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DRAGEN Single Cell RNA
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DRAGEN Single Cell RNA
  • Introduction
  • Get Started
    • Prerequisites
  • Run Set Up
    • Sample Sheet Introduction
    • Run Planning in BaseSpace Sequence Hub
    • Sample Sheet Requirements
    • Manual Launch on BaseSpace
  • Analysis Methods
    • FASTQ Processing
    • Error Correction
    • Transcript Counting
    • PIPseq CRISPR Mode
  • Analysis Results
    • Accessing Results
    • DRAGEN Report
      • Single-Cell RNA
      • Single-Cell Clustering
      • Trimmer
      • DRAGEN FastQC
      • Mapping
    • Secondary Analysis Results
  • Tertiary Analysis
    • Illumina Connected Multiomics
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On this page
  • Select Input Data
  • Configuration
  • Demultiplexing
  • Cell Hashing and Feature Counting
  • Advanced Settings
  • Additional Arguments
  • Launch Application

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  1. Run Set Up

Manual Launch on BaseSpace

PreviousSample Sheet RequirementsNextFASTQ Processing

Last updated 3 months ago

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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 .

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.

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 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

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

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 for Illumina Single Cell 3' RNA Prep Kits.

Poly-A Trimming

Optional

Select the poly-A trimming method. Disabled for Illumina Single Cell 3' RNA Prep Kits.

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.

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 <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.

Advanced Settings

Parameter Name
Required?
Description

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.

Additional Arguments

Use the Additional Arguments section to define 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.

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 .

BaseSpace Data Model
Prepare a Reference Genome