# Multi-Assay Flow Cells

## 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 analysis software (v2.6.0 and above) 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.

## Planning a Run with a Multi-Assay Flow Cell

1. In the BaseSpace Sequence Hub home page, click "Runs" -> "New Run" -> "Run Planning".
2. In the "Create a Run" page, select "NovaSeq X Series" as instrument platform.
3. 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 "[Starts from FASTQ](https://help.tso500software.illumina.com/dragen-tso-500-guides/dragen-tso-500-v2.6/launching-analysis/analysis-launch-on-ica/autolaunch/auto-launch-with-fastqs-generated-by-standalone-bcl-convert-pipeline-start-from-fastq)" to True
4. In the "Run Review" page, click "Add another configuration" to add the sample prep info for the second assay.
5. A sample sheet containing multiple data sections (one per assay) will be available for exporting at the end of the 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 library prep kit determined from the input sample sheet.
* The intermediate sample sheet includes only samples listed in the TSO 500 Data section.
* BCL Convert (inside the TSO 500 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 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 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.

{% hint style="success" %}
For more information about auto-launching BCL Convert before starting TSO 500 analysis from FASTQ, see [Auto-Launch with FASTQs generated by Standalone BCL Convert Pipeline (Start from FASTQ)](https://help.tso500software.illumina.com/dragen-tso-500-guides/dragen-tso-500-v2.6/launching-analysis/analysis-launch-on-ica/autolaunch/auto-launch-with-fastqs-generated-by-standalone-bcl-convert-pipeline-start-from-fastq)
{% endhint %}

### 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 `+`                          | Expands flexibility across assays                          |
| **Override Cycles**       | Auto-calculated or user-provided depending on workflow               | Maintains correct cycle definitions per sample             |