Use Case: Single Cell

This section describes the cubi-tk use case for the analysis of single cell data. It provides an outline of how cubi-tk helps in connecting

  • Sea-Snap (the CUBI pipeline for the processing of RNA sequencing, including scRNA-seq),

  • SODAR (the CUBI system for meta and mass data storage and management).

Overview

_images/seasnap-data-flow.png
1 FASTQ and ISA-tab files are uploaded to SODAR.

  • ISA-tab files can be created with the help of cubi-tk isa-tpl isatab-single_cell.

  • FASTQ files can be uploaded with the help of cubi-tk sodar ingest-fastq

2 FASTQ and ISA-tab files are pulled from SODAR.

  • FASTQ files can be downloaded using cubi-tk sodar pull-raw-data or iRods icommands.

  • ISA-tab files can be downloaded using cubi-tk sea-snap pull-isa.

3 A results folder is created on the HPC cluster and the config files are edited. A sample info file is created.

  • A results folder can be created with cubi-tk sea-snap working-dir.

  • The sample_info.yaml file can be created with cubi-tk sea-snap write-sample-info. This combines information from the parsed FASTQ folder structure and ISA-tab meta information.

4 Running the Sea-snap pipeline.

  • This is done as usual via ./sea-snap sc --slurm c.

5 The results are uploaded to SODAR.

  • Create a landing zone on SODAR with cubi-tk sodar lz-create.

  • Create a blueprint of which files to upload with ./sea-snap sc l export.

  • Upload the results using the blueprint and cubi-tk itransfer-results.

6 Check whether all files have been uploaded to SODAR correctly.

  • This can be done via cubi-tk sea-snap check-irods.

Processing Commands

Hint: Also see the Seasnap single cell pipeline documentation here.

First, you can pull the meta data from SODAR with the command:

$ cubi-tk sea-snap pull-isa <project_uuid>

This will create a folder with ISA-tab files. Alternatively, you can omit this step and automatically pull the files later.

The next step is to fetch the raw data from SODAR/iRODS. You first have to authenticate with iRODS using iinit. Internally, cubi-tk will use the iRODS icommands and you will be shown the commands it is about to execute.

$ iinit
$ cubi-tk sodar pull-raw-data <project_uuid>

Create a working directory for the project results:

$ cubi-tk sea-snap working-dir <path_to_seasnap_pipeline>

This will also copy relevant files and a config template into the new directory. Edit the config files to adjust the pipeline execution to your needs.

Create a sample info file. This is equivalent to a sample sheet and summarizes information about the samples in yaml format. A path pattern to the downloaded FASTQ files is needed, see Sea-snap doku: https://cubi-gitlab.bihealth.org/CUBI/Pipelines/sea-snap/blob/master/documentation/prepare_input.md#fastq-files-folder-structure

$ cubi-tk sea-snap write-sample-info --isa-assay <path_to_assay_file> <path_pattern_to_fastq>

This combines information from both the FASTQ folder structure (given via path pattern) and the ISA-tab meta data (given via ISA-assay file). If ISA-tab files have not been downloaded yet, you can use the option --project-uuid <project_uuid> instead of --isa-assay to download them on-the-fly.

Now you can start the processing. Run the Sea-snap pipeline as usual:

$ ./sea-snap sc --slurm c <any snakemake options>
$ ./sea-snap sc --slurm c export

After the pipeline has finished, you can create a new landing zone with the following command. This will print the landing zone properties as JSON. You will need the landing zone UUID (ZONE) in the next step.

$ cubi-tk sodar landing-zone-create <project_uuid>

You can then transfer the data using the following commands. You will have to specify the blueprint file generated by the export rule of sea-snap.

$ cubi-tk sea-snap itransfer-results <blueprint_file> <landing_zone_uuid>

Finally, you can validate and move the landing zone to get the data into SODAR:

$ cubi-tk sodar landing-zone-move <landing_zone_uuid>

You may check, whether everything was uploaded correctly using the following command:

$ cubi-tk sea-snap check-irods <path_to_local_results_folder> <irods_path_to_results_on_sodar>