# 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 
# 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 
# Single Cell PIPseq 
--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](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html)

### Input options

DRAGEN input sources include: fastq list, fastq, bam, or cram. For BCL input, first create FASTQs using [BCL conversion](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/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`        | Optionally disable map & align (default=true).  |
| `--enable-map-align-output true` | Optionally save the output BAM (default=false). |

### Single-cell RNA PIPseq options

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

By default the barcode/BI is read 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 UMI would be set to: `--scrna-barcode-position 0_8+21_29+43_51`, and `--scrna-umi-position 52_59`.

The following table list some optional settings:

| Option                          | Description                                                                                                                                                                    |
| ------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| `--scrna-enable-pipseq-mode`    | Option to enable PIPseq mode.                                                                                                                                                  |
| `--scrna-barcode-position`      | See example above or refer to [scRNA PIPseq](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna-pipseq) |
| `--scrna-umi-position`          | See example above or refer to [scRNA PIPseq](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-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 inclde \['read1', 'read2', 'qname', 'fastq'].                                                                  |

For more details on PIPseq pipeline options, refer to the [scRNA PIPseq Pipeline User Guide](https://help.connected.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna-pipseq)