Sample Sheet Creation in BaseSpace Run Planning tool
How to Create TSO 500 ctDNA Sample Sheets in BaseSpace Run Planning tool
The BaseSpace Sequence Hub Run Planning tool is available, and is used to generate a valid sample sheet in v2 format for use on a TSO 500 ctDNA supported sequencer for both ICA and Standalone DRAGEN Server analysis options. Filling out the form on the user interface will produce a exportable sample sheet with the required fields filled in. Refer to ICA Auto-launch Sample Sheet Requirements for descriptions of fields that appear in ICA sample sheets.
The sections below represent each step in the BaseSpace Run Planning tool.
Note that NovaSeq X Series has a different run set up screen than other instrument platforms, as it allows the user to include multiple assay configurations in one run. DRAGEN TSO 500 ctDNA supports multi-assay flow cell analysis starting in version 2.6.3.
For ctDNA v2.6.0 and above, in order to run TSO 500 ctDNA on NovaSeq X Series, enter the appropriate Read 1, Read 2, Index 1 and Index 2 described in the instructions below.
BaseSpace Run Planning tool cannot generate a valid sample sheet for the NovaSeq 6000Dx TSO 500 ctDNA Analysis Application on Illumina Run Manager. Refer to to create a valid sample sheet.
Step 1: Run Settings
Parameter Name
Required
Description
Step 2: Configuration
Note: On NovaSeq X Series, this page is called "Configuration 1". The 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
Step 3: Sample Settings
Users can manually enter sample information, or download a template file to bulk upload sample information. Users can import the completed template or a compatible sample sheet.
Parameter Name
Required
Description
Step 4: Run Review
Once all details are captured and pass validation, the user can review the details on the Run Review screen. From here they can choose to edit details in previous screens or export the sample sheet. Once completed, press the Cancel button to finish run planning.
Note: once leaving this screen, the run and sample sheet will not be accessible.
For NovaSeqX Plus users, 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. There is no option to export the sample sheet on this screen.
Planned Runs Screen (NovaSeq X Series only)
The Planned Runs screen lists all planned or drafted runs. Users can set drafted runs to planned, export the sample sheet, and edit or delete a run on this screen.
Once the run is saved as Planned, it will appear on the NovaSeq X Series instrument where it can be selected for sequencing.
For more information on run planning, refer to the .
Guided Examples
Please review these guided examples of analysis workflows that include a step of setting up a run in BaseSpace Run Planning tool:
Read 1
Required on Instrument Platform NovaSeq X Series
Fill with value 151 for TSO 500 ctDNA analysis
Index 1
Required on Instrument Platform NovaSeq X Series
Fill with value 10 for TSO 500 ctDNA analysis
Index 2
Required on Instrument Platform NovaSeq X Series
Fill with value 10 for TSO 500 ctDNA analysis
Read 2
Required on Instrument Platform NovaSeq X Series
Fill with value 151 for TSO 500 ctDNA analysis
Sample Container ID
Optional
Unique Identifier for the container that holds the sample
Index ID
Required
Index set ID options are based on selected Index Adapter Kit
Project
Optional
Optional field to describe the associated project
Starts from Fastq
Required
True or False
If auto-launching TSO 500 ctDNA from BCL files, set the value to False.
If auto-launching TSO 500 ctDNA from FASTQ after auto-launching BCL Convert, set the value to True.
DNA Barcode Mismatches Index 1
DNA Barcode Mismatches Index 2
Required on NovaSeq X
Default value is set to 1.
These fields are required by NovaSeq X and represent BCL Convert settings for index diversity checks when demultiplexing. These values are not used in TSO 500 ctDNA analysis.
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.
BaseSpace/Illumina Connected Analytics (to generate sample sheet for cloud analysis. May also be used for analysis on a DRAGEN server.)
Local (only for analysis onboard the sequencer— not applicable to TSO 500 ctDNA)
None (to generate a sample sheet for analysis on a DRAGEN server. Note that cloud sample sheets may also be used for local analysis.)
TSO 500 ctDNA:
TruSight Oncology 500 ctDNA (NovaSeq6000Dx)
TruSight Oncology 500 ctDNA (NovaSeq6000)
TSO 500 ctDNA v2:
Specify lanes for each sample. The unmarked checkbox at the top of the dropdown selects all lanes.
TruSight Oncology 500 ctDNA Index Set A and B (UDP 1-192) (NovaSeq6000Dx, NovaSeqX Series)
TruSight Oncology 500 ctDNA Index Set A and B (UDP 1-192) (NovaSeq6000)
NovaSeq 6000Dx Run Set Up
The following instructions describe steps to set up a run on NovaSeq 6000Dx Analysis Application.
Use the following steps to configure a TruSight™ Oncology 500 ctDNA run in Illumina Run Manager.
Go to the "Runs" section of Illumina Run Manager by selecting "Runs" on the left-hand side.
Enter sample data manually or by importing a sample sheet
To enter sample data run manually, select “Create Run”.
Choose "DRAGEN TruSight™ Oncology 500 ctDNA Analysis Application" from the "Create Run" screen to set-up and analyze runs for TruSight Oncology 500 ctDNA assay.
Run Settings
On the "Run Settings" screen, enter a run name with the following criteria:
1 - 40 characters.
Alphanumeric characters, underscores, or dashes only.
Depending on the library prep kit selected, additional fields will be populated for run settings and are not editable. Read and index lengths will differ between library prep kit type.
Sample Data
Use the table on the "Sample Data" screen to enter sample information manually.
Alternately, select Import Samples to upload sample information. Refer to for sample sheet requirements.
Select lane information. Options include one to four, or all lanes.
Enter a unique sample ID in the sample ID field with the following criteria:
Controls should be added first.
Depending on the options selected for index set ID, additional fields will be auto-populated for sample data and are not editable.
Sample Settings
Use the table on the "Sample Settings" screen to enter additional sample information.
[Optional] Enter a sample name with the following criteria:
1 - 50 characters.
Alphanumeric characters, dashes, underscores, or spaces.
Additional fields will be auto-populated based on selections made in the Sample Data screen, which are not editable.
Before starting your run, review that the information entered is correct in the “Run Review” page before saving.
Sample Sheet Requirements
DRAGEN TSO 500 ctDNA Analysis Software has optional and required fields that are required in addition to general sample sheet requirements. Follow the steps below to create a valid samplesheet.
TSO 500L Data Section header changes depending on the deployment:
Standalone DRAGEN Server and ICA with Manual Launch: TSO500L_Data
ICA with Auto-launch: Cloud_TSO500L_Data
[TSO500L_Data] Section
Parameter
Required
Details
To ensure a successful analysis, follow these guidelines:
Avoid any blank lines at the end of the sample sheet; these can cause the analysis to fail.
When running local analysis using the command line save the sample sheet in the sequencing run folder with the default name SampleSheet.csv, or choose a different name and specify the path in the command-line options.
ICA with Auto-launch: Sample Sheet Requirements
Refer to the following requirements to create sample sheets for running the analysis on ICA with Auto-launch. For sample sheet requirements common between deployments see . Samples sheets can be created using BaseSpace Run Planning Tool or manually by downloading and editing a sample sheet template
To auto-launch analysis from the sequencer run folder, ensure the StartsFromFastq and SampleSheetRequested fields are set to FALSE. To auto-launch analysis from FASTQs after BCL Convert auto-launch, StartsFromFastq and SampleSheet Requested fields must be set to TRUE
This section describes fields specific for sample sheets for NovaSeq 6000Dx Analysis Application. For more information on DRAGEN TSO 500 ctDNA Analysis Software sample sheet requirements, refer to the sections above.
Mismatches between the samples and index primers can cause incorrect results due to loss of positive sample identification. Enter sample IDs and assign indexes in the sample sheet before beginning library preparation. Record sample IDs, indexes, and plate well orientation for reference during library preparation.
[BCLConvert_Settings] Section
Parameter Name
Required
Sample Sheet Introduction
Overview
A sample sheet is required for each analysis with DRAGEN TruSight Oncology 500 ctDNA Analysis Software. A sample sheet is a comma-separated value (*.csv) file format used by Illumina instruments, platforms, and analysis pipelines to store settings and data for sequencing and analysis. The DRAGEN TruSight Oncology 500 ctDNA Analysis Software is compatible with the sample sheet v2. For general information on the sample sheet v2, refer to Illumina Connected Software - Sample Sheet.
The sample sheet includes a list of samples and their index sequences, along with additional information required to run DRAGEN TruSight Oncology 500 ctDNA Analysis Software. For example, the library prep kit used for analysis will need to be listed in the sample sheet. Appropriate index adapter sequences are determined by the assay used to perform analysis.
When running analysis on a standalone DRAGEN server or on ICA, a valid sample sheet can be created by:
BaseSpace Run Planner (preferred), see for details
Downloading and modifying a sample sheet template following the requirements, see for details
When running analysis using a NovaSeq 6000Dx Analysis Application, a valid sample sheet can be created by:
Using the user interface of the DRAGEN TruSight Oncology 500 ctDNA Analysis Application, see for details
Downloading and modifying a sample sheet template following the requirements (see for details), then importing it to Illumina Run Manager.
The run set up section of this guide includes specific instructions to plan a run and set up a valid sample sheet for each deployment of DRAGEN TruSight Oncology 500 ctDNA Analysis Software.
Unique across all runs on the instrument.
The run name identifies the run from sequencing through analysis.
[Optional] Enter a run description. The run description must have the following criteria:
1 - 50 characters.
Alphanumeric characters or spaces only.
Spaces must be preceded and followed by an alphanumeric character.
Select kit used during library preparation:
TruSight Oncology 500 ctDNA
TruSight Oncology 500 ctDNA v2
Index adapter kit will be automatically selected based on the library prep kit selection
[Optional] Enter a library tube ID.
1 - 40 characters.
Alphanumeric characters, underscores, or dashes only.
Underscores and dashes must be preceded and followed by an alphanumeric character.
Select an index set ID for the DNA library prepared from the sample.
[Optional] Enter a library name.
Spaces, underscores, and dashes must be preceded and followed by an alphanumeric character.
[Optional] Enter a sample description with the following criteria:
1 - 50 characters.
Alphanumeric characters, dashes, underscores, or spaces.
Spaces, underscores, and dashes must be preceded and followed by an alphanumeric character.
The unique ID to identify a sample. The sample ID is included in the output file names. Sample IDs are not case sensitive. Sample IDs must have the following characteristics:
- Unique for the run.
- 1–40 characters.
- No spaces.
- Alphanumeric characters with underscores and dashes. If you use an underscore or dash, enter an alphanumeric character before and after the underscore or dash. eg, Sample1-T5B1_022515.
- Cannot be called all, default, none, unknown, undetermined, stats, or reports.
- Must match a Sample_ID listed in the BCL Convert Data section.
- Each sample must have a unique combination of Lane (if applicable), sample ID, and index ID or the analysis will fail.
Sample_Type
Required
Enter DNA
Sample_Description
Not Required
Sample description must meet the following requirements:
- 1–50 characters.
- Alphanumeric characters with underscores, dashes and spaces. If you enter a underscore, dash, or space, enter an alphanumeric character before and after. eg, Liquid-Sample_213.
SoftwareVersion
Not Required
The TSO500S software version
StartsFromFastq
Required
Set the value to TRUE or FALSE. To auto-launch from BCL files, set to FALSE. To auto-launch from FASTQ files after auto-launch of BCL Convert, set to TRUE.
SampleSheetRequested
Required
Set the value to TRUE or FALSE.
To auto-launch from BCL files, set to FALSE. To auto-launch from FASTQ files after auto-launch of BCL Convert, set to TRUE.
Sample_ID
Not Required
The same sample ID used in the Cloud_TSO500L_Data section.
ProjectName
Not Required
The BaseSpace project name.
LibraryName
Not Required
Combination of sample ID and index values in the following format: sampleID_Index_Index2
LibraryPrepKitName
Required
The Library Prep Kit used.
GeneratedVersion
Not Required
The cloud GSS version used to create the sample sheet. Optional if manually updating a sample sheet.
CloudWorkflow
Not Required
Ica_workflow_1
Cloud_TSO500L_Pipeline
Required
This value is a universal record number (URN) . The valid value is: urn:ilmn:ica:pipeline:850b4fe6-2a2f-4a85-ae57-cf082dfbefd4#DRAGEN_TruSight_Oncology_500_ctDNA_v2_6_1_4
BCLConvert_Pipeline
Required
The value is a URN in the following format: urn:ilmn:ica:pipeline: <pipeline-ID>#<pipeline-name>
How to plan a run and analyze data with multiple assays loaded individually per lane on one flow cell.
Introduction
The NovaSeq X platform supports loading samples from different assays into different lanes within a single sequencing run. To ensure compatibility, the DRAGEN TSO 500 ctDNA v2.6.3 analysis software supports sample sheets containing multiple data sections, with one section per assay (for example, TSO500S_Data and TSO500L_Data).
This section describes how the software validates and processes multi-assay sample sheets and details the specific rules and logic applied during validation.
Sample Sheet Templates
Sample Sheet templates for TSO 500 ctDNA standalone DRAGEN server and ICA manual launch analysis can be found in the table below. For auto-launch compatible sample sheets, use BaseSpace Run Planner.
DRAGEN TSO 500 ctDNA analysis software is compatible with several instruments and assay workflows, each of which have implications for the sample sheet.
Sample sheet templates contain all required fields, including index sequences in the proper orientation for all indexes from a given library prep kit. The templates are provided as a starting point for creating a sample sheet manually when launching analysis on a standalone DRAGEN server or on ICA using manual launch.
For interactive run planning or to create a sample sheet for ICA Autolaunch, use to create valid sample sheets for either local or cloud analysis. To set up a run in BaseSpace run planner, refer to .
Planning a Run with a Multi-Assay Flow Cell
In the BaseSpace Sequence Hub home page, click "Runs" -> "New Run" -> "Run Planning".
In the "Create a Run" page, select "NovaSeq X Series" as instrument platform.
Provide information for the first assay until the "Run Review" page.
For TSO 500 or TSO 500 ctDNA assays, to auto-launch multiple pipelines at once, set "" to True
In the "Run Review" page, click "Add another configuration" to add the sample prep info for the second assay.
A sample sheet containing multiple data sections (one per assay) will be available for exporting at the end of run planning.
Analyzing Data from a Multi-Assay Flow Cell
Sample Sheet Validation
The sample sheet validator determines which data section(s) to validate based on the active workflow type. The list of samples to process is generated only from the relevant section (for example, Solid or ctDNA). This ensures that only valid samples for the selected workflow are validated.
The workflow’s data section may contain fewer samples than the BCLConvert_Data section. However, all samples listed in the workflow data section must exist in BCLConvert_Data section. The downstream pipeline will only process samples found in the workflow-specific data section.
Library Prep Kits
Multi-assay sample sheets may include multiple library prep kits values separated by semicolons (";").
Validation Logic:
Workflow Type
Validation Logic
Example of Accepted Values
Solid
One and only one value must match the predefined Solid kit set: TSO500, TSO500HT, or TSO500_v2. The validation fails if none or more than one valid Solid kit is provided. The validated value will be written into the intermediate sample sheet for baseline selection.
TSO500HT
ctDNA
The library prep kit and the Sequencing_Settings section are not required. The library prep kit validation rule is skipped, and the Sequencing_Settings section is excluded from the intermediate sample sheet.
N/A
Adapter Reads
When working with multiple assays, the "AdapterRead" field in the sample sheet may include multiple adapter sequences separated by "+". The adapter read of the selected workflow will be filled in the intermediate sample sheet.
Override Cycles
Override cycles are determined by the index and read length specified in the "Reads" section of the RunInfo.xml file and assay specifics. For multi-assay flow cells, the index and read length provided in RunInfo.xml should be the longer of the sequencing cycles required by the two assays. Override cycles for assays requiring shorter indexes and read lengths will be padded to match the cycle lengths used in the run.
BCL Convert Settings and Data
Starting from BCL:
Sample Sheet Validator recalculates and inserts the correct override cycles into the BCLConvert_Data section of the intermediate sample sheet.
If index lengths vary, the logic has been enhanced to automatically pad shorter indexes with "N" to standardize cycle lengths.
Sample Sheet Validator also writes in values for: Adapter Reads, MaskShortReads, AdapterBehavior, MinimumTrimmedReadLength. These values are based on the TSO 500 ctDNA library prep kit determined from the input sample sheet.
The intermediate sample sheet includes only samples relevant to the workflow type (Solid or ctDNA).
BCL Convert (inside the TSO 500 ctDNA workflow) then generates FASTQ files only for those samples.
Starting from FASTQ:
If users run BCL Convert separately to generate FASTQ files:
The input sample sheet must already contain valid override cycles, adapter reads, and other BCL Convert Settings.
The resulting TSO 500 ctDNA workflow run will start from FASTQ input files.
BCLConvert_Settings and BCLConvert_Data sections will be excluded from the intermediate sample sheet created by the TSO 500 ctDNA workflow.
BaseSpace Autolaunch
Analyses of samples in multi-assay flow cells can be launched simultaneously from BaseSpace. As long as "Starts from FASTQ" is set to True in Analysis Settings, all samples from a multi-assay flow cell will be demultiplexed first to generate FASTQ files. Then, the ICA pipelines (e.g. DRAGEN TSO 500, DRAGEN TSO 500 ctDNA) will be launched simultaneously to process the samples for each of the assays.
For more information about auto-launching BCL Convert before starting TSO 500 analysis from FASTQ, see
Summary of Rule Changes
Component
Update Summary
Effect
Sample Index Rule
Filtered to process samples only from workflow-specific data section
Prevents validation failures for multi-assay sample sheets
Sample Parity Rule
Enforces that workflow samples exist in BCLConvert_Data
Ensures downstream consistency
Library Prep Kit Rule
Supports multiple values separated by ;
Allows multi-assay sample sheets
Adapter Read Rule
Supports multiple adapters separated by +
Users can visit the Sample Sheet guidelines section to learn additional details on required fields and values as they fill in sample information. Use the lookup tables below to select and download the sample sheet template that matches your instrument, assay, and workflow configuration.
Note: Templates are not instrument-specific. Users can fill in InstrumentPlatform and InstrumentType according to the Instrument Lookup table provided.