Getting Started

Prerequisite

Make sure that LANG and LC_ALL environment variables have been set. You can use the following command to check this:

locale

If some are not set, you can set them to the default language for instance:

export LANG="C"
export LC_ALL="C"

Dependencies

Make sure you have the following software installed,

• Nextflow

  • Currently VSN-Pipelines requires Nextflow version 20.10.0 or higher.

• A container system, either of:

  • Docker
  • Singularity

NOTE: Due to licensing restrictions, to use the cellranger components of VSN you must build and/or provide a container with cellranger and bcl2fastq2 installed yourself. A sample Dockerfile can be found in ./src/cellranger/, you must download bcl2fastq2 from the Illumina website, and cellranger from the 10x Genomics website yourself to build this container.

Quick start

To run a quick test of the single sample analysis pipeline, we can use the 1k PBMC datasets provided by 10x Genomics. This will take only ~3min to run.

1. The data first needs to be downloaded (instructions can be found here).

2. Next, update to the latest pipeline version:

   nextflow pull vib-singlecell-nf/vsn-pipelines
   

3. Next, generate a config file using the standard settings for the test data, and the appropriate profiles (e.g., replace singularity with docker if necessary):

   nextflow config vib-singlecell-nf/vsn-pipelines \
       -profile tenx,singularity,single_sample > single_sample.config
   

4. The test pipeline can now be run using the config file just generated, specifying the single_sample workflow as an entrypoint:

   nextflow -C single_sample.config \
       run vib-singlecell-nf/vsn-pipelines \
           -entry single_sample
   

Example Output

$ nextflow -C nextflow.config run $VSN -entry single_sample
N E X T F L O W  ~  version 20.10.0
Launching `/staging/leuven/stg_00002/lcb/dwmax/documents/aertslab/GitHub/vib-singlecell-nf/vsn-pipelines/main.nf` [silly_pare] - revision: 77be3ba59d
WARN: DSL 2 IS AN EXPERIMENTAL FEATURE UNDER DEVELOPMENT -- SYNTAX MAY CHANGE IN FUTURE RELEASE
executor >  local (83)
[44/e02c9e] process > single_sample:SINGLE_SAMPLE:SC__FILE_CONVERTER (1)                                                                                [100%] 2 of 2 ✔
[22/723593] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:QC_FILTER:SC__SCANPY__COMPUTE_QC_STATS (2)                                      [100%] 2 of 2 ✔
[2e/10d845] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:QC_FILTER:SC__SCANPY__CELL_FILTER (2)                                           [100%] 2 of 2 ✔
[d6/fbe4b6] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:QC_FILTER:SC__SCANPY__GENE_FILTER (2)                                           [100%] 2 of 2 ✔
[22/d4a31b] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:QC_FILTER:GENERATE_DUAL_INPUT_REPORT:SC__SCANPY__GENERATE_DUAL_INPUT_REPORT (2) [100%] 2 of 2 ✔
[20/b43313] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:QC_FILTER:GENERATE_DUAL_INPUT_REPORT:SC__SCANPY__REPORT_TO_HTML (2)             [100%] 2 of 2 ✔
[e3/ee3f9c] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:NORMALIZE_TRANSFORM:SC__SCANPY__NORMALIZATION (2)                               [100%] 2 of 2 ✔
[79/7f4e25] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:NORMALIZE_TRANSFORM:PUBLISH_H5AD_NORMALIZED:COMPRESS_HDF5 (2)                   [100%] 2 of 2 ✔
[40/370971] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:NORMALIZE_TRANSFORM:PUBLISH_H5AD_NORMALIZED:SC__PUBLISH (2)                     [100%] 2 of 2 ✔
[f1/aa0726] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:NORMALIZE_TRANSFORM:PUBLISH_H5AD_NORMALIZED:SC__PUBLISH_PROXY (2)               [100%] 2 of 2 ✔
[76/e42ef9] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:NORMALIZE_TRANSFORM:SC__SCANPY__DATA_TRANSFORMATION (2)                         [100%] 2 of 2 ✔
[04/11b8b8] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:SC__SCANPY__FIND_HIGHLY_VARIABLE_GENES (2)                        [100%] 2 of 2 ✔
[1e/e1d058] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:SC__SCANPY__SUBSET_HIGHLY_VARIABLE_GENES (2)                      [100%] 2 of 2 ✔
[07/b3580a] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:SC__SCANPY__FEATURE_SCALING (2)                                   [100%] 2 of 2 ✔
[b4/00bf5e] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:PUBLISH_H5AD_HVG_SCALED:COMPRESS_HDF5 (2)                         [100%] 2 of 2 ✔
[8f/4d5d49] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:PUBLISH_H5AD_HVG_SCALED:SC__PUBLISH (2)                           [100%] 2 of 2 ✔
[9a/3c5d0d] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:PUBLISH_H5AD_HVG_SCALED:SC__PUBLISH_PROXY (2)                     [100%] 2 of 2 ✔
[dc/40cda6] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:GENERATE_REPORT:SC__SCANPY__GENERATE_REPORT (2)                   [100%] 2 of 2 ✔
[62/9dc791] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:HVG_SELECTION:GENERATE_REPORT:SC__SCANPY__REPORT_TO_HTML (2)                    [100%] 2 of 2 ✔
[8c/ed79b8] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:DIM_REDUCTION_PCA:SC__SCANPY__DIM_REDUCTION__PCA (2)                            [100%] 2 of 2 ✔
[be/ed9c2e] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:NEIGHBORHOOD_GRAPH:SC__SCANPY__NEIGHBORHOOD_GRAPH (2)                           [100%] 2 of 2 ✔
[01/ec367e] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:DIM_REDUCTION_TSNE_UMAP:SC__SCANPY__DIM_REDUCTION__TSNE (2)                     [100%] 2 of 2 ✔
[ea/7fbf7c] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:DIM_REDUCTION_TSNE_UMAP:SC__SCANPY__DIM_REDUCTION__UMAP (2)                     [100%] 2 of 2 ✔
[e5/a5a70a] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:DIM_REDUCTION_TSNE_UMAP:GENERATE_REPORT:SC__SCANPY__GENERATE_REPORT (2)         [100%] 2 of 2 ✔
[dd/b38b9b] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:DIM_REDUCTION_TSNE_UMAP:GENERATE_REPORT:SC__SCANPY__REPORT_TO_HTML (2)          [100%] 2 of 2 ✔
[5f/5bcb4d] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:CLUSTER_IDENTIFICATION:SC__SCANPY__CLUSTERING (2)                               [100%] 2 of 2 ✔
[fa/9765a9] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:CLUSTER_IDENTIFICATION:GENERATE_REPORT:SC__SCANPY__GENERATE_REPORT (2)          [100%] 2 of 2 ✔
[aa/7b6adb] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:CLUSTER_IDENTIFICATION:GENERATE_REPORT:SC__SCANPY__REPORT_TO_HTML (2)           [100%] 2 of 2 ✔
[0f/82f171] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:CLUSTER_IDENTIFICATION:SC__SCANPY__MARKER_GENES (2)                             [100%] 2 of 2 ✔
[96/04fc81] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:UTILS__GENERATE_WORKFLOW_CONFIG_REPORT                                          [100%] 1 of 1 ✔
[ee/7fe3fa] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:SC__SCANPY__MERGE_REPORTS (2)                                                   [100%] 2 of 2 ✔
[6f/7cbcb5] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:SC__SCANPY__REPORT_TO_HTML (2)                                                  [100%] 2 of 2 ✔
[87/7e681b] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:FINALIZE:SC__H5AD_TO_FILTERED_LOOM (2)                                          [100%] 2 of 2 ✔
[f0/176c0c] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:FINALIZE:FILE_CONVERTER_TO_SCOPE:SC__H5AD_TO_LOOM (1)                           [100%] 2 of 2 ✔
[b3/608cde] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:FINALIZE:FILE_CONVERTER_TO_SCANPY:SC__H5AD_MERGE (2)                            [100%] 2 of 2 ✔
[d1/43da78] process > single_sample:SINGLE_SAMPLE:SCANPY__SINGLE_SAMPLE:PUBLISH:SC__PUBLISH_PROXY (2)                                                   [100%] 2 of 2 ✔
[c3/6209f8] process > single_sample:PUBLISH_SINGLE_SAMPLE_SCOPE:COMPRESS_HDF5 (2)                                                                       [100%] 2 of 2 ✔
[d5/e1a0c3] process > single_sample:PUBLISH_SINGLE_SAMPLE_SCOPE:SC__PUBLISH (2)                                                                         [100%] 2 of 2 ✔
[4b/2e236a] process > single_sample:PUBLISH_SINGLE_SAMPLE_SCOPE:SC__PUBLISH_PROXY (2)                                                                   [100%] 2 of 2 ✔
[87/f3f350] process > single_sample:PUBLISH_SINGLE_SAMPLE_SCANPY:COMPRESS_HDF5 (2)                                                                      [100%] 2 of 2 ✔
[d4/2c09af] process > single_sample:PUBLISH_SINGLE_SAMPLE_SCANPY:SC__PUBLISH (2)                                                                        [100%] 2 of 2 ✔
[da/3817b5] process > single_sample:PUBLISH_SINGLE_SAMPLE_SCANPY:SC__PUBLISH_PROXY (2)                                                                  [100%] 2 of 2 ✔
WARN: To render the execution DAG in the required format it is required to install Graphviz -- See http://www.graphviz.org for more info.
Completed at: 12-Nov-2020 10:55:52
Duration    : 2m 36s
CPU hours   : 0.6
Succeeded   : 83

Output

The pipelines will generate 3 types of results in the output directory (params.global.outdir), by default out/

• data: contains the workflow output file (in h5ad format), plus symlinks to all the intermediate files.
• loom: contains final loom files which can be imported inside SCope visualization tool for further visualization of the results.
• notebooks: contains all the notebooks generated along the pipeline (e.g.: Quality control report)
• pipeline_reports: Nextflow dag, execution, timeline, and trace reports

For a full list of the pipelines available please see the pipelines page.

---

Further pipeline configuration details

This pipeline can be fully configured and run on custom data with a few steps. The recommended method is to first run nextflow config ... to generate a complete config file (with the default parameters) in your working directory. The tool-specific parameters, as well as Docker/Singularity profiles, are included when specifying the appropriate profiles to nextflow config.

1. First, update to the latest pipeline version (this will update the Nextflow cache of the repository, typically located in ~/.nextflow/assets/vib-singlecell-nf/):

   nextflow pull vib-singlecell-nf/vsn-pipelines
   

2. Next, a config file needs to be generated. This step will merge parameters from multiple profiles together to create a master config which specifies all parameters used by the pipeline. In this example, these are tenx for the input data, singularity to use the Singularity system (replace with docker if necessary), and single_sample to load the defaults for the single sample pipeline. In your working directory, run nextflow config ... with the appropriate profiles:

   nextflow config vib-singlecell-nf/vsn-pipelines \
       -profile tenx,singularity,single_sample > single_sample.config
   

3. Now, edits can be made to single_sample.config. Generally, the default values are acceptable to use for a first pass, but certain variables (input directory, etc.) need to be changed.

   In particular, the following parameters are frequently modified in practice:

   • params.global.project_name: a project name which will be included in some of the output file names.
   • params.data.tenx.cellranger_mex, which should point to the outs/ folder generated by Cell Ranger (if using 10x data). See Information on using 10x Genomics datasets for additional info.
   • Filtering parameters (params.sc.scanpy.filter): filtering parameters, which will be applied to all samples, can be set here: min/max genes, mitochondrial read fraction, and min cells. See Multi-sample parameters for additional info on how to specify sample-specific parameters.
   • Louvain cluster resolution: params.sc.scanpy.clustering.resolution.
   • Cell- and sample- level annotations are also possible.

4. Run the workflow using the new config file (using -C is recommended to use only this file), specifying the proper workflow as the entry point:

   nextflow -C single_sample.config \
       run vib-singlecell-nf/vsn-pipelines \
       -entry single_sample
   

Additional resources for running on custom data

• Input file formats available.
• Available pipelines.
• Advanced features for customizing pipelines.

Finally, see the list of case studies with specific examples and full config files at VSN-Pipelines-examples.