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 appear in the AVITI OS based on compatibility with the AVITI System. Factors that affect compatibility are 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	AVITI OS and Instrument Compatibility	Add-On Requirement
Two Library Pools	2.3.0 or later - AVITI LT, AVITI, AVITI24™	Individually Addressable Lanes
Low-Diversity High-Multiplex w/ 2 × 300 Cloudbreak™ + Adept™ library	2.4.0 or later - AVITI, AVITI24	No add-on required
Low-Diversity High-Multiplex w/ other kits or library types	2.5.0 or later - AVITI LT, AVITI, AVITI24	No add-on required
Cloudbreak Freestyle™ Sequencing Kit	2.5.0 or later - AVITI LT, AVITI, AVITI24	No add-on required
Cloudbreak UltraQ™ Sequencing Kit	2.6.0 or later - AVITI, AVITI24	No add-on required
Custom Recipe	2.6.0 or later - AVITI LT, AVITI, AVITI24	Add-on required for AVITI OS 3.0.0 or earlier
Filter Mask	2.6.0 or later - AVITI LT, AVITI, AVITI24	Filter Mask
High Density or Expert Mode HD (including 2 × 300 Cloudbreak sequencing kit)	2.6.0 to 3.3.0 - AVITI, AVITI24 3.4.0 or later - AVITI, AVITI24 only with 2 x 300 Cloudbreak sequencing kit	Earlier than 3.0.0 - Add-on required 3.0.0 or later - No add-on required
PMG Shift	3.0.0 or later - AVITI LT, AVITI, AVITI24	PMG Shift
Trinity™ Sequencing Kit	3.0.0 or later - AVITI, AVITI24	No add-on required
Cloudbreak 2 × 75 Low Sequencing Kit	3.1.0 or later - AVITI LT, AVITI, AVITI24	No add-on required
Trinity Sequencing Kit with Third Party	3.4.0 or later - 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 Cloudbreak.

3. Enter a unique name.

4. (Optional) Provide a description of your run.

5. If you have a prepared sequencing run manifest, upload the CSV file.

6. Select Adept, Elevate™, or Third Party.

7. Select one of the following library structures:

   • Circular: (For Adept only) Sequences libraries that complete circularization before loading.
   • Linear: (For Adept, Elevate, and Third Party) Sequences libraries prepared for on-instrument circularization.

8. Select your sequencing kit.

9. (For Adept and Third Party) In the Low-Diversity High-Multiplex field, select Yes or No.

   Select Yes to sequence low-diversity high-multiplex libraries, which requires at least 4 cycles for Index 1. Select No to sequence other libraries.

10. In the Library Pools field, if you use Individually Addressable Lanes add-on, then select 1 or 2.

11. In the Cycles field, 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. (Optional) Select Advanced Run Settings, and then proceed to Configure Advanced Run Settings.

    Planned runs that use an Advanced Run Setting might only be available on instruments that have the specific feature enabled. For more information, see the planned runs compatibility matrix.

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 for your run.

4. (Optional) Provide a description of your run.

5. If you have a prepared sequencing run manifest, upload the CSV file.

6. In the Library Type field, select Elevate or Third Party.

7. In the Sequencing Kit field, select your sequencing kit.

8. In the Panel field, select one of the following panels:

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

9. In the Cycles field, 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
   Third Party	2 x 75	0	0	5	0	184
   Third Party	2 x 150	0	0	5	0	334
   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

10. (Optional) Select Advanced Run Settings, and then proceed to Configure Advanced Run Settings.

    Planned runs that use an Advanced Run Setting might only be available on instruments that have the specific feature enabled. For more information, see the planned runs compatibility matrix.

11. 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 the Expert Mode HD setting (High Density), relaxes certain quality filters to increase the total number of polonies in a run for increased read output. For AVITI, High Density or Expert Mode HD is available with v2.6.0 or later. For AVITI24, High Density or Expert Mode HD is available from v2.6.0 to v3.3.0. Starting with AVITI OS 3.4.0, AVITI24 computational enhancements maximize read output for 2 x 75 and 2 x 150 kits, and Expert Mode HD is available only for 2 x 300 kits on AVITI24.

To apply the Expert Mode HD setting, select one of the following options:

• 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 specific applications. When you apply this feature, run output data and on-instrument run metrics account for the filter mask. To apply a filter mask, complete the following:

• 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

When you use the Custom Recipe feature, a run is executed in coordination with Element Biosciences™ and tailored to your needs. A recipe governs the stages of a run and can impact specifications and significantly increase run times. To apply a custom recipe, select the drop-down menu in the planned run, and then select a recipe based on the following 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) for compatibility with specific sequencing runs for Adept or third-party libraries. This setting is beneficial for libraries that have low diversity at the start of read 1. Also, skipped cycles do not affect data output when an Index Read is included in the run. Using PMG shift without any Index Reads excludes the specified number of shifted bases from Read 1.

To use PMG shift, complete the following:

• Enter the number of cycles (0-20) to shift PMG. 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 PMG Shift, then the default value is 0 cycles.
• PMG Shift is available for the following:
  • Cloudbreak with Adept
  • Cloudbreak Freestyle with third-party libraries
  • Trinity with 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, make sure that you resolve the errors and re-upload the run manifest. For more information 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.