Illumina scRNA Perturb seq

A DRAGEN recipe, like this one, is a predefined set of analysis parameters and workflow settings tailored to a specific type of genomic analysis. For clarity, some default parameters are explicitly included and annotated with comments.

  
/opt/dragen/$VERSION/bin/dragen         #DRAGEN install path 
--ref-dir $REF_DIR                      #path to DRAGEN linear hashtable 
--output-directory $OUTPUT 
--intermediate-results-dir $PATH        #e.g. SSD /staging 
--output-file-prefix $PREFIX 
# Inputs 
--fastq-list $PATH                      #see 'Input Options' for FQ, BAM or CRAM 
--fastq-list-sample-id $STRING 
# Mapper 
--enable-rna true 
--annotation-file $GTF                  #GTF or GFF3 format 
--enable-map-align true                 #required for RNA/scRNA 
--enable-map-align-output true          #optionally save the output BAM 
--enable-sort true                      #default=true 
# Illumina Single Cell Perturb-seq 
--scrna-enable-pipseq-mode true 
--single-cell-threshold ratio           #['fixed', 'ratio', inflection'] 
--scrna-enable-pipseq-crispr-mode true 
--scrna-feature-barcode-groups $FEATURE_RGIDS 
--scrna-feature-barcode-reference $FEATURE_REF_CSV 

Notes and additional options

Hashtable

For DRAGEN RNA/scRNA runs, it is recommended to use the linear hashtable.

See: Product Files

Input options

DRAGEN input sources include: fastq list, fastq, bam, or cram. For BCL input, first create FASTQs using BCL conversion.

FQ list Input

--fastq-list $PATH 
--fastq-list-sample-id $STRING 

FQ Input

--fastq-file1 $PATH 
--fastq-file2 $PATH 
--RGSM $STRING 
--RGID $STRING 

BAM Input

--bam-input $PATH 

CRAM Input

--cram-input $PATH 

Mapping and Aligning

Option	Description
--enable-map-align true	For RNA/scRNA pipelines, map-align should always be turned on.
--enable-map-align-output true	Optionally save the output BAM (default=false).

Illumina single-cell RNA Prep Perturb-seq options

PIPseq mode batch option automatically sets the barcode/BI source, the barcode and binning index positions and the barcode sequence list options.

By default the barcode/BI is obtained from read 1 and the transcript is obtained from read 2.

PIPseq CRISPR mode option configures DRAGEN to automatically process CRISPR feature reads.

CRISPR mode enables feature counting, transforming of CRISPR cell-barcodes to match transcript barcodes, guide RNA calling, and additional metrics.

CRISPR mode requires a feature barcode reference CSV file to be provided with the CRISPR 'hook' and 'grab' sequences and positions. Refer to Illumina scRNA CRISPR Mode for details.

CRISPR mode also requires the feature barcode groups to be provided, i.e. the RGIDs of the CRISPR input FASTQs (multiple read groups should be comma-separated).

To change the barcode or binning index positions, use --scrna-barcode-position and --scrna-umi-position. These settings should be provided in the form <startPos>_<endPos> for each barcode. Connect multiple barcode sequence positions with a '+'.

For example, a library with the cell-barcode split into three blocks of 9 bp separated by fixed linker sequences and an 8 bp BI would be set using: --scrna-barcode-position 0_8+21_29+43_51, and --scrna-umi-position 52_59.

The following table lists some optional settings:

Option	Description
--scrna-enable-pipseq-mode	Option to enable Illumina PIPseq mode.
--scrna-barcode-position	See example above or refer to Illumina scRNA PIPseq.
--scrna-umi-position	See example above or refer to Illumina scRNA PIPseq.
--scrna-enable-pipseq-crispr-mode	Option to enable Illumina PIPseq CRISPR mode.
--scrna-feature-barcode-groups	Comma-separated list of CRISPR read groups (RGID strings), required in PIPseq CRISPR mode.
--scrna-feature-barcode-reference	CSV-file of feature barcode reference sequences. Refer to Illumina scRNA CRISPR Mode.
--single-cell-threshold	Cell filtering can be set to ['fixed', 'ratio', or 'inflection'].
--scrna-barcode-sequence-list	A known barcode sequence list can be optionally provided.
--umi-source	Optionally override the default barcode/BI source, valid option include ['read1', 'read2', 'qname', 'fastq'].

For more details on Illumina single cell perturb-seq pipeline options, refer to the Illumina scRNA CRISPR Mode User Guide