Run Planning in BaseSpace Sequence Hub

The BaseSpace Sequence Hub Run Planning tool is used to generate a valid sample sheet in v2 format for use on a supported sequencer. Filling out the form on the user interface will produce a sample sheet with the required fields filled in that can be used to auto-launch a DRAGEN Single Cell RNA analysis. Refer to Cloud Analysis Auto-launch for more information about the Run Planning workflow and auto-launch.

For NextSeq 2000, the samplesheet created by the Run Planning tool needs to be exported and uploaded to the instrument. When using the NovaSeq X, the samplesheet will automatically be on the instrument.

Use the steps below to create a DRAGEN Single Cell RNA run with the BaseSpace Run Planning tool. To get to the Run Planning tool, open BaseSpace Sequence Hub and navigate to the Runs page by using the navigation bar or by opening the menu on the left-hand side. From the New Run dropdown menu select Run Planning.

Step 1: Run Settings

Parameter Name	Required?	Description
Run Name	Required	Run Name can contain 255 alphanumeric characters, dashes, underscores, periods, and spaces; and must start with an alphanumeric, a dash or an underscore.
Run Description	Optional	Run Description can contain 255 characters except square brackets, asterisks, and commas.
Instrument Platform	Required	Choose from DRAGEN Single Cell RNA software supported instruments: NovaSeq X Series NovaSeq 6000/6000Dx NextSeq 1000/2000
Secondary Analysis	Required	Select BaseSpace / Illumina Connected Analytics.
Read 1	Required only for Instrument Platform NovaSeq X Series	45 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.
Index 1	Required only for Instrument Platform NovaSeq X Series	10 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.
Index 2	Required only for Instrument Platform NovaSeq X Series	10 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.
Read 2	Required only for Instrument Platform NovaSeq X Series	72 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.
Sample Container ID	Optional	Unique identifier for the container that holds the sample.

Step 2: Configuration

On NovaSeq X Series, this page is called "Configuration 1". The top right-hand corner of the UI displays the Read 1, Read 2, Index 1 and Index 2 entered on the previous run settings screen.

Parameter Name	Required?	Description
Application	Required	Select DRAGEN Single Cell RNA - 4.4.4
Description	Optional	Optional Text Field
Library Prep Kit	Required	Select Illumina Single Cell 3’ RNA Prep
Index Adapter Kit	Required	Select a supported index adapter kit: Illumina Single Cell UD 8 Indexes Illumina Single Cell UD Indexes Set A
Reference Genome	Required only for Instrument Platform NovaSeq X Series	Select the appropriate genome reference for the sample type. When selecting human, it is recommended to use linear references for RNA analysis. See DRAGEN Reference Support for more information.

Step 3: Run Configuration and Analysis Settings

Parameter Name	Required?	Description
Description	Optional	Optional Text Field
Library Prep Kit	Required	Auto-populated from previous step
Index Adapter Kit	Required	Auto-populated from previous step
Index Reads	Required	Defaults to 2 indexes
Read Type	Required	Defaults to Paired End
Read Lengths	Required	Defaults to 45:10:10:72. May be different if running multiple applications in a single run. The default is compatible with 150 cycle SBS kits. If using a larger kit, the Read 2 cycle information can be increased. There are diminishing returns for increased read lengths as the insert will read through the cDNA sequence into the poly-A region with longer read lengths. Read 1 should not be updated as it contains the cell barcode and binning index. Longer read lengths will need to be trimmed. Shorter read lengths will impact cell barcode identification
Sample Table	Required	The Lanes, Sample ID, and Index ID should be filled out based on how the sample will be prepared based on the library preparation kit used. See <link to prep docs , but need direct link> for more information. The optional Project field is used to specify the associated BaseSpace Project to output data to. If left empty, Project will default to the Project name derived from the Experiment/Run name.
Override Cycles	Required	Defaults to U45;I10;I10;Y72. May be different if running multiple applications in a single run.
Reference Genome	Required	Select the appropriate genome reference for the sample type. When selecting human, it is recommended to use linear references for RNA analysis. See DRAGEN Reference Support for more information.
RNA Annotation File	Optional	For custom references, use this field to select the corresponding GTF file to use for annotation. For built in references, use this field to override default annotations. The following list shows the default GTFs being used for annotation. 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
Configuration Type	Optional	Defaults to Illumina Single Cell 3’ RNA
Barcode Read	Required	Defaults to Read 1
RNA Library Type	Required	Defaults to Stranded Forward
Barcode Position	Required	Defaults to 0_7+11_16+20_25+31_38
UMI/BI Position	Required	Defaults to 39_41

Step 4: Run Review

Once all details are captured and pass validation, review the run information and choose the Edit option to correct any information.

For NovaSeq 6000/6000Dx and NextSeq 1000/2000, Export the sample sheet to be uploaded to the instrument.

For NovaSeq X Series, the run can be saved as a draft or as a planned run (via Save as Draft and Save as Planned buttons respectively). Either selection will save the run to the Planned Runs screen on BaseSpace. Once a run is saved as Planned, it will appear on the NovaSeq X Series instrument where it can be selected for sequencing.

The sample sheet for Planned runs can be downloaded by selecting the planned run and File -> Download -> SampleSheet.

For more information about the auto-launch, refer to Cloud Analysis Auto-launch. For additional information on run planning, refer to Plan Runs on Basespace Sequence Hub.