matching config selector: PIXELATOR_ADAPTERQC
WARN: There's no process matching config selector: PIXELATOR_DEMUX
WARN: There's no process matching config selector: PIXELATOR_COLLAPSE
WARN: There's no process matching config selector: PIXELATOR_CLUSTER
Error executing process > 'NFCORE_PIXELATOR:PIXELATOR:INPUT_CHECK:SAMPLESHEET_CHECK (test_samplesheet.csv)'

Caused by:
  Process `NFCORE_PIXELATOR:PIXELATOR:INPUT_CHECK:SAMPLESHEET_CHECK (test_samplesheet.csv)` terminated with an error exit status (1)

Command executed:

  check_samplesheet.py \
      test_samplesheet.csv \
      samplesheet.valid.csv

  cat <<-END_VERSIONS > versions.yml
  "NFCORE_PIXELATOR:PIXELATOR:INPUT_CHECK:SAMPLESHEET_CHECK":
      python: $(python --version | sed 's/Python //g')
  END_VERSIONS

Command exit status:
  1

Command output:
  ERROR: Please check samplesheet header -> sample,design,barcodes,fastq_1,fastq_2 != sample,fastq_1,fastq_2

Command error:
  WARNING: The requested image's platform (linux/amd64) does not match the detected host platform (linux/arm64/v8) and no specific platform was requested

Work dir:
  /Users/ambarrio/WORK/IDEO/Colibri/Pixelgen_Technologies/src/nf-core-pixelator/work/2c/e83c807c6ddc6596cbfea924b12a4a

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

Update once Bioconda recipe is live

Prepare for public release of `nf-core-pixelator`

Putting an Issue as a placeholder for a bunch of activities to be discussed and delineated here:

• Support channel(s)? nf-core? Others?
• Synergies with nf-core community? Webinars? Etc?
• Sync with pixelator
• Activities: Hackatons?
• Others?

Replace/hide all the Github related stuff

Create a repository to store test files and stuff (mimicking what nf-core has for modules)

In GitLab by @jfnavarro on Sep 5, 2022, 15:06

Revamp modules/local/rename_reads.nf

I think we can do better than that, but not blocking for the release

Originally posted by @maxulysse in #63 (comment)

Nf-core pixelator prototype

Create module for concatenate command

As of now use the same arguments as the command

Configure CI

Setup internal AWS Batch infrastructure

• Setup AWS Batch environment.
• Setup S3 bucket for input data (or reuse existing?).
• Setup S3 bucket for nextflow temp results.
• Setup S3 bucket for nextflow output data.

New nextflow_schema.json validation issues with nf-validation

The upcoming meta schema in nf-validation raises some issues:

Schema validation errors were encountered.
  pixelator_schema.json::$.definitions.adapterqc_options: 'type' is a required property
  pixelator_schema.json::$.definitions.adapterqc_options.properties.adapterqc_mismatches.maximum: 0.9 is not of type 'integer'
  pixelator_schema.json::$.definitions.demux_options: 'type' is a required property
  pixelator_schema.json::$.definitions.demux_options.properties.demux_mismatches.maximum: 0.9 is not of type 'integer'
  pixelator_schema.json::$.definitions.collapse_options: 'type' is a required property
  pixelator_schema.json::$.definitions.graph_options: 'type' is a required property
  pixelator_schema.json::$.definitions.annotate_options: 'type' is a required property
  pixelator_schema.json::$.definitions.analysis_options: 'type' is a required property
  pixelator_schema.json::$.definitions.report_options: 'type' is a required property
  pixelator_schema.json::$.definitions.global_config_options.properties.pixelator_container.format: 'url' is not one of ['file-path', 'directory-path', 'path']

See: nextflow-io/nf-validation#133

Investigate ways to fetch the design file from pixelator

In GitLab by @jfnavarro on Sep 5, 2022, 15:05

Create module for preqc command

As of now use the same arguments as the command

Investigate ways to fetch the barcodes files from pixelator

In GitLab by @jfnavarro on Sep 5, 2022, 15:05

Finalise test data setup

Create module for adapterqc command

As of now use the same arguments as the command

Configure GitHub Machine user account

See PixelgenTechnologies/pixelator#178

initial template build from nf-core/tools, version 2.2 - [merged]

Merges master -> main