Illumina scRNA

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 Prep 
--scrna-enable-pipseq-mode true 
--single-cell-threshold ratio           #['fixed', 'ratio', inflection'] 

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 PIPseq 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.

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.
--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 prep pipeline options, refer to the Illumina scRNA PIPseq Pipeline User Guide