# 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. To create a sample sheet manually, see [Sample Sheet Requirements](https://help.connected.illumina.com/dragen-single-cell-rna/run-set-up-in-bssh/run-planning/sample-sheet-requirements) for details. Refer to [Cloud Analysis Auto-launch](https://help.connected.illumina.com/analysis/analysis_autolaunch) for more information about the Run Planning workflow and auto-launch.

{% hint style="info" %}
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.
{% endhint %}

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**.

<figure><img src="https://2107948471-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FqVEYIKB8JFfdScsTocFN%2Fuploads%2FlYmtfUUu69GO9MY2T3yK%2Fimage.png?alt=media&#x26;token=327105ff-ca82-4a77-95dc-752ee94916da" alt=""><figcaption></figcaption></figure>

## Step 1: Run Settings

<table><thead><tr><th width="206">Parameter Name</th><th width="123">Required?</th><th>Description</th></tr></thead><tbody><tr><td>Run Name</td><td>Required</td><td>Run Name can contain 255 alphanumeric characters, dashes, underscores, periods, and spaces; and must start with an alphanumeric, a dash or an underscore.</td></tr><tr><td>Run Description</td><td>Optional</td><td>Run Description can contain 8192 characters except square brackets, asterisks, and commas.</td></tr><tr><td>Instrument Platform</td><td>Required</td><td><p>Choose from DRAGEN Single Cell RNA software supported instruments:</p><ul><li>NovaSeq X Series</li><li>NovaSeq 6000/6000Dx</li><li>NextSeq 1000/2000</li></ul></td></tr><tr><td>Secondary Analysis</td><td>Required</td><td>Select BaseSpace / Illumina Connected Analytics.</td></tr><tr><td>Read 1</td><td>Required only for Instrument Platform NovaSeq X Series</td><td>45 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.</td></tr><tr><td>Index 1</td><td>Required only for Instrument Platform NovaSeq X Series</td><td>10 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.</td></tr><tr><td>Index 2</td><td>Required only for Instrument Platform NovaSeq X Series</td><td>10 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.</td></tr><tr><td>Read 2</td><td>Required on this step only for runs on NovaSeq X Series</td><td>72 for DRAGEN Single Cell RNA analysis. May be different if running multiple applications in a single run.</td></tr><tr><td>Sample Container ID</td><td>Optional</td><td>Unique identifier for the container that holds the sample.</td></tr></tbody></table>

## Step 2: Configuration

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

<table><thead><tr><th width="193">Parameter Name</th><th width="129">Required?</th><th>Description</th></tr></thead><tbody><tr><td>Application</td><td>Required</td><td>Select "DRAGEN Single Cell RNA - 4.5.0" or the latest version available</td></tr><tr><td>Description</td><td>Optional</td><td>Optional Text Field</td></tr><tr><td>Library Prep Kit</td><td>Required</td><td>Select Illumina Single Cell 3’ RNA Prep or Illumina Single Cell CRISPR Prep</td></tr><tr><td>Index Adapter Kit</td><td>Required</td><td><p>Select a supported index adapter kit:</p><ul><li>Illumina Single Cell UD 8 Indexes</li><li>Illumina Single Cell UD Indexes Set A</li></ul></td></tr><tr><td>Reference Genome</td><td>Required on this step only for runs on NovaSeq X Series</td><td><p>Select the appropriate genome reference for the sample type.</p><p>When selecting human, it is recommended to use linear references for RNA analysis. See <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-reference-support">DRAGEN Reference Support</a> for more information.</p></td></tr></tbody></table>

## Step 3: Run Configuration and Analysis Settings

The run configuration and analysis settings options differ slightly depending on the instrument platform chosen for the run in step 1. The below subsections describe the fields available for each option.

{% tabs %}
{% tab title="NovaSeq X" %}

<table><thead><tr><th width="202">Parameter Name</th><th width="122">Required?</th><th>Description</th></tr></thead><tbody><tr><td>Description</td><td>Optional</td><td>Optional Text Field</td></tr><tr><td>Library Prep Kit</td><td>Required</td><td>Auto-populated from previous step</td></tr><tr><td>Index Adapter Kit</td><td>Required</td><td>Auto-populated from previous step</td></tr><tr><td>Reference Genome</td><td>Required</td><td>Auto-populated from previous step</td></tr><tr><td>RNA Annotation File</td><td>Optional</td><td><p>For custom references, use this field to select the corresponding GTF file to use for annotation. </p><p></p><p>For built in references, use this field to override default annotations. The following list shows the default GTFs being used for annotation. </p><ul><li><p>GENCODE v19</p><ul><li>Homo sapiens [UCSC] hg19 v5</li><li>Homo sapiens [UCSC] hg19 v5 Pangenome</li><li>Homo sapiens [NCBI] hs37d5 v5</li><li>Homo sapiens [NCBI] hs37d5 v5 Pangenome</li></ul></li><li><p>GENCODE v44</p><ul><li>Homo sapiens [1000 Genomes] hg38 v5</li><li>Homo sapiens [1000 Genomes] hg38 v5 Pangenome</li></ul></li><li><p>GENCODE vM23</p><ul><li>Mus musculus [UCSC] mm10</li></ul></li><li><p>ENSEMBL 98</p><ul><li>Rattus norvegicus [UCSC] rn6 </li></ul></li></ul></td></tr><tr><td>Feature Barcode Reference</td><td>Required for Illumina Single Cell CRISPR Library Prep</td><td>Specify a CSV feature reference file that contains feature barcode information as specified in the <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna-pipseq#inputs">DRAGEN documentation</a>.</td></tr><tr><td>Custom Adapters</td><td>Optional</td><td>Select if custom adapter reads will be specified instead of those in the index adapter kit.</td></tr><tr><td>Adapter Read 1</td><td>Optional</td><td>Use this field to specify custom adapter reads.</td></tr><tr><td>Adapter Read 2</td><td>Optional</td><td>Use this field to specify custom adapter reads.</td></tr><tr><td>Override Cycles</td><td>Required</td><td>Defaults to U45;I10;I10;Y72. May be different if running multiple applications in a single run.</td></tr><tr><td>Lane Usage</td><td>Optional</td><td>Select the checkbox if samples are loaded in all lanes. If selected, the generated sample sheet will not contain the Lane column.</td></tr><tr><td>Sample Table</td><td>Required</td><td><p>The sample table should be filled out based on how the sample will be prepared based on the library preparation kit used. See <a href="https://support.illumina.com/sequencing/sequencing_kits/illumina-single-cell-prep.html">Illumina Single Cell 3' RNA Prep Documentation</a> for more information. The following fields are included in the table:</p><ul><li>Sample Name</li><li>Expression Lanes</li><li>Expression Index ID</li><li>Expression Barcode Mismatches Index 1 - the allowed number of index read 1 mismatches. The default is 1.</li><li>Expression Barcode Mismatches Index 2 - the allowed number of index read 2 mismatches. The default is 1.</li><li>Feature Lanes*</li><li>Feature Index ID*</li><li>Feature Barcode Mismatches Index 1* - the allowed number of index read 1 mismatches. The default is 1.</li><li>Feature Barcode Mismatches Index 2* - the allowed number of index read 1 mismatches. The default is 1.</li><li>Thresholding Method - specify the method for determining the count threshold value. See <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna#cell-filtering">DRAGEN documentation</a> for more details.</li><li>Expected Number of Cells</li><li>Project - 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.</li></ul><p>*Feature fields are required for Illumina Single Cell CRISPR Library Prep</p></td></tr><tr><td>Configuration Type</td><td>Optional</td><td>Specify either Illumina Single Cell 3’ RNA or Illumina Single Cell CRISPR</td></tr><tr><td>Barcode Read</td><td>Required</td><td>Defaults to Read 1</td></tr><tr><td>RNA Library Type</td><td>Required</td><td>Defaults to Stranded Forward</td></tr><tr><td>Barcode Sequence File</td><td>Optional</td><td>Not required for Illumina Single Cell Prep Kits. Specify a file containing valid cell barcode sequences. Maps to --single-cell-barcode-sequence-whitelist in command line arguments. </td></tr><tr><td>Barcode Position</td><td>Required</td><td>Defaults to 0_7+11_16+20_25+31_38</td></tr><tr><td>UMI/BI Position</td><td>Required</td><td>Defaults to 39_41</td></tr></tbody></table>
{% endtab %}

{% tab title="NovaSeq 6000" %}

<table><thead><tr><th width="202">Parameter Name</th><th width="122">Required?</th><th>Description</th></tr></thead><tbody><tr><td>Description</td><td>Optional</td><td>Optional Text Field</td></tr><tr><td>Library Prep Kit</td><td>Required</td><td>Auto-populated from previous step</td></tr><tr><td>Index Adapter Kit</td><td>Required</td><td>Auto-populated from previous step</td></tr><tr><td>Reference Genome</td><td>Required</td><td><p>Select the appropriate genome reference for the sample type.</p><p>When selecting human, it is recommended to use linear references for RNA analysis. See <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-reference-support">DRAGEN Reference Support</a> for more information.</p></td></tr><tr><td>RNA Annotation File</td><td>Optional</td><td><p></p><p>For custom references, use this field to select the corresponding GTF file to use for annotation. </p><p></p><p>For built in references, use this field to override default annotations. The following list shows the default GTFs being used for annotation. </p><ul><li><p>GENCODE v19</p><ul><li>Homo sapiens [UCSC] hg19 v5</li><li>Homo sapiens [UCSC] hg19 v5 Pangenome</li><li>Homo sapiens [NCBI] hs37d5 v5</li><li>Homo sapiens [NCBI] hs37d5 v5 Pangenome</li></ul></li><li><p>GENCODE v44</p><ul><li>Homo sapiens [1000 Genomes] hg38 v5</li><li>Homo sapiens [1000 Genomes] hg38 v5 Pangenome</li></ul></li><li><p>GENCODE vM23</p><ul><li>Mus musculus [UCSC] mm10</li></ul></li><li><p>ENSEMBL 98</p><ul><li>Rattus norvegicus [UCSC] rn6 </li></ul></li></ul></td></tr><tr><td>Feature Barcode Reference</td><td>Required for Illumina Single Cell CRISPR Library Prep</td><td>Specify a CSV feature reference file that contains feature barcode information as specified in the <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna-pipseq#inputs">DRAGEN documentation</a>.</td></tr><tr><td>Custom Adapters</td><td>Optional</td><td>Select if custom adapter reads will be specified instead of those in the index adapter kit.</td></tr><tr><td>Adapter Read 1</td><td>Optional</td><td>Use this field to specify custom adapter reads.</td></tr><tr><td>Adapter Read 2</td><td>Optional</td><td>Use this field to specify custom adapter reads.</td></tr><tr><td>Index Reads</td><td>Required</td><td>Defaults to 2 indexes</td></tr><tr><td>Read Type</td><td>Required</td><td>Defaults to Paired End</td></tr><tr><td>Read Lengths</td><td>Required</td><td><p>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.</p><p>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.</p><p>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</p></td></tr><tr><td>Lane Usage</td><td>Optional</td><td>Select the checkbox if samples are loaded in all lanes. If selected, the generated sample sheet will not contain the Lane column.</td></tr><tr><td>Sample Table</td><td>Required</td><td><p>The sample table should be filled out based on how the sample will be prepared based on the library preparation kit used. See <a href="https://support.illumina.com/sequencing/sequencing_kits/illumina-single-cell-prep.html">Illumina Single Cell 3' RNA Prep Documentation</a> for more information. The following fields are included in the table:</p><ul><li>Sample Name</li><li>Expression Lanes</li><li>Expression Index ID</li><li>Feature Lanes*</li><li>Feature Index ID*</li><li>Thresholding Method - specify the method for determining the count threshold value. See <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna#cell-filtering">DRAGEN documentation</a> for more details.</li><li>Expected Number of Cells</li><li>Project - 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.</li></ul><p>*Feature fields are required for Illumina Single Cell CRISPR Library Prep</p></td></tr><tr><td>Barcode Mismatches Index 1</td><td>Required</td><td>The allowed number of index read 1 mismatches. The default is 1 and the maximum value is 2.</td></tr><tr><td>Barcode Mismatches Index 2</td><td>Required</td><td>The allowed number of index read 2 mismatches. The default is 1 and the maximum value is 2.</td></tr><tr><td>Override Cycles</td><td>Required</td><td>Defaults to U45;I10;I10;Y72. May be different if running multiple applications in a single run.</td></tr><tr><td>Configuration Type</td><td>Optional</td><td>Specify either Illumina Single Cell 3’ RNA or Illumina Single Cell CRISPR</td></tr><tr><td>Barcode Read</td><td>Required</td><td>Defaults to Read 1</td></tr><tr><td>RNA Library Type</td><td>Required</td><td>Defaults to Stranded Forward</td></tr><tr><td>Barcode Sequence File</td><td>Optional</td><td>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.</td></tr><tr><td>Barcode Position</td><td>Required</td><td>Defaults to 0_7+11_16+20_25+31_38</td></tr><tr><td>UMI/BI Position</td><td>Required</td><td>Defaults to 39_41</td></tr></tbody></table>
{% endtab %}

{% tab title="NextSeq 1000/2000" %}

<table><thead><tr><th width="202">Parameter Name</th><th width="122">Required?</th><th>Description</th></tr></thead><tbody><tr><td>Description</td><td>Optional</td><td>Optional Text Field</td></tr><tr><td>Library Prep Kit</td><td>Required</td><td>Auto-populated from previous step</td></tr><tr><td>Index Adapter Kit</td><td>Required</td><td>Auto-populated from previous step</td></tr><tr><td>Reference Genome</td><td>Required</td><td><p>Select the appropriate genome reference for the sample type.</p><p>When selecting human, it is recommended to use linear references for RNA analysis. See <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-reference-support">DRAGEN Reference Support</a> for more information.</p></td></tr><tr><td>RNA Annotation File</td><td>Optional</td><td><p>For custom references, use this field to select the corresponding GTF file to use for annotation. </p><p></p><p>For built in references, use this field to override default annotations. The following list shows the default GTFs being used for annotation. </p><ul><li><p>GENCODE v19</p><ul><li>Homo sapiens [UCSC] hg19 v5</li><li>Homo sapiens [UCSC] hg19 v5 Pangenome</li><li>Homo sapiens [NCBI] hs37d5 v5</li><li>Homo sapiens [NCBI] hs37d5 v5 Pangenome</li></ul></li><li><p>GENCODE v44</p><ul><li>Homo sapiens [1000 Genomes] hg38 v5</li><li>Homo sapiens [1000 Genomes] hg38 v5 Pangenome</li></ul></li><li><p>GENCODE vM23</p><ul><li>Mus musculus [UCSC] mm10</li></ul></li><li><p>ENSEMBL 98</p><ul><li>Rattus norvegicus [UCSC] rn6 </li></ul></li></ul></td></tr><tr><td>Feature Barcode Reference</td><td>Required for Illumina Single Cell CRISPR Library Prep</td><td>Specify a CSV feature reference file that contains feature barcode information as specified in the <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna-pipseq#inputs">DRAGEN documentation</a>.</td></tr><tr><td>Custom Adapters</td><td>Optional</td><td>Select if custom adapter reads will be specified instead of those in the index adapter kit.</td></tr><tr><td>Adapter Read 1</td><td>Optional</td><td>Use this field to specify custom adapter reads.</td></tr><tr><td>Adapter Read 2</td><td>Optional</td><td>Use this field to specify custom adapter reads.</td></tr><tr><td>Index Reads</td><td>Required</td><td>Defaults to 2 indexes</td></tr><tr><td>Read Type</td><td>Required</td><td>Defaults to Paired End</td></tr><tr><td>Read Lengths</td><td>Required</td><td><p>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.</p><p>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.</p><p>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</p></td></tr><tr><td>Sample Table</td><td>Required</td><td><p>The sample table should be filled out based on how the sample will be prepared based on the library preparation kit used. See <a href="https://support.illumina.com/sequencing/sequencing_kits/illumina-single-cell-prep.html">Illumina Single Cell 3' RNA Prep Documentation</a> for more information. The following fields are included in the table:</p><ul><li>Sample Name</li><li>Expression Index ID</li><li>Feature Index ID*</li><li>Thresholding Method - specify the method for determining the count threshold value. See <a href="https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-single-cell-pipeline/dragen-scrna#cell-filtering">DRAGEN documentation</a> for more details.</li><li>Expected Number of Cells</li><li>Project - 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.</li></ul><p>*Feature fields are required for Illumina Single Cell CRISPR Library Prep</p></td></tr><tr><td>Barcode Mismatches Index 1</td><td>Required</td><td>The allowed number of index read 1 mismatches. The default is 1 and the maximum value is 2.</td></tr><tr><td>Barcode Mismatches Index 2</td><td>Required</td><td>The allowed number of index read 2 mismatches. The default is 1 and the maximum value is 2.</td></tr><tr><td>Override Cycles</td><td>Required</td><td>Defaults to U45;I10;I10;Y72. May be different if running multiple applications in a single run.</td></tr><tr><td>Configuration Type</td><td>Optional</td><td>Specify either Illumina Single Cell 3’ RNA or Illumina Single Cell CRISPR</td></tr><tr><td>Barcode Read</td><td>Required</td><td>Defaults to Read 1</td></tr><tr><td>RNA Library Type</td><td>Required</td><td>Defaults to Stranded Forward</td></tr><tr><td>Barcode Sequence File</td><td>Optional</td><td>Not required for Illumina Single Cell 3' RNA Prep Kits. Specify a file containing valid cell barcode sequences. Maps to --single-cell-barcode-sequence-whitelist in command line arguments. </td></tr><tr><td>Barcode Position</td><td>Required</td><td>Defaults to 0_7+11_16+20_25+31_38</td></tr><tr><td>UMI/BI Position</td><td>Required</td><td>Defaults to 39_41</td></tr></tbody></table>
{% endtab %}
{% endtabs %}

## 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, **Export** the sample sheet to be uploaded to the instrument.

For NovaSeq X Series and NextSeq 1000/2000, the run can be saved as a draft or as a planned run (via **Save as Draf**t 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 instrument where it can be selected for sequencing.

{% hint style="info" %}
The sample sheet for Planned runs can be downloaded by selecting the planned run and **File** -> **Download** -> **SampleSheet**.
{% endhint %}

For more information about the auto-launch, refer to [Cloud Analysis Auto-launch](https://help.connected.illumina.com/analysis/analysis_autolaunch). For additional information on run planning, refer to [Plan Runs on Basespace Sequence Hub](https://help.connected.illumina.com/basespace-sequence-hub/sequence/plan-runs).