Manual Launch on BaseSpace
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DRAGEN Single Cell RNA
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The DRAGEN Single Cell RNA analysis can be manually launched to analyze previously sequenced 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.
The DRAGEN Single Cell RNA app only supports Biosample inputs. For more information on Biosamples refer to the .
Analysis Name
Required
Name of the analysis
Save Results To
Required
Select the project that will store the analysis results.
Biosample(s)
Required
Browse and select the biosamples to be analyzed.
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.
Custom Reference Files
Optional
Ensure "Include RNA Data in Reference" is enabled
Gene Annotation File
Optional
For custom references, select a GTF file if the custom reference does not contain a default GTF. For built-in references, select a custom GTF to override the default one.
Map/Align Output
Required
Select whether to output the alignments in BAM or CRAM format.
Library Kit
Required
Select your Illumina Single Cell 3' RNA Prep Kit.
Barcode Position
Required
Defaults to 0_7+11_16+20_25+31_38 for Illumina Single Cell 3' RNA Prep Kits.
UMI Position
Required
Defaults to 39_41 for Illumina Single Cell 3' RNA Prep Kits.
Barcode/UMI Read
Required
Defaults to Read 1 for Illumina Single Cell 3' RNA Prep Kits.
Barcode/UMI 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.
Barcode Sequence List File
Required
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 for Illumina Single Cell 3' RNA Prep Kits.
Poly-A Trimming
Required
Select the poly-A trimming method. Disabled for Illumina Single Cell 3' RNA Prep Kits.
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.
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 <start index>_<end index>. 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 or FASTA feature reference file that contains feature barcodes. Maps to --single-cell-feature-barcode-reference in command line arguments.
Expected Number of Cells
Optional
Specify the expected number of cells. The DRAGEN default 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.
Use the Additional Arguments section to define any custom settings. Below are some commonly used additional arguments.
--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".
Accept the BaseSpace Labs disclaimer and Launch Application to begin your analysis.
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 .