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10x Genomics scRNA-Seq (cellranger) mkfastq Pipeline

Introduction

This pipeline is a wrapper for the cellranger mkfastq tool from 10x Genomics (which uses Illumina's bcl2fastq). It takes demultiplexes samples from 10x Genomics Single Cell Gene Expression libraries into fastqs.

FastQC is run on the resulting fastq and those reports and bcl2fastq reports are collated with the MultiQC tool.

The pipeline uses Nextflow, a bioinformatics workflow tool.

This pipeline is primarily used with a SLURM cluster on the BioHPC Cluster. However, the pipeline should be able to run on any system that Nextflow supports.

Additionally, the pipeline is designed to work with Astrocyte Workflow System using a simple web interface.

Cloud Compatibility

This pipeline is also capable of being run on AWS. To do so:

• Build a AWS batch queue and environment either manually or with aws-cloudformantion
• Edit one of the aws configs in workflow/config/
  • Replace workDir with the S3 bucket generated
  • Change region if different
  • Change queue to the aws batch queue generated
• The user must be have awscli configured with an appropriate authentication (with aws configure and access keys) in the environment which nextflow will be run
• Add -profile with the name aws config which was custamized
  • eg. nextflow run workflow/main.nf -profile aws_ondemand

To Run:

• Available parameters:
  • --name
    • run name, puts outputs in a directory with this name
    • eg: --name 'test'
  • --bcl
    • Base call files (tarballed [.tar] +/- gunzipping [.tar.gz] from a sequencing of 10x single-cell expereiment, supports pigr parallelization).
    • There can be multiple basecall files, but they all will be demultiplexed by the same design file.
    • eg: --bcl '/project/shared/bicf_workflow_ref/workflow_testdata/cellranger/cellranger_mkfastq/simple1/cellranger-tiny-bcl-1_2_0.tar.gz'
  • --designFile
    • path to design file (csv format) location
    • column 1 = "Lane" (number of lanes to demultiplex, * for all lanes)
    • column 2 = "Sample" (sample name)
    • column 3 = "Index" (10x sample index barcode, eg SI-GA-A1)
    • Last character set in Index references the well position of the 96-well plate that the sample barcode kit is sold in.
    • Current sample barcode IDs
    • can have repeated "Sample" if there are multiple fastq R1/R2 pairs for the samples
    • can be downloaded HERE
    • eg: --designFile '/project/shared/bicf_workflow_ref/workflow_testdata/cellranger/cellranger_mkfastq/simple/cellranger-tiny-bcl-simple-1_2_0.csv'
  • --outDir
    • optional output directory for run
    • eg: --outDir 'test'
• FULL EXAMPLE:

  nextflow run workflow/main.nf --name 'test' --bcl '/project/shared/bicf_workflow_ref/workflow_testdata/cellranger/cellranger_mkfastq/simple1/cellranger-tiny-bcl-1_2_0.tar.gz' --designFile '/project/shared/bicf_workflow_ref/workflow_testdata/cellranger/cellranger_mkfastq/simple1/cellranger-tiny-bcl-simple-1_2_0.csv' --outDir 'test'

• Design example:

CHANGELOG

Credits

This worklow is was developed jointly with the Bioinformatic Core Facility (BICF), Department of Bioinformatics

Please cite in publications: Pipeline was developed by BICF from funding provided by Cancer Prevention and Research Institute of Texas (RP150596).