Run Planning for Sequencing

Planned runs allow team members to create and manage runs in ElemBio Cloud for use on any connected AVITI System. A team member can plan a run in ElemBio Cloud, then select it during run setup. The Planned Runs table displays saved configuration information for planned runs. You can only edit or delete planned runs through ElemBio Cloud.

Run Progression

After an instrument starts processing a planned run, the following rules apply:

• During the run setup and before priming begins, the status of a planned run appears as In Use.
• If you discard a planned run before priming, the In Use status is removed and the run becomes available for selection on the instrument.
• After priming, the run moves from the Planned Runs table to the Active Runs and All Runs pages.
• If you discard the run or the run fails after priming, the run is no longer available as a planned run. To make the run available again, you must re-create the planned run.

Compatibility

Planned runs only appear in the AVITI OS if they are compatible with the AVITI System. Compatibility is based on on the AVITI OS version, AVITI System type, and available add-ons. When you add or edit a planned run, a statement at the bottom of the form identifies the run compatibility requirements.

The following table defines the compatibility requirements for different configurations of a planned sequencing run.

Planned Run Configuration	Compatible AVITI OS Version	Compatible AVITI Systems	Add-On Requirement
Two library pools	2.3.0+	AVITI LT, AVITI, AVITI24	Individually Addressable Lanes
Low-Diversity High-Multiplex with a 2 x 300 Cloudbreak™ sequencing kit and an Adept library	2.4.0+	AVITI, AVITI24	No add-on required
Low-Diversity High-Multiplex with other kits or library types	2.5.0+	AVITI LT, AVITI, AVITI24	No add-on required
Cloudbreak Freestyle™ sequencing kit	2.5.0+	AVITI LT, AVITI, AVITI24	No add-on required
High Density or Expert Mode HD	2.6.0+	AVITI, AVITI24	Add-on required below AVITI OS v3.0.0
Filter Mask	2.6.0+	AVITI LT, AVITI, AVITI24	Advanced Run Settings: Filter Mask
Custom Recipe	2.6.0+	AVITI LT, AVITI, AVITI24	Add-on required below AVITI OS v3.0.0
Cloudbreak UltraQ™ sequencing kit	2.6.0+	AVITI, AVITI24	No add-on required
PMG Shift	3.0.0+	AVITI LT, AVITI, AVITI24	Advanced Run Settings: PMG Shift
Trinity™ sequencing kit	3.0.0+	AVITI, AVITI24	No add-on required
Cloudbreak 2 x 75 Low sequencing kit	3.1.0+	AVITI LT, AVITI, AVITI24	No add-on required

Add a Planned Run

Sequencing with Cloudbreak

1. On the Planned Runs page, select Plan a Run.

2. Select Sequencing with Cloubreak.

3. Enter a unique name.

4. If you have prepared a run manifest, then upload the CSV file.

5. (Optional) Enter a description for your planned run.

6. Select a Library Type: Adept, Elevate, or Third Party.

7. If you use an Adept library, then select a Library Structure.

   • Circular: Sequences libraries that complete circularization before loading.
   • Linear: Sequences libraries prepared for on-instrument circularization.

8. In the Sequencing Kit drop-down menu, select your kit.

9. If you use an Adept or third-party library, then select a Low-Diversity High-Multiplex option.

   • Yes: Sequence low-diversity high-multiplex libraries. Selecting Yes requires at least 4 cycles for Index 1.
   • No: Sequence other libraries.

10. If you use the Individually Addressable Lanes add-on, then select the number of libraries.

11. In the Cycles fields, enter the number of cycles to perform in each read.

    • To skip a read, enter 0.
    • For bioinformatics purposes, add one cycle to Read 1 and Read 2. For example, enter 151 in the Read 1 field to perform 150 cycles in Read 1.
    • See the following table for minimum cycle values for each read and the maximum number of total cycles. Excluding these limitations, you can distribute the available cycles among reads as necessary.

    Library Type	Kit	Index 1 Minimum	Index 2 Minimum	Read 1 Minimum	Read 2 Minimum	Maximum Total Cycles
    Adept or Third Party	2 x 75	0	0	5	0	184
    Adept or Third Party	2 x 150	0	0	5	0	334
    Adept or Third Party	2 x 300	0	0	5	0	634
    Elevate	2 x 75	4	0	5	0	184
    Elevate	2 x 150	4	0	5	0	334
    Elevate	2 x 300	4	0	5	0	634

12. If you use the Advanced Run Settings add-on, then select Advanced Run Settings and proceed to Configure Advanced Run Settings.

    Planned runs that use an Advanced Run Setting are only available on instruments with the specific feature enabled.

13. Select Save Run.

    ElemBio Cloud saves the run, which appears in the Sequencing Planned Runs table.

Sequencing with Trinity

1. On the Planned Runs page, select Plan a Run.

2. Select Sequencing with Trinity.

3. Enter a unique name.

4. If you have prepared a run manifest, then upload the CSV file.

5. (Optional) Enter a description for your planned run.

6. In the Sequencing Kit drop-down menu, select your kit.

7. In the Panel drop-down menu, select your panel.

   • Twist for Element, Trinity Exome Workflow: For use with Twist Exome standard or fast hybridization protocols.
   • xGen Exome Kit for Trinity: For use with the IDT xGen Exome hybridization protocol.
   • Other: For use with custom panel.

8. In the Cycles fields, enter the number of cycles to perform in each read.

   • To skip a read, enter 0.
   • For bioinformatics purposes, add one cycle to Read 1 and Read 2. For example, enter 151 in the Read 1 field to perform 150 cycles in Read 1.
   • See the following table for minimum cycle values for each read and the maximum number of total cycles. Excluding these limitations, you can distribute the available cycles among reads as necessary.

   Library Type	Kit	Index 1 Minimum	Index 2 Minimum	Read 1 Minimum	Read 2 Minimum	Maximum Total Cycles
   Elevate	2 x 75	4	0	5	0	184
   Elevate	2 x 150	4	0	5	0	334

9. (Optional) Select Advanced Run Settings, and then configure the Advanced Run Settings.

   Some Advanced Run Settings require an add-on.

10. Select Save Run.

    ElemBio Cloud saves the run, which appears in the Sequencing Planned Runs table.

Configure Advanced Run Settings

At the bottom of the sequencing run planning form, Advanced Run Settings can be opened. When you select a kit in the Sequencing Kit drop-down menu, ElemBio Cloud enables the settings for the available features.

For more information about the features that require add-ons, see the compatibility table.

Polony Density

The Polony Density feature, also known as Expert Mode HD, relaxes certain quality filters to increase the total number of polonies in a run for increased read output. High Density is not available for AVITI LT Systems. To apply Expert Mode HD, select an option:

• Standard: Uses the standard read output (default option)
• High Density: Increases the read output.

Filter Mask

The Filter Mask feature modifies the cycles that are used for filtering, which is beneficial for certain applications. Applying the feature causes run output data and on-instrument run metrics to account for the filter mask. To apply a filter mask:

• Enter the filter mask in base mask format.
• If you do not use the Filter Mask feature, then the default filter mask is R1:Y15N*-R2:Y15N*.

Custom Recipes

The Custom Recipe feature tailors a run execution in consultation with Element by enabling a custom recipe. A recipe governs the stages of a sequencing run and custom recipes can impact specifications and significantly increase run times. To apply a custom recipe, use the drop-down menu in the planned run, and select a recipe based on two options:

• Preloaded recipe: Select a recipe already loaded onto AVITI Systems, such as short insert and long insert custom recipes. Before you use a preloaded recipe, contact Element Technical Support to verify the run compatibility. The following pre-loaded recipes are available:
  • Short insert recipe: If you use Cloudbreak Freestyle chemistry and libraries with ≤ 100 bp DNA inserts, such as miRNA libraries, then use the short insert recipe. The recipe is only compatible with 2 x 75 and 2 x 150 sequencing kits.
  • Long insert recipe: If you use Cloudbreak or Cloudbreak Freestyle chemistry and libraries with > 1000 bp DNA inserts, then use the long insert recipe. The recipe is only applicable for 2 x 75 and 2 x 150 sequencing kits, as 2 x 300 sequencing kits already account for long insert conditions.
  • AVITI OS 3.0.0 recipe: For use with AVITI OS 3.0.0 Cloudbreak planned runs.
• Uploaded recipe: Element prepares an encrypted, custom recipe package as a .rec file for upload to a planned run. To obtain a custom recipe for a specific use case, contact Element Technical Support.

PMG Shift

The PMG Shift feature allows a user to shift (dark-cycle) up to 20 cycles of PMG (Polony Map Generation). To use PMG shift:

• Enter the number of cycles (0-20) to shift Polony Map Generation.
• This value counts toward the total number of cycles. If the kit maximum is exceeded, then then an error is returned.
• If you do not use the PMG Shift feature, then the default value is 0 cycles.
• Available for Cloudbreak and Cloudbreak Freestyle Adept and Third Party libraries.

Run Manifest Validation

When you add or edit a planned run, ElemBio Cloud validates any attached run manifest. Validation ensures that the run manifest is compatible with the planned run setup and settings that are specified inside the manifest do not result in demultiplexing errors. If the run manifest contains mistakes or invalid data, then ElemBio Cloud displays a list of errors and warnings.

You can save a planned run with warnings, but not with errors. Before you proceed, resolve the errors and re-upload the run manifest. For help on how to set up a cytoprofiling run manifest, see the Run Manifest Documentation.

Edit a Planned Run

1. On the Planned Runs page, locate the run that you want to edit.
2. In the Actions column, select More, and then select Edit.
3. Update the run information as needed.
4. Select Save Run.

Delete a Planned Run

1. On the Planned Runs page, locate the run that you want to delete.
2. Select the Run Name. Or, in the Actions column, select More, and then select Delete.
3. When prompted, enter the name of the planned run.
4. Select Delete to immediately remove the run.