perllb / ctg-sc-mkfastq-10x

demux (mkfastq) + qc of 10x data

ctg-sc-mkfastq-10x

Nextflow pipeline for demultiplexing and qc of 10x data with mkfastq.

• Designed to handle multiple projects in one sequencing run (but also works with only one project)

Pipeline steps:

Cellranger version: cellranger v6.0

• Demultiplexing (cellranger mkfastq): Converts raw basecalls to fastq, and demultiplex samples based on index (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/6.0/using/mkfastq).
• FastQC: FastQC calculates quality metrics on raw sequencing reads (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). MultiQC summarizes FastQC reports into one document (https://multiqc.info/).
• multiQC: Compile fastQC and demux metrics in multiqc report
• md5sum: md5sum of all generated files

Input files

The following files must be in the runfolder to start pipeline successfully.

1. Samplesheet (CTG_SampleSheet.sc-mkfastq-10x.csv)

Samplesheet requirements:

Note: no header! only the rows shown below, starting with the column names.

Sample_ID	index	Sample_Project
Si1	SI-GA-D9	proj_2021_012
Si2	SI-GA-H9	proj_2021_012
Sample1	SI-GA-C9	proj_2021_013
Sample2	SI-GA-C9	proj_2021_013

The nf-pipeline takes the following Columns from samplesheet to use in channels:

- `Sample_ID` : ID of sample. Sample_ID can only contain a-z, A-Z and "_".  E.g space and hyphen ("-") are not allowed! If 'Sample_Name' is present, it will be ignored. 
- `index` : Must use index ID (10x ID) if dual index. For single index, the index sequence works too.
- `Sample_Project` : Project ID. E.g. 2021_033, 2021_192.

Samplesheet template (.csv)

Name : CTG_SampleSheet.sc-mkfastq-10x.csv

Sample_ID,index,Sample_Project
Si1,Sn1,SI-GA-D9,2021_012
Si2,Sn2,SI-GA-H9,2021_012
Sample3,S3_1,SI-GA-C9,2021_013
Sample4,S2_3,SI-GA-C9,2021_013

USAGE NEXTFLOW

1. Clone and build the Singularity container for this pipeline: https://github.com/perllb/ctg-sc-rna-10x/tree/master/container/sc-rna-10x.v6
2. Edit your samplesheet to match the example samplesheet. See section SampleSheet below
3. Edit the nextflow.config file to fit your project and system.
4. Run pipeline

nohup nextflow run pipe-sc-mkfastq-10x.nf > log.pipe-sc-mkfastq-10x.txt &

USAGE DRIVER

• Execute from within runfolder!

Run with default

Assumes

• CTG_SampleSheet.sc-mkfastq-10x.csv is in runfolder
• Dual index

sc-mkfastq-10x-driver 

Run with single index

Assumes

• CTG_SampleSheet.sc-mkfastq-10x.csv is in runfolder

sc-mkfastq-10x-driver -a

Run with specific samplesheet

sc-mkfastq-10x-driver -s /path/to/my_special_samplesheet.csv

Output:

• ctg-PROJ_ID-output
  • qc: Quality control output.
    • fastqc output (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
    • multiqc output: Summarizing FastQC output and demultiplexing (https://multiqc.info/)
  • fastq: Contains raw fastq files from cellranger mkfastq.
  • ctg-md5.PROJ_ID.txt: text file with md5sum recursively from output dir root

Container

https://github.com/perllb/ctg-sc-rna-10x/tree/master/container/sc-rna-10x.v6