Required Software¶

This section will provide instructions for installing the required software to run the demultiplexing and doublet detecting pipeline.

Here is a table listing all the required software to run this pipeline:

Software	Requires Additional Set up Steps?
Singularity Image	Yes
Snakemake	No
scipy	No

Singularity Image¶

You will need the Singularity image (has all softwares needed for the pipeline installed for consistency across different systems). Most HPCs have Singularity installed so you should be able to run Singularity images without any issue. If you are unsure, try running singularity --help. If Singularity is not installed on your HPC, reach out to your system administrators and see if they can install it. Otherwise, feel free to open an issue and we can find another solution. To install the singularity image with the pipeline:

Make a directory where you want to house the pipeline
Move to the directory that you just made to house the pipeline
Download the singularity bucket from singularity hub.
```
wget https://www.dropbox.com/s/t8txw3yttj14we2/WG1-pipeline-QC_wgpipeline.sif
wget https://www.dropbox.com/s/n2vugd6w7glyuft/WG1-pipeline-QC_wgpipeline.sif.md5
```
After downloading the image, it is best to make sure the md5sum of the WG1-pipeline-QC_wgpipeline.sif file matches the md5sum in the WG1-pipeline-QC_wgpipeline.sif.md5:
```
md5sum WG1-pipeline-QC_wgpipeline.sif > downloaded_WG1-pipeline-QC_wgpipeline.sif.md5
diff -s WG1-pipeline-QC_wgpipeline.sif.md5 downloaded_WG1-pipeline-QC_wgpipeline.sif.md5
```
which should return:
```
Files WG1-pipeline-QC_wgpipeline.sif.md55 and downloaded_WG1-pipeline-QC_wgpipeline.sif.md5 are identical
```
Note

With the implementation of newer versions of the pipeline, it is important to make sure your singularity image alignes with the version of the pipeline documentation that you are currently using. To check the version of your singluation image plase run:
```
singularity inspect WG1-pipeline-QC_wgpipeline.sif
```
which will tell you the image version you are currenlty using and, therefore, the relevant documentation for that image.
Set up the pipeline with the following command; you will need to provide an absolute directory path to allow singularity to find the correct directories - this must be a directory somewhere above where you current working directory or the current working directory itself:
```
singularity run --bind <absolute_directory_path> --app setup WG1-pipeline-QC_wgpipeline.sif .
```
- If you want to use the smaller test dataset to test the pipeline and installation, please also run:
```
singularity run --bind <absolute_directory_path> --app test_dataset WG1-pipeline-QC_wgpipeline.sif .
tar xzvf TestData4PipelineSmall.tar.gz
```
- The directory structure is exactly the same as the full test dataset but the parent directory is called TestData4PipelineSmall
- This should copy all the files from the singularity bucket that are needed to run the pipeline to the current directory
Note

The pipeline assumes that the files are in the same directory as the singularity image so it is important that you do not copy them to a different location - follow the instructions above and you shouldn’t have any issues

Snakemake¶

You will also need Snakemake and scipy to run the pipeline. You can either use a conda environment that we have prepared with all the requirements (recommended) or install these yourself. You likely already created an environment when preparing for the SNP Genotype Imputation steps. If you didn’t, you can find the steps for the Snakemake conda environment here.

Next Steps¶

Now you have prepared the required data and installed the required software, we can move on to running the pipeline. If you are familiar and comfortable with Snakemake pipelines and structures, you can use the Quick Run instructions. However, they contain far less detail than the main sections so we only recommend using the Quick Run instructions if you have quite a bit of experience with Snakemake pipelines.