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Setup.md

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SETUP

Requirements

Before proceeding with the installation, make sure you have the following requirements installed on your system:

Native Dependencies

  • nextflow (version > 22.10.6.5843)
  • Singularity
  • openjdk (version > 11.0.13)
  • git (only needed to download the repository)

Singularity Containers

We have our singularity recipes in the singularity folder of this project. See Sylabs official documentation on how to build singularity containers.

Installation

To set up the project, follow these steps:

  1. Clone the repository by running the following command:

    git clone https://github.com/ctg-lund/singleCellWorkflows

  2. After cloning the repository, you need to configure the nextflow.config file. Open the file and locate the following settings that require configuration:

    • refdir: Specify the directory where all the references used by cellranger are stored, such as vdj libraries, gex libraries, probe sets, etc. You will need to specify each reference separately.
    • container_dir: Set the directory path where all your singularity containers, including cellranger, spaceranger, multiqc, fastqc, etc., are stored.
    • process: Each process needs to have its corresponding container linked. Also, review the CPU and memory specifications to ensure they are appropriate for your system.
    • outdir: Where your project folders with your data are.

Make the necessary changes to the nextflow.conf file according to your system setup and requirements.

Once you have completed these steps, you will have successfully set up the project.

For more information on how to configure the nextflow.config file for the analysis you are interested in, see the docs folder

Outdir structure

The pipeline assumes the following structure when you process the data:

outdir (Set from nextflow.config)
 ┣ project1 (Sample_Project from samplesheet)
 ┃ ┣ fastq
 ┣ project2
 ┃ ┣ fastq

After processing it should look something like this:

output
 ┣ project1
 ┃ ┣ fastq
 ┃ ┣ 1_qc
 ┃ ┃ ┣ fastqc
 ┃ ┃ ┃ ┣ sample-1.fastqc.html
 ┃ ┃ ┃ ┣ sample-1.fastqc.zip
 ┃ ┃ ┃ ┣ sample-2.fastqc.html
 ┃ ┃ ┃ ┗ sample-2.fastqc.zip
 ┃ ┃ ┗ multiqc
 ┃ ┃ ┃ ┗ multiqc_report.html
 ┃ ┣ 2_count
 ┃ ┃ ┣ sample-1
 ┃ ┃ ┃ ┗ outs
 ┃ ┃ ┃ ┃ ┣ cloupe.cloupe
 ┃ ┃ ┃ ┃ ┣ metrics_summary.csv
 ┃ ┃ ┃ ┃ ┗ web_summary.html
 ┃ ┃ ┗ sample-2
 ┃ ┃ ┃ ┗ outs
 ┃ ┃ ┃ ┃ ┣ cloupe.cloupe
 ┃ ┃ ┃ ┃ ┣ metrics_summary.csv
 ┃ ┃ ┃ ┃ ┗ web_summary.html
 ┃ ┣ 3_summaries
 ┃ ┃ ┗ cellranger
 ┃ ┃ ┃ ┗ web_summaries.tar
 ┃ ┗ ctg-md5.project1.txt
 ┣ project2
 ┃ ┣ fastq
 ┃ ┣ 1_qc
 ┃ ┃ ┣ fastqc
 ┃ ┃ ┃ ┣ sample-3.fastqc.html
 ┃ ┃ ┃ ┗ sample-3.fastqc.zip
 ┃ ┃ ┗ multiqc
 ┃ ┃ ┃ ┗ multiqc_report.html
 ┃ ┣ 2_count
 ┃ ┃ ┗ sample-3
 ┃ ┃ ┃ ┗ outs
 ┃ ┃ ┃ ┃ ┣ cloupe.cloupe
 ┃ ┃ ┃ ┃ ┣ metrics_summary.csv
 ┃ ┃ ┃ ┃ ┗ web_summary.html
 ┃ ┣ 3_summaries
 ┃ ┃ ┗ cellranger
 ┃ ┃ ┃ ┗ web_summaries.tar
 ┃ ┗ ctg-md5.project2.txt

10X References

We use the prebuilt references from 10X genomics which can be found here. To make custom references, we use our add2ref script, which is hardcoded to work on our systems.

A very simple download script can be found in /bin/download-references.sh, which will download all the references used in the workflows. You will still need to untar the references though.