Configure the settings for the analysis type selected for your run.

To perform a secondary analysis, an Application may require certain types of files, such as:

• AuxCnvPanelOfNormalsFile - for Enrichment workflow

• AuxNoiseBaselineFile - for Enrichment workflow

• BedFile - for Enrichment workflow

• RnaGeneAnnotationFile - for RNA workflow

Reference files for NovaSeq X analysis are managed from Resources page.

• Select Resources from the user menu on the top right of Sequence Hub page

• Select Reference Files tab to see the list of reference files available for use in the run planning.

• Both standard and custom files are included in the list.

Import a Custom Reference File

1. Select Import Custom Reference File to upload a custom file.

2. After file upload is completed,

   • Select the correct File Type. Run Planning tool will associate the new custom file with the Application based on the selected File Type.

   • Select one or more reference genome(s) that should be associated with the file.

   • [Optional] Enter a description for the custom reference file.

3. Select Save.

Edit a Custom Reference File

To edit a custom reference file's metadata, go to the listing page and select the file. Update the information on the Edit page and save it upon completion.

To update the file content, select Import Custom Reference File from the listing page and upload the new file.

Delete a Custom Reference File

To delete a custom reference file, go to the listing page, and select the delete icon beside the file.

Import Samples from CSV file is an alternative way to populate Sample table and sample-level analysis settings on the Configuration page.

CSV Template

The CSV template only includes editable columns in the Sample Table and the sample-level analysis settings if applicable (e.g. for RNA Seq application with Differential Expression). Import will auto-populate the derived columns from the input data, e.g. Index names and sequences are derived from the respective Well Position.

• For custom or standard kit with fixed plates:

  • Sample ID*, Well Position*, Project, and sample-level analysis settings (if applicable)

• For custom or standard kit with fixed indexes:

  • If single index strategy is selected: Sample ID*, I7 Index* , Project, and sample-level analysis settings (if applicable)

  • If dual indexes strategy is selected: Sample ID*, I7 Index* , I5 Index*, Project, and sample-level analysis settings (if applicable)

• For Not Specified kit:

  • If single index strategy is selected: Sample ID*, Index 1*, Project, and sample-level analysis settings (if applicable)

  • If dual indexes strategy is selected: Sample ID*, Index 1*, Index 2*, Project, and sample-level analysis settings (if applicable)

Columns marked with asterisks are mandatory.

Import behaviors

• Import will fail if an incorrect CSV template is used (columns do not match the Sample table).

• All rows in the input CSV file will be imported.

• Invalid inputs will result in blank cells in the Sample table.

  • Invalid well position or Index name when custom or standard kit is used

  • Incorrect data type, e.g. string input when a number is expected

  • Out-of-range value

  • Invalid option for a select or dropdown list field

• If there are multiple rows defining the same Sample ID, the sample-level analysis settings must be consistent.

  • If different values are specified, the resulting sample-level analysis settings will be blank.

Lane numbers

For instruments that support loading separate lanes, the CSV template also includes Lanes column.

• Enter one or more lane numbers in this column. When multiple lane numbers are specified, separate the values by commas.

• If a Lanes cell contains an invalid lane number, the resulting cell will be blank.

Overview

Use Run Planning tool in BaseSpace Sequence Hub to create and configure your run settings.

If your instrument is configured for Cloud mode or Hybrid mode, submit the run configuration to your BaseSpace Sequence Hub account. The run becomes available in the planned runs control software of the instrument.

If your instrument is configured for Local mode, use Run Planning tool to create your sample sheet in v2 file format. Alternatively, create a sample sheet without BaseSpace Sequence Hub using a provided template.

If your instrument is not configured for Cloud mode integration, you can export the sample sheet in v2 file format. Placing the sample sheet in the instrument run folder and sequencing with Run Monitoring and Storage enabled will allow the specified analysis to launch automatically when the upload to BaseSpace Sequence Hub completes.

Planning a run in BaseSpace Sequence Hub is available for the following sequencing systems:

• NextSeq 1000 and NextSeq 2000 Sequencing Systems - see Plan a NextSeq 1000/2000 Run

• NovaSeq X Series Sequencing Systems - see Plan a NovaSeq X Series Run

1. Select the Runs tab, and then select the New Run drop-down.

2. Select Run Planning.

3. Run Settings wizard will be loaded.

4. In the Run Name field, enter a unique name of your preference to identify the current run. The run name can contain a maximum of 255 alphanumeric characters, spaces, dashes, and underscores.

5. [Optional] In the Run Description field, enter a description of the current run. The run description can contain a maximum of 255 alphanumeric characters.

6. Select the Instrument Platform.

7. Select the analysis location. Depending on the selected instrument type, not all options may be available.

   • BaseSpace - Analyze sequencing data in the cloud.

   • Local - Analyze sequencing data on-instrument or generate a Sample Sheet v2 for Local or Hybrid mode.

8. [Optional] In the Library Tube ID field, optionally enter the library tube ID of the current run. The library tube id can contain a maximum of 255 alphanumeric characters.

9. Select Next

10. Configuration wizard will be loaded

11. Select an analysis type and version. For more information about secondary analyses, see DRAGEN Secondary Analysis Output Files on the system guide for your instrument or the BaseSpace Sequence Hub app documentation. If you selected DRAGEN Single Cell RNA analysis, see the NextSeq 1000/2000 Products Files page for information on third-party single cell RNA library prep kit compatibility.

    For on-instrument analysis, the version selected must match the version of DRAGEN installed on the instrument. To confirm the version of DRAGEN installed on the instrument, see DRAGEN Workflow and License Updates on the system guide for your instrument.

12. [Optional] Set up custom index kits as follows. If you are using more than one library, the libraries must have the same index read lengths.

    1. Select Add Custom Index Adapter Kit under the Index Adapter Kit dropdown.

    2. Select a template type and enter the kit name, adapter sequences, index strategies, and index sequences. Make sure the second index (i5) adapter sequences are in forward orientation.

    3. Select Create New Kit.

13. [Optional] Set up custom library prep kit as follows.

    1. Select Add Custom Library Prep Kit under the Library Prep Kit dropdown.

    2. Enter the name, read types, default read cycles, and compatible index adapter kits for your custom library prep kit.

    3. Select Create New Kit.

14. Select the following instrument settings. Depending on the library prep kit, recommended options are automatically selected. Some library prep kits have hard-coded number of indexes reads and read types, which cannot be changed.

    • Library prep kit

    • Index adapter kit

    • Number of index reads

    • Read type

    • Number of sequencing cycles per read

    If Not Specified is selected for library prep kit, enter the number of Index sequencing cycles to be used in the run.

15. Enter sample information into the Sample Data spreadsheet using one of the following options. To group samples for data aggregation during downstream analysis, assign a name for the group in the Project column.

    • Select Import Samples and the type of the import source file in the dropdown, either CSV or Sample Sheet, and then select your source file. If CSV file is selected, make sure that your file follows the template that can be downloaded by selecting Download Template button.

      If Sample Sheet is selected, make sure that your sample sheet meets the formatting requirements. If CSV is selected, make sure to use the correct template as the template can be different depending on the selected index adapter kit and index strategy. For more details, see Import Samples.

    • Paste sample IDs and either index plate well positions or i7 and i5 indexes directly from an external file. Before pasting, enter the number of sample rows in the Rows field, and then select +. Sample IDs can contain up to 100 alphanumeric characters, hyphens, and underscores.

      Fixed-layout index plates require entries for well position. Indexes that do not have a fixed layout require entries for i7 and i5 indexes. i5 indexes must be entered in the forward orientation.

    • Manually enter sample IDs and corresponding well positions or indexes. If Not Specified is selected for the library prep kit, enter Index 2 (i5) sequences in the forward orientation.

NovaSeq X Series systems allow you to perform multiple DRAGEN analyzes in a single sequencing run. Before setting up secondary analysis, make sure you have installed the appropriate DRAGEN application on your instrument. For more information on installing DRAGEN applications, refer to the NovaSeq X Series Product Documentation.

If storing data in the cloud, you can create up to seven analysis application and reference genome combinations with an additional BCL Convert-only application. If storing data locally, you can create up to three analysis application and reference genome combinations with an additional BCL Convert-only application. For each combination, you can use up to eight configurations using a different library prep kit, index adapter kit, or configuration settings for an analysis application and reference genome combination already used.

The following combinations are included in the seven or three configuration limit:

• The same analysis application and application version with a different reference genome

• The same reference genome with a different application or application version

• A different application or application version with a different reference genome

Analysis Configuration Template is a template containing configuration/settings for a secondary analysis to allow planning a run on Clarity LIMS. Analysis Configuration Templates created on BaseSpace Sequence Hub can be retrieved and used in a workflow on Clarity Lab Information Management System (LIMS).

To use Analysis Configuration Template, please turn on the Advanced LIMS Run Planning from the User Account Settings page (Note that Workgroup admin privilege is required). Once the setting is enabled, go to the Resources page by selecting Resources from the User Account menu and select the Analysis Configuration Templates tab.

Create an Analysis Configuration Template

1. In the Template Name field, enter a unique name of your preference to identify the template.

2. [Optional] In the Template Description field, enter a description for the template.

3. Select the Instrument Platform

   Analysis Configuration Template is currently only supported for NovaSeq X Series.

4. Select the analysis location.

   • Cloud - Analyze sequencing data using Illumina pipeline in the cloud.

   • Local - Analyze sequencing data on-instrument.

5. Select an analysis type and version from the Application dropdown.

6. Select the Reference Genome (only applicable if analysis type is not BCL Convert).

7. Enter other settings that are applicable for the selected analysis.

   RNA Differential Expression settings is not supported in Analysis Configuration Template. Please setup the Differential Expression on BaseSpace Sequence Hub after creating the planned run on Clarity.

8. Select Save to save the template.

9. The saved template shall be available on Clarity LIMS. After a run is planned on Clarity LIMS, it can be opened on BaseSpace Sequence Hub Run Planning for further editing. Please note that sample and kit information are not editable.

Edit an existing Analysis Configuration Template

1. To begin editing, click on the template name hyperlink

2. Update the settings

3. Select the Save button to save the changes or the Cancel button otherwise

Delete an existing Analysis Configuration Template

To delete a template, select the trash button located at the right most column.

If using somatic mode, you can generate a custom noise baseline file. The noise baseline file is built using normal samples that do not match to the subject the samples are from. The recommended number of normal samples is 50.

To generate a custom noise baseline file, use one of the following methods:

• Use the DRAGEN Bio-IT Platform server. See the DRAGEN Bio-IT Platform Online Help for instructions.

• Use DRAGEN Baseline Builder App on BaseSpace Sequence Hub. Use the BCL Convert pipeline in BaseSpace Sequence Hub Run Planning to generate FASTQ files. After the sequencing run is complete and 50 samples are available, input the FASTQ files into the DRAGEN Baseline Builder App.

The MiniSeq, NextSeq, and NeoPrep systems provide an option to set up a run in BaseSpace Sequence Hub using the Prep tab.

When you complete run setup in BaseSpace Sequence Hub, the run becomes available in the control software of the instrument. For instructions to complete the run, see the system guide for your instrument:

• MiniSeq System Guide (document # 1000000002695)

• NextSeq 550 System Guide (document # 15069765)

• NextSeq 500 System Guide (document # 15046563)

• NeoPrep System Guide (document # 15049720)

Use the following instructions to plan a run for the NovaSeq X series systems in BaseSpace Sequence Hub.

1. Select the Runs tab, and then select the New Run drop-down.

2. Select Run Planning.

3. In the Run Name field, enter a unique name of your preference to identify the current run. The run name can contain a maximum of 255 alphanumeric characters, spaces, dashes, and underscores.

4. [Optional] Enter a description for the run. The run description can contain a maximum of 255 characters.

5. Select your sequencing system as the instrument platform.

6. Select one of the following analysis locations.

   • BaseSpace — Analyze sequencing data in the cloud.

   • Local — Analyze sequencing data on-instrument. When this option is selected, the planned run can only be exported to a sample sheet v2 file.

7. If analyzing data locally, select one of the following FASTQ output format options: The output format is not available for cloud storage and only applicable if you select to keep FASTQ files when setting up secondary analysis.

   • gzip — Save the FASTQ files in gzip format.

   • DRAGEN — Save FASTQ files in ora format. See for more details.

8. Enter the number of cycles performed in each read: If using multiple analysis configurations, use the longest read length required by the configuration. When setting up a configuration, override automatically trims the length based on the recommended lengths for the selected library prep kit.

   • Read 1 — Enter up to 151 cycles.

   • Index 1 — Enter the number of cycles for the Index 1 (i7) primer. For a PhiX-only run, enter 0 in both index fields.

   • Index 2 — Enter the number of cycles for the Index 2 (i5) primer.

   • Read 2 — Enter up to 151 cycles. This value is typically the same as the Read 1 value.

9. [Optional] Enter the ID for your library tube. The library tube ID is located on the label of your library tube strip.

10. Select Next.

Please take note of the following when setting up a configuration.

• Instrument Platform and Analysis location in Run Settings page are not editable once a Configuration is created.

• Application version cannot be changed once a Configuration is saved. You need to delete the configuration and create a new one instead.

1. Select your analysis application.

2. [Optional] Enter a description for the configuration.

3. Select a library prep kit or add a new custom library prep kit as follows.

   • Select Add Custom Library Prep Kit under the Library Prep Kit dropdown.

   • Enter the name, read types, default read cycles, and compatible index adapter kits for your custom library prep kit.

   • Select Create New Kit.

4. Select an index adapter kit or add a new a custom index kits as follows. If you are using more than one library, the libraries must have the same index read lengths.

   • Select Add Custom Index Adapter Kit under the Index Adapter Kit dropdown.

   • Select a template type and enter the kit name, adapter sequences, index strategies, and index sequences. Make sure the second index (i5) adapter sequences are in forward orientation.

   • Select Create New Kit.

5. If applicable to your application, select a reference genome.

6. Select Next to configure secondary analysis settings.

1. In the Libraries page, select the Library Prep Kit drop-down arrow, and select a kit.

2. In the Plate ID field, enter a unique plate ID.

3. [Optional] In the Notes field, enter any notes. For Nextera Rapid Capture libraries, specify that the plate contains multiple enrichments.

4. [Optional] Assign the sample to a different project.

   1. Select the checkbox of each library to assign to a project.

   2. Select Set Project.

   3. In the Select Project dialog, select or create a project to assign libraries to, and then select Select.

5. Select Auto Prep to automatically assign each library to a well and index location. For Nextera Rapid Capture libraries, samples belonging to the same enrichment must be in the same row. Each plate has a maximum layout of 12 indexes by 8 indexes. If you require more wells or indexes, create multiple plates.

6. [Optional] To change indexes, select an index on the plate and select a new index from the drop-down list. NOTE: This option is not available for fixed layout plates.

7. [Optional] Move libraries to a different well as follows. Select the checkbox of the library to move. Drag the selected library from the Libraries area to the appropriate well.

8. Select Export to save your library prep settings in a .csv file. This file serves as a reference when preparing samples in the lab.

9. [Optional] Select additional plates as follows.

   1. SelectSave and Continue Later to return to Libraries.

   2. Select the checkbox of each library you want to use.

10. Select Pool Libraries.

1. On the Pools page, drag a library from the plate to a pool. For Nextera Rapid Capture libraries, assign samples from the same enrichment to 1 pool.

2. In the Pool ID field, enter a unique name for the pool.

3. [OptionalSelect Add Pool to add a pool.

4. [Optional] Use 1 of the following methods to pool libraries from multiple plates:

   • Select the Plate ID drop-down arrow to switch between plates.

   • Select Save & Continue Later, select the checkbox of each pool to merge, and then select Merge Pools.

5. Select Plan Run.

Use the following steps to import biological samples from an external .csv file.

1. From the runs page, select New Run, and then select Prep Tab.

2. From the Prep page, select Biological Samples.

3. Select Import.

4. [Optional] Create a .csv file as follows.

   1. Select the spreadsheet image to download a template.

   2. Complete the following fields for each sample:

      • Sample ID—Enter a unique sample ID.

      • Name—Enter a descriptive name for the biological sample.

      • [Optional] Species—Enter the appropriate species.

      • Project—Enter the name of the project to save samples to. The selected project is the default project that contains biosample data output.

      • Nucleic Acid—Enter RNA or DNA.

   3. Save the file.

5. Select Choose .csv File.

6. Browse to the appropriate file and select Open. Information from the .csv file populates the Biological Samples page.

7. [Optional] Select additional samples as follows.

   1. Select Save & Continue Later.

   2. Select the checkbox for each sample you want to use.

8. Select Prep Libraries.

Complete the following steps to create biological samples one at a time. For information about adding multiple samples, see Import Biological Samples.

1. Select the Prep tab, and then select Biological Samples.

2. Select Create.

3. Provide the following information:

   • Sample ID—Enter a unique sample ID.

   • Name—Enter a descriptive name for the biological sample.

   • [Optional] Species—Select a species from the drop-down list.

   • Project—Select Select Project to select or create a project to add your samples to. The selected project is the default project that contains biosample data output.

   • Nucleic Acid—Select the type of nucleic acid.

4. [Optional] Select additional samples as follows.

   1. Select Save & Continue Later.

   2. Select the checkbox for each sample you want to use.

5. Select Prep Libraries.

1. On the Plan Run page, click the Select Instrument drop-down arrow, and select a sequencing system, either MiniSeq or NextSeq.

2. In the Name field, enter a name for the sequencing run.

3. [Optional] In the Reagent Barcode field, enter the barcode ID of the reagent kit used for the run. Entering the barcode ID links the reagent kit to the run.

4. [Optional] Select Use Custom Primer options:

   • R1 — Use custom primer for Read 1.

   • R2 — Use custom primer for Read 2.

5. Select a read type, either Single Read or Paired End.

6. Enter the number of cycles for each read in the sequencing run:

   • Read 1 Cycles — Enter a value up to 151 cycles.

   • Read 2 Cycles — Enter a value up to 151 cycles. This value is typically the same number of cycles as Read 1.

7. Review the indexing scheme for the run. To make changes, override the defaults as follows.

   1. Select the Override default indexing scheme checkbox.

   2. Select an indexing scheme:

      • Single Index — Performs a run with 1 index read.

      • Dual Index — Performs a run with 2 index reads.

      • No Index — Performs a non-indexed run.

   3. Enter the number of cycles for each index read*:

      • Index 1 Cycles — Enter the number of cycles required for the Index 1 (i7) primer.

      • Index 2 Cycles — Enter the number of cycles required for the Index 2 (i5) primer.

8. Make sure that a pool is present.

9. Select 1 of the following buttons to continue:

   • Sequence — The run appears on the Planned Runs list with a status of Ready. The run becomes available from the control software of your sequencing system.

   • Save and Continue Later — The run appears on the Planned Runs list with a status of Planning. When you are ready to sequence, select the checkbox for the run and select Sequence. The run then becomes available from the control software of your sequencing system.

1. In the Review Run Details page, review the run parameters.

2. If the parameters are acceptable, select Finish.

3. [Optional] Select the run setup details or library card mapping to view or print details.

4. Select Done to return to the NeoPrep Runs page.

The new run is listed with a status of Ready, which means the run is listed in the NeoPrep control software.

If your library prep kit is not available on the Libraries page, use the following instructions to create it.

1. When prepping a library, select + Custom Library Prep Kit in the Library Prep Kit dropdown menu. The Custom Library Prep Kit Definition page opens.

2. Fill out the name of the custom prep. It has the following requirements:

   • Unique for your account.

   • Characters: only alphanumeric, hyphen, underscores, and spaces accepted.

   • Less than or equal to 50 characters.

3. Select at least 1 of the supported read types.

4. Select at least 1 of the indexing strategies. Only selecting None is not allowed.

5. Fill out the default number of cycles.

6. Select template to download the index definition file template.

7. Fill out the Settings section the following way:

   • For single read only: no adapter (blank), or 1 adapter sequence for Read 1.

   • For paired-end: no adapter (blank), or 2 adapter sequences, 1 for Read 1 and 1 for Read 2.

   • Each adapter sequence meets the following criteria:

     • Sequence of A, T, C, or G character.

     • Length from 1 to 20 characters.

8. Fill out the Index1Sequences and Index2Sequences sections the following way:

   • For Single Index, with or without None: 1 to 100 Index 1 names

   • For each Index 1 name an associated Index 1 sequence

   • For Dual Index, with or without None and Single Index: 1 to 100 Index 1 names

   • For each Index 1 name an associated Index 1 sequence: 1 to 100 Index 2 names

   • For each Index 2 name an associated Index 2 sequence

   • Each index name meets the following criteria:

     • Unique within the file

     • Length from 1 to 8 characters alphanumeric, hyphen, or underscore characters.

     • Each index sequence meets the following criteria

     • Sequence of A, T, C, or G characters

     • Length from 1 to 20 characters

     • All index sequence lengths (Read 1 and Read 2) are equal

     • Index 1 sequences are unique within the file set of Index 1 sequences

     • Index 2 sequences are unique within the file set of Index 2 sequences

9. If the supported indexing strategy specifies Single Index, you can set up Default Layout By Well the following way:

   • Each well unique from A01 to H12

   • For each well, an associated index name exists in the specified Index1Sequences section

10. If the supported indexing strategy specifies Single Index or Dual Index, you can set up Default Layout By Column the following way:

    • Each column number unique from 1 to 12

    • For each column, an associated index name exists in the specified Index1Sequences section

11. If the supported indexing strategy specifies Dual Index, you can set up Default Layout By Row the following way:

    • Each row letter unique from A to H

    • For each row, an associated index name exists in the specified Index2Sequences section

12. Select the Choose.csv File button to select and upload your custom index file.

13. Select Create New Kit to complete the process.

Your custom library prep has been added to the library kit drop-down!

1. From the Runs page, select New Run, and then select Prep Tab.

2. From the Prep page, select NeoPrep.

3. From the NeoPrep Runs page, select Create New Run to open the Configure Run screen.

4. Select the Protocol drop-down arrow, and select a protocol, either TruSeq Nano DNA or TruSeq Stranded mRNA.

5. Select the Version drop-down arrow, and select a version of NeoPrep control software.

6. [Optional] In the Notes field, enter any notes.

7. In the Run Name field, enter a name for the run.

8. Select the Default Project field, select the project to configure for the run, and then select Confirm.

9. Select the checkbox for each process you want to run:

   • Prep Library—Requires preparation of libraries.

   • Quantify—Quantifies libraries after library prep is complete.

   • Normalize—Normalizes libraries after quantification is complete.

10. Complete the following fields as applicable. Read-only or unavailable fields are default selections for the run.

    • Select the Sample Count drop-down arrow, and select the number of samples to include in the run.

    • Select the Index Type drop-down arrow, and select the indexing scheme for the samples.

    • Select the Insert Size drop-down arrow, and select the insert size of the libraries. Mixed insert sizes are specified on the next page.

    • Select the PCR Cycles drop-down arrow, and select the number of PCR cycles for the run.

11. Select Next.

Set Up a Library Prep Run for NeoPrep

The library prep run setup for NeoPrep includes the following three parts:

1. Create and configure the run.

2. Assign samples to wells.

3. Review the run setup.

At any point during this process, you can select the Save button to save your work. Saved runs appear on the NeoPrep Runs page with a status of Planning. When you are ready to resume work, select the run name.

Run Planning provides a list of Illumina index adapter kits used for sequencing. If your index adapter kit is not available, please follow the following instructions to create a custom one.

Creating a custom index adapter kit can be done from within Run Planning (when creating a Configuration) or from the Resources page (select Index Adapter Kit tab).

A custom Index kit can be configured in yaml or tsv.

Configuring in yaml

The following are basic rules to follow when configuring in yaml

• 3 dashes indicates the start of the definition

• Begin a comment line with '#' character

• Each line is typically in the format of SettingName: SettingValue. Setting which value is a string has to be enclosed in double quotes. Other types like numeric or boolean do not require double quotes.

• When a setting contains more complex information, it is usually defined in multiple lines. Ensure the right indentation to maintain the structure. Use two space characters instead of a tab character.

Three yaml templates are provided.

1. Non-fixed Layout: for non-fixed layout kit where any index can be selected for any sample.

2. Fixed Layout - Single Plate: for fixed layout kit with single plate, where each well has a defined index combination.

3. Fixed Layout - Multi Plate: for fixed layout kit with multi-plate, where each well has a defined index combination.

Setting Details

AllowedIndexStrategies

The supported values are "Dual", "Single", and "NoIndex". Create a list below this setting. Each list item should be preceded with a dash (-) character and enclosed in double quotes. Use two spaces for indentation. See Example 1.

Example 1: Defining AllowedIndexStrategies for a kit that support single-index and dual-index, with dual-index as the default.

AllowedIndexStrategies:
  - "Dual"
  - "Single"
Settings:
  DefaultIndexStrategy: "Dual"

IndexSequences

• i7Index1 Create a list of Index1 sequences below this setting. Each index should be in the format of IndexName: "IndexSequence". See example 2.

• i5Index2 Create a list of Index2 sequences below this setting. Each index should be in the format of IndexName: "IndexSequence". See example 2.

Example 2: Defining Index1 and Index2 sequences.

IndexSequences: #Enter index sequences for both Index1 and Index2
  i7Index1: #Index1
    D701: "ATTACTCG" #<IndexName>: <IndexSequence>
    D702: "TCCGGAGA"
    D703: "CGCTCATT"
  i5Index2: #Index2
    D501: "TATAGCCT" #<IndexName>: <IndexSequence>
    D502: "ATAGAGGC"
    D503: "CCTATCCT"

Example 3: Settings for non-fixed-layout kit (FixedIndexPositions is not defined).

IndexSequences: #Enter index sequences for both Index1 and Index2
  i7Index1: #Index1
    D701: "ATTACTCG" #<IndexName>: <IndexSequence>
    D702: "TCCGGAGA"
    D703: "CGCTCATT"
  i5Index2: #Index2
    D501: "TATAGCCT" #<IndexName>: <IndexSequence>
    D502: "ATAGAGGC"
    D503: "CCTATCCT"
# Settings for non-fixed-layout kit with dual-index
Settings:
  FixedLayout: false
  Multiplate: false

Settings - FixedIndexPositions

The mapping should be defined in the format of "WellPosition/Index1Name-Index2Name" or "Plate-WellPosition/Index1Name-Index2Name". The allowed well positions are A01 - H12. If the kit requires well positions defined in different format, define FixedLayoutPositionKeyByIndexId: true. See example 4, example 5, example 6.

Example 4: Settings for single-plate fixed-layout kit (Note that WellPositions are in the A01 - H12 range).

# Settings for fixed-layout kit with single plate
Settings:
  FixedLayout: true
  Multiplate: false
  FixedIndexPositions: #Format: '- "WellPosition/Index1Name-Index2Name"'
    - "A01/D701-D501"
    - "B01/D702-D502"
    - "H01/D703-D503"
    - "H12/D704-D504"

Example 5: Settings for single-plate fixed-layout kit (Note that FixedLayoutPositionKeyByIndexId is true because WellPositions do not follow A01 - H12 format).

# Settings for single-plate fixed-layout kit, where well positions do not follow A01 - H12 format.
Settings:
  FixedLayout: true
  Multiplate: false
  FixedLayoutPositionKeyByIndexId: true
  FixedIndexPositions: #Format: '- "WellPosition/Index1Name-Index2Name"'
    - "UDP0001/UDP0001-UDP0001"
    - "UDP0002/UDP0002-UDP0002"

Example 6: Settings for multi-plate fixed-layout kit.

# Settings for multi-plate fixed-layout kit 
Settings:
  FixedLayout: true
  Multiplate: true
  FixedIndexPositions: #Format: '- "Plate-WellPosition/Index1Name-Index2Name"'
    - "A-A01/D701-D501"
    - "C-B01/D703-D502"

Settings - AllowVariableLengthIndexSequences

The value is true or false. However, the usage is currently restricted to Instrument Platforms which allow multi-configuration. As each configuration only allows one Override Cycles, when setting up a run, samples with different index lengths should be separated into different configurations.

Override Cycles

• EnableCustomIndexCycles

  • The value is true or false. If the setting is set to false or if the setting is not defined, the Override Cycles used is Y;I;I;Y pattern.

• NumCyclesIndex1Override The value should be a numeric value. If this setting is not defined, the number of Index1 cycles follows the number of bases in the Index1 sequences.

• NumCyclesIndex2Override The value should be a numeric value. If this setting is not defined, the number of Index2 cycles follows the number of bases in the Index2 sequences. See example 7.

• OverrideCycles

  • The value should be defined in this format: "Y{{Read1Length}};I{{Index1Length}};I{{Index2Length}};Y{{Read2Length}}?", where:

    • {{Read1Length}} is the number of cycles for Read1,

    • {{Read2Length}} is the number of cycles for Read2,

    • {{Index1Length}} is the number of cycles for Index1, and

    • {{Index2Length}} is the number of cycles for Index2.

  • If UMI is used, update the pattern accordingly.

    • E.g. if Read1 and Read2 cycles include 7 UMI cycles and 1 skipped-cycle: U7N1Y{{Read1Length-8}};I{{Index1Length}};I{{Index2Length}};U7N1Y{{Read2Length-8}}?"

    • E.g. if the kit is a single index kit, with UMI cycles instead of Index2: "Y50N{{Read1Length-50}};I8N{{Index1Length-8}};N{{Index2Length-16}}U16;Y50N{{Read2Length-50}}?". See example 7.

Settings - CustomBclConvertSettings

This section contains custom BCL Convert settings. The settings will be included in the sample sheet generated by Run Planning.

• TrimUMI Indicates if the UMI should be excluded from fastq files. The value is "0" or "1". Set to "0" if BCL Convert should still output UMI cycles to fastq files. See example 8.

• CreateFastqForIndexReads Indicates if the UMI in Index cycles should be trimmed or not. The value is "0" or "1". Set to "1" if BCL Convert should still output UMI cycles in Index to fastq files. Note that TrimUMI should also be set to "0".

Example 7: Settings to override Index2 cycles and custom override cycles.

# Settings for non fixed-layout kit using single-index, with UMI in-place of Index2 :
# - NumCyclesIndex2Override is set to 16
# - Custom OverrideCycles is defined
# - CustomBclConvertSettings is defined so that fastq will be output for index reads.
IndexSequences:
  i7Index1:
    SI_NA_A1_1: "AAACGGCG"
    SI_NA_A1_2: "CCTACCAT"
    SI_NA_A1_3: "GGCGTTTC"
Settings:
  FixedLayout: false
  Multiplate: false
  EnableCustomIndexCycles: true
  NumCyclesIndex2Override: "16"
  OverrideCycles: "Y50N{{Read1Length-50}};I8N{{Index1Length-8}};N{{Index2Length-16}}U16;Y50N{{Read2Length-50}}?"
  CustomBclConvertSettings:
    TrimUMI: "0"
    CreateFastqForIndexReads: "1"

Example 8: Settings to disable TrimUMI, i.e. BCL Convert should output fastq files for UMI cycles.

# Settings for single-plate fixed-layout kit using dual-index, with UMI in Read1 :
# - Custom OverrideCycles is defined
# - CustomBclConvertSettings is defined so that fastq will be output for reads.
IndexSequences:
  i7Index1:
    SI_NT_A1: "ATTTACCGCA"
    SI_NT_A2: "TTGTCGTAGA"
    SI_NT_A3: "AGTCCTGCGG"
  i5Index2:
    SI_NT_A1: "GACAATAAAG"
    SI_NT_A2: "CAATGTAGCA"
    SI_NT_A3: "TGACACAAGT"
Settings:
  FixedLayout: true
  Multiplate: false
  FixedIndexPositions:
    - "A01/SI_NT_A1-SI_NT_A1"
    - "A02/SI_NT_A2-SI_NT_A2"
    - "A03/SI_NT_A3-SI_NT_A3"
  OverrideCycles: "U28N{{Read1Length-28}};I10N{{Index1Length-10}};N{{Index2Length-10}}I10;Y90N{{Read2Length-90}}?"
  CustomBclConvertSettings:
    TrimUMI: "0"

Configuring in tsv

Similar to yaml, three tsv templates are provided. Please note that currently .tsv file supports fewer custom kit settings (as compared to .yaml file).

1. Non-fixed Layout: for non-fixed layout kit where any index can be selected for any sample.

2. Fixed Layout - Single Plate: for fixed layout kit with single plate, where each well has a defined index combination.

3. Fixed Layout - Multi Plate: for fixed layout kit with multi-plate, where each well has a defined index combination.

A tsv file contains of three sections, namely [IndexKit], [Resources], [Indices], where each section contains rows of tab-separated values.

IndexKit Section

Resources Section

Each row in the Resources section consists of four columns: Name, Type, Format, and Value. It is used to define Adapter Read settings and the type of index kit (whether a fixed layout with single- or multi- plate or non fixed layout). In addition, the mappings of well positions and index names (only for a fixed layout kit) should be included in this section (see No 5 in the table below).

[Resources]
Name	Type	Format	Value
Adapter	Adapter	string	ACCTCCCCGTGA
AdapterRead2	AdapterRead2	string	ACCTCCCCGTGA
FixedLayout	FixedLayout	bool	true
Multiplate	Multiplate	bool	false
A01 FixedIndexPosition  string  D701-D501
B01 FixedIndexPosition  string  D702-D502

Indices Section

Index1 and Index2 sequences should be defined in this section. Each row consists of three columns: Name, Sequence, IndexReadNumber.

[Indices]
Name	Sequence	IndexReadNumber
D701	ATTACTCG	1
D702	TCCGGAGA	1

D501	TATAGCCT	2
D502	ATAGAGGC	2

Use the following instructions to import libraries to the Libraries page from a CSV file.

This method is appropriate for the following:

• Prepared libraries

• Samples with assigned indexes If your samples do not have indexes assigned and are not in libraries, use .

1. On the Libraries page, select Import.

2. [Optional] Create a CSV file as follows.

   1. Select the spreadsheet image to download a template.

   2. For each sample, enter the following information.

   3. Sample ID—The unique sample ID.

   4. Name—A descriptive name for the biological sample.

   5. [Optional] Species—The appropriate species.

   6. [Optional] Project—The name of the project to save samples to. Although optional at this step, a project is required later to store the data.

   7. NucleicAcid—The nucleid acid, either RNA or DNA.

   8. Well—The plate well.

   9. Index1Name—The Index 1 name.

   10. Index1Sequence—The Index 1 sequence.

   11. Index2Name—The Index 2 name.

   12. Index2Sequence—The Index 2 sequence.

   13. Save the file.

3. Select Choose .csv File.

4. Browse to and select the appropriate CSV file, and then select Open. The information from the file populates the Import Sample Libraries page.

5. [Optional] Select additional libraries as follows.

   1. Select Save & Continue Later.

   2. Select the checkbox of each library you want to use.

6. Select Pool Libraries.

1. In the Assign Samples to Wells page, set up each well as follows.

   1. In the Sample ID field, select Select Sample,

   2. In the Select a Biological Sample dialog box, select a sample, and then select Select.

   3. In the Library ID field, enter a unique name for the library.

   4. Select the Index drop-down arrow, and select a unique index to add to the sample.

   5. If you selected mixed insert sizes on the previous page, select the Insert Size drop-down arrow, and select the insert size of the library.

2. Select Next.

The Run Planning tool can be used to requeue planned analyses for supported Cloud applications.

• Illumina DRAGEN Apps version 3 or higher

• DRAGEN TruSight Oncology 500 Apps version 2.1 or higher

To initiate a requeue, navigate to the Run Summary page and select Requeue > Planned Run from the Status menu.

You can use the sample sheet from the selected run or select a new sample sheet file to be load. This will load the analysis configurations contained in the selected Sample Sheet into the Run Planning tool, and allow edits before requeuing the analysis.

Depending on the instrument platform and analysis specified, some fields may be non-editable, including Run Name and Library Tube ID. See the page for specific instructions on using the Run Planning tool.

For instruments that support multiple analysis configurations, existing configurations can be edited or deleted. New configurations can also be added before requeueing.

When all changes have been made (if desired), review the settings on the Run Review page and click "Requeue" to initiate the requeue.