# Manual Launch on BaseSpace
The DRAGEN Single Cell RNA analysis can be manually launched to analyze previously generated FASTQ files by using a BaseSpace App.
Use the steps below to create a manually launch the DRAGEN Single Cell RNA app in BaseSpace. To get to the app, open BaseSpace Sequence Hub and navigate to the **Apps** page by using the navigation bar or by opening the menu on the left-hand side. Select or search for the **DRAGEN Single Cell RNA** app from the list of available apps. Select **Launch Application** to provide details for your analysis. Detailed steps are provided below.
### Select Input Data
The DRAGEN Single Cell RNA app only supports Biosample inputs. For more information on Biosamples refer to the [BaseSpace Data Model](https://help.connected.illumina.com/basespace-sequence-hub/overview/data-model).
### Configuration
| Parameter Name | Required? | Description |
| ---------------------- | --------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| Analysis Name | Required | Name of the analysis |
| Save Results To | Required | Select the project that will store the analysis results. |
| Library Kit | Required | Select either Illumina Single Cell 3' RNA or Illumina Single Cell CRISPR |
| Input Type | Required | Select "Expression input only" for samples with only gene expression libraries. Select "Paired expression and feature inputs" for samples with gene expression and feature inputs. Depending on the option chosen, the biosamples section below will disable either the "Select Biosample(s)" option or the table with the option to pair expression and feature biosamples. |
| Biosample(s) | Required | Depending on the Input Type chosen browse for and select either of the following:
- A set of gene expression biosamples
- A set of paired gene expression and feature biosamples. Use the Sample Name field to designate the name of the paired sample containing both gene expression and feature information
|
| Map/Align Output | Required | Select whether to output the alignments in BAM or CRAM format. The default is to not output an alignments file, which decreases compute time. |
| Barcode/MI Source | Required | Select the appropriate setting that matches how FASTQ files were generated.
- FASTQ Header – the FASTQ files were generated with the OverrideCycles sample sheet setting writing the R1 sequence to the FASTQ header
- Barcode/UMI Read - the Read 1 FASTQ files were created without setting OverrideCycles in the sample sheet so the Read 1 FASTQ file contains the full sequencing read.
|
| Reference | Required | Select the reference genome to use in the analysis. The app provides support for common human, mouse, and rat genomes in addition to supporting custom references built by the DRAGEN Reference Builder app. When selecting human, it is recommended to use linear references for RNA analysis. See [DRAGEN Reference Support](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-reference-support) for more information. |
| Custom Reference Files | Optional | Custom references can be generated from a FASTA file and optionally a GTF file with the DRAGEN Reference Builder app. For more information, refer to Prepare a Reference Genome.
- Ensure "Include RNA Data in Reference" is enabled
|
| Gene Annotation File | Optional | For custom references, select the corresponding GTF file to use. For built in references, the following list shows the default GTFs being used. This can be overridden for custom annotations by using this field.
GENCODE v19
- Homo sapiens \[UCSC] hg19 v5
- Homo sapiens \[UCSC] hg19 v5 Pangenome
- Homo sapiens \[NCBI] hs37d5 v5
- Homo sapiens \[NCBI] hs37d5 v5 Pangenome
GENCODE v44
- Homo sapiens \[1000 Genomes] hg38 v5
- Homo sapiens \[1000 Genomes] hg38 v5 Pangenome
GENCODE vM23
- Mus musculus \[UCSC] mm10
ENSEMBL 98
- Rattus norvegicus \[UCSC] rn6
|
### Library Kit Configuration
| Parameter Name | Required? | Description |
| -------------------------- | --------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| Barcode Position | Required | Defaults to 0\_7+11\_16+20\_25+31\_38 for Illumina Single Cell Prep Kits. |
| UMI/BI Position | Required | Defaults to 39\_41 for Illumina Single Cell 3' RNA Prep Kits. |
| Barcode/MI Read | Required | Defaults to Read 1 for Illumina Single Cell 3' RNA Prep Kits. |
| Barcode Sequence List File | Optional | Specify a file containing valid cell barcode sequences. Maps to --single-cell-barcode-sequence-whitelist in command line arguments. Not required for Illumina Single Cell 3' RNA Prep Kits. |
| RNA Library Type | Required | Auto-populated with forward for Illumina Single Cell 3' RNA Prep Kits. |
### Cell Hashing and Feature Counting
| Parameter Name | Required? | Description |
| --------------------------------- | --------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| Cell Hashing and Feature Counting | Optional | Use the checkboxes to enable cell hashing and feature counting using feature barcode UMI. |
| Feature Barcode UMI Position | Optional | Feature barcode UMI position is in the format of \\_\. ex: 11\_18 specifies an 8 bp sequence from positions 11 to 18 (inclusive). The first position is 0. |
| Cell Hashing Reference | Optional | Specify a CSV or FASTA cell-hashing reference file that contains sample-specific oligo-tags. Maps to --single-cell-cell-hashing-reference in command line arguments. |
| Detect Doublets | Optional | Select the checkbox to enable doublet detection in cell-hashing sample demultiplexing. Maps to --single-cell-demux-detect-doublets in command line arguments. |
| Feature Barcode Reference | Optional | Specify a CSV feature reference file that contains feature barcode information as specified in the [DRAGEN documentation](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna-pipseq#inputs). Maps to --single-cell-feature-barcode-reference in command line arguments. |
### Demultiplexing
| Parameter Name | Required? | Description |
| --------------------- | --------- | ------------------------------------------------------------------------------------------------------------------------------------- |
| Demultiplexing Method | Optional | Select genotype-based or genotype-free sample demultiplexing. |
| Sample VCF | Optional | Specify a VCF file for genotype-based demultiplexing. Maps to --single-cell-demux-sample-vcf in command line arguments. |
| Reference VCF | Optional | Specify a VCF file for genotype-free demultiplexing. Maps to --single-cell-demux-reference-vcf in command line arguments. |
| Number of Samples | Optional | Specify the number of samples for genotype-free demultiplexing. Maps to --single-cell-demux-number-samples in command line arguments. |
| Detect Doublets | Optional | Enable doublet detection in sample demultiplexing. Maps to --single-cell-demux-detect-doublets in command line arguments. |
### Advanced Settings
| Parameter Name | Required? | Description |
| ------------------------- | --------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| Poly-A Trimming | Optional | Disabled for Illumina Single Cell 3' RNA Prep Kits. |
| Expected Number of Cells | Optional | Specify the expected number of cells. The DRAGEN default of 400 is used if not set. Adjust only if the expected number of cells is so far from the default that DRAGEN does not call the correct cell filtering threshold automatically. |
| Thresholding Method | Optional | Specify the method for determining the count threshold value.
- Ratio: DRAGEN estimates the count threshold as max(Te, Tm). Tm is 10% of the count seen in the cell at the 10th percentile of the expected cells. Te is 50% of the count seen in the least abundant expected cell.
- Inflection: DRAGEN estimates the count threshold by analyzing inflection points in the cumulative distribution of counts.
- Fixed: The count threshold is set to force the expected number of cells.
Maps to --single-cell-threshold in command line arguments. See DRAGEN documentation for more details.
|
| ICA Scratch Size Per Node | Optional | Scratch size for DRAGEN process. Defaults to "8 TiB". |
### Additional Arguments
Use the Additional Arguments section to acknowledge disclaimer prior to defining any custom settings. Below are some commonly used additional arguments.
| Argument | Description |
| -------------------------------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| --annotation-file-ignore-biotypes=none | When selecting the Illumina Single Cell 3’ RNA Library Prep Kit, the pipeline will automatically ignore pseudogenes, shortRNA, and rRNA biotypes during mapping. This behavior can be disabled by adding "--annotation-file-ignore-biotypes=none". |
### Launch Application
Accept the BaseSpace Labs disclaimer and **Launch Application** to begin your analysis.