Illumina Connected Software

MiSeq i100 Series Sequencing v2.0 Workflow User Interaction, Validation and Troubleshooting

This section explains how to validate the installation of the Illumina MiSeq i100 Series Integration Package (hosted or on-premise) using the MiSeq i100 Sequencing v2.0 Workflow.

The validation process involves the following actions:

  • Running samples through the Library Prep Validation v2.3.6 workflow.

    • The workflow contains a single-step protocol that models the library prep required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to MiSeq i100 Series Sequencing v2.0 workflow.

  • Running libraries through the MiSeq i100 Series Sequencing v2.0 workflow validates the following items:

    • Successful sequential step advancement of samples through the steps of the workflow.

    • Automated creation of a planned run. The control software retrieves the planned run created. The sample sheet is automatically generated and attached to Load to Dry Cartridge step.

    • Automated tracking of the MiSeq i100 Series sequencing run and parsing of run statistics from Instrument to Clarity LIMS.

    • Automated tracking of the MiSeq i100 Series analysis run (if analysis is configured) from Instrument to Clarity LIMS.

The validation steps assume the following conditions have been met:

  • The user has a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.

  • The appropriate integration package is installed and you have imported the default Clarity LIMS configuration.

    • For cloud integration, MiSeq i100 Series Integration Package v1.1.0 is installed

    • For on-premise integration, MiSeq i100 Series On-Prem Integration Package v1.1.0 is installed

  • Analysis configuration templates (ACTs) are created in BaseSpace Sequence Hub (for cloud integration) or Illumina Run Manager (for on-premise integration) for your run.

  • The index adapter kit label group in the selected ACT is created in Clarity LIMS and used in the Run Library Prep Validation v2.3.6 step to index the samples.

    For more information on creating a reagent label group, refer to Add and Configure Labels and Label Groups in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation. For the adapter sequences for your library prep kits, refer to Illumina Adapter Sequences.

Analysis Configuration Template Creation in BaseSpace Sequence Hub or Illumina Run Manager

You must create the Analysis Configuration Templates (ACTs) that are required for configuring secondary analysis in the MiSeq i100 Series Sequencing v2.0 workflows. For instructions to create and delete ACTs, refer to Online Help for BaseSpace Sequence Hub or Illumina Run Manager

Activate Workflow, Create Project, Add and Assign Samples

The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.6 and MiSeq i100 Series Sequencing v2.0 workflows.

  1. On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.6 and MiSeq i100 Series Sequencing v2.0 workflows.

  2. On the Projects and Samples screen, create a project and add samples to it.

    ⚠ Sample and library names must use only alphanumeric, dash, or underscore characters. Invalid characters can cause a sample sheet validation failure in the Load to Dry Cartridge step.

  3. Assign the samples to the Library Prep Validation workflow.

Library Prep Protocol

This single-step protocol models the library prep required to produce libraries tagged with index sequences that are ready for the MiSeq i100 Series Sequencing v2.0 workflow.

Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:

  • Label Group = same as the Index Adapter Kit selected in the ACT that is being used

  • Sequencing Instrument = MiSeq i100 Series

On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq i100 Series Sequencing v2.0 workflow, Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v2.0).

Protocol: MiSeq i100 Series Sequencing v2.0

The MiSeq i100 Series Sequencing v2.0 protocol consists of the following steps:

  • Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v2.0)

  • Make Bulk Pool (MiSeq i100 Series Sequencing v2.0)

  • Load To Dry Cartridge (MiSeq i100 Series Sequencing v2.0)

  • AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v2.0)

  • AUTOMATED - Analysis Run (MiSeq i100 Series Sequencing v2.0)

Step 1: Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v2.0)

  1. In Lab View, locate the MiSeq i100 Series Sequencing v2.0 protocol. The samples are queued for the Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v2.0) step.

  2. Add the samples to the Ice Bucket and select View Ice Bucket.

  3. On the Ice Bucket screen, select Begin Work to start the Validate Sample Names and Register Step Started automation.

    The automation does the following:

    • checks that the sample names do not use restricted characters and are within character limits,

    • registers the start time of the step by publishing messages to CLPA through ICA. The part of the script for start time registration is used only for CLPA support.

  4. Select the connected MiSeq i100 instrument to integrate with if applicable.

  5. On Record Details screen, select 1. Retrieve ACT List to retrieve the list of ACTs created on Illumina Run Manager (for on-premise integration) or BaseSpace Sequence Hub (for cloud integration).

  6. Select the applicable ACT to assign to the samples. The index adapter kit specified by the ACT must correspond with the label group used in Library Prep Protocol.

  7. [Optional] In Step Details, select 2. Retrieve ACT Information to trigger the Retrieve ACT Information automation.

    This automation retrieves ACT information (e.g., Library Prep Kit, Index Adapter Kit, Reference Genome, and so on) and populates the fields in Clarity LIMS. These details are saved to the ACTMetadata.csv file that you can download to view details of the selected ACT.

  8. Set the Skip Pooling toggle switch to Yes if the input sample is one or more pools and no further pooling is required. Otherwise, leave it to the default value of No.

    ⚠ By setting Skip Pooling toggle to No, input samples (one or more pools) can be further combined into a bigger pool in the subsequent Make Bulk Pool step.

  9. Select Next Steps to assign the ACT to the samples.

    This action triggers the Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step. This automation:

    • validates that Molarity (nM) custom field for each sample is greater than 0,

    • validates that the indexes applied to the samples are valid for the selected ACT. If all are valid, set the ACT Name custom field for each sample to the selected ACT.

    • sets the next step for each sample based on the logics below:

      • If Skip Pooling is set to Yes, set next step to Load to Dry Cartridge.

      • Otherwise, set next step to Make Bulk Pool step.

  10. Select Finish Step. Samples are then routed according to the next step set above.

Step 2: Make Bulk Pool (MiSeq i100 Series Sequencing v2.0)

This step supports pooled libraries as inputs. If a pool consists of input pooled libraries, each pooled library will be considered as a single unit in the Calculate Volumes automation script.

  1. In Lab View, locate the MiSeq i100 Series Sequencing v2.0 protocol. The samples are queued for the Make Bulk Pool (MiSeq i100 Series Sequencing v2.0) step.

  2. Add the samples to the Ice Bucket and select View Ice Bucket.

  3. On the Ice Bucket screen, select Begin Work.

  4. Create a pool of samples as follows.

    1. On the Pooling screen, create a pool by dragging samples into the Pool Creator.

    1. Enter a name for your pool or accept the default name (Pool #1).

    1. [Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.

    ℹ Only a single pool is allowed in the Load to Dry Cartridge step for each run. Create multiple pools if required for subsequent runs.

    1. [Optional] To remove a pool, select the X in the top right corner of the pool.

  5. Select Record Details to trigger the Validate Analysis Configurations automation.

    This automation performs the following checks on the analysis configuration for each pool:

    • Pooled samples are within the maximum configuration limit.

    • Samples having no analysis are not allowed to combine with samples having analysis.

    • Pooled samples assigned with the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).

  6. [Optional] In the Sample Table, select the pool next to the sample name to view details on the pool composition.

  7. On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.

    [Optional] Create a new lot. For more information, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

  8. In the Step Details area, complete the following fields:

    • Final Loading Concentration (pM) — The final loading concentration of the pool in the flow cell.

    • Final Loading Volume (ul) — The final loading volume of the pool in the flow cell.

    • Bulk Pool Volume (ul) — The bulk pool volume (default = 30 µl) is used in the Calculate Volumes script to determine RSB volumes required for pooling.

    • Minimum Per Sample Volume (ul) — The minimum volume for each sample. This field is prepopulated with the configured default value (2 µl) and can be edited. If the per sample volume is below the value set in this field, the Calculate Volumes script applies a rounding factor to affected samples so that the volume reaches the minimum volume.

    • Flowcell Type — The flow cell type that the run uses. Select from the following options:

      • 5M

      • 25M

      • 50M

      • 100M

    • [Optional] % PhiX Spike In — Percentage of PhiX Spike-In.

  9. Select Calculate Volumes to trigger the Calculate Volumes automation.

    This automation calculates the volumes required for each library to form a pool that has the concentration and volume specified in the Step Details field. It also generates the calculation file in a CSV format and attaches it to the step. Select the file name to download it.

    If % PhiX Spike In is specified, the automation also calculates the volumes required to spike in the desired PhiX percentage. The volumes and instructions are included in the calculation file as well. For information on the calculation, refer to the MiSeq i100 protocol.

  10. Select Next Steps to trigger the Set Next Step automation.

    This automation sets the next step for samples to ADVANCE, which moves them to the Load to Dry Cartridge (MiSeq i100 Series Sequencing v2.0) step.

  11. Select Finish Step.

Step 3: Load to Dry Cartridge (MiSeq i100 Series Sequencing v2.0)

  1. In Lab View, locate the MiSeq i100 Series Sequencing v2.0 protocol. The pool of samples are queued for the Load to Dry Cartridge (MiSeq i100 Series Sequencing v2.0) step.

  2. Add the pool to the Ice Bucket and select View Ice Bucket.

    ⚠ Only single pool is allowed at this step.

  3. On the Ice Bucket screen, select the destination container type:

    • MiSeq i100 Series Dry Cartridge

  4. Select Begin Work to trigger the Validate Single Pool and Register Step Started automation. The automation

    • ensures that only one pool is selected for loading to the dry cartridge in this step,

    • registers the start time of the step by publishing messages to CLPA through ICA. The part of the script for start time registration is used only for CLPA support.

  5. On the Placement screen, do as follows.

    1. Drag the pool into the Placed Samples area on the right.

    2. Scan or type the barcode of the dry cartridge into the container name field.

    3. Select Record Details.

    After exiting the Placement screen, the Validate Dry Cartridge Barcode automation makes sure that the dry cartridge barcode conforms to the barcode mask. The barcode mask is SC[0-9]{7}-SC3.

  6. The fields displayed on the Record Details screen are used to create the planned run and generate the sample sheet.

    The analysis related information for the planned run comes from the ACT associated with the samples. Refer to the following table for details.

    Fields Displayed on Record Details Screen of Load to Dry Cartridge (MiSeq i100 Series Sequencing v2.0) Step

    Field

    Description

    Run Name

    Experiment name.

    • Only alphanumeric characters, dashes, periods, spaces and underscores are permitted.

    • Maximum of 255 characters

    Read 1 Cycles

    Presets

    • 51

    • 151

    • 301

    • 501

    • Type a custom value

    Read 2 Cycles

    Presets

    • 51

    • 151

    • 301

    • 501

    • Type a custom value

    Index 1 Cycles

    Presets

    • 0

    • 6

    • 8

    • Type a custom value¹

    Index 2 Cycles

    Presets

    • 0

    • 6

    • 8

    • Type a custom value¹

    Create Planned Run

    Specifies if a planned run² should be created.

    ¹ The custom value must correspond to the longest index sequence of the samples in the pools in the library tube strip. Otherwise, the planned run creation fails and an error message displays.

    ² Planned run is created and available on BaseSpace Sequence Hub (for cloud integration) or Illumina Run Manager (for on-premise integration). This configuration is downloaded to the MiSeq i100 Series instrument to start the run. The downstream secondary analysis is done in the cloud or in the on-premise DRAGEN module depending on the run mode.

  7. Select Validate Run Setup and Create Sample Sheet to trigger the automation script. The script performs the following actions:

    • Validates the parameters entered on the Record Details screen.

    • (Optional) Creates the planned run.

    • Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen.

  8. Select Next Steps.

  9. Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v2.0) step.

For more information on how to start the sequencing run for different run modes, refer to the relevant Configuration guide:

Step 4: AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v2.0)

Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v2.0) step. The integration does this automatically.

When no analysis is configured, samples will be removed from the workflow upon completion of AUTOMATED - Sequencing Run step instead of being routed into the AUTOMATED - Analysis Run step.

The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. User interaction is not required, but you can review the stages of the step in Clarity LIMS.

For cloud hosted Clarity LIMS setup, make sure that the MiSeq i100 Control Software is configured properly. Refer to MiSeqi100 Series Integration Protocol for more information

Under Run Settings, check the boxes next to Cloud run monitoring and Cloud run storage for Cloud hosted Clarity LIMS Integration to work.

If there are issues after using the configuration settings on the MiSeq i100 Control Software, contact Illumina Support.

Review Run Data

Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics in the Step Details section and the Sample Details table.

Step Details Section

The following values populate the master step fields:

  • Run Name

  • Run Status

  • Output Folder

  • Current Read

  • Current Cycle

  • Dry Cartridge Barcode Serial Number

  • Dry Cartridge Expiration Date

  • Wet Cartridge Expiration Date

  • Wet Cartridge Serial Number

  • Flow Cell ID

  • Flow Cell Type

  • Instrument ID

  • Instrument Type

  • Instrument Control Software Version

  • Sequencing Log

Sample Details Table

ℹ Sequencing run metrics is displayed in terms of per-run and per-lane. All per-run metrics will be displayed to the left of the Sample Details table followed by the per-lane metrics.

Summary metrics populate the following global custom fields:

  • % Bases >=Q30 R1

  • % Bases >=Q30 R2

  • % Error Rate R1

  • % Error Rate R2

  • Yield (Gb) R1

  • Yield (Gb) R2

  • Reads PF

  • % PF

  • % Aligned R1

  • % Aligned R2

  • % Occupied

  • % Phasing R1

  • % Phasing R2

  • % Prephasing R1

  • % Prephasing R2

  • Intensity Cycle 1 R1

  • Intensity Cycle 1 R2

Step 5: AUTOMATED - Analysis Run (MiSeq i100 Series Sequencing v2.0)

Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Analysis Run (MiSeq i100 Series Sequencing v2.0) step. The integration does this automatically.

The integration starts the step automatically and parses run data back into Clarity LIMS. User interaction is not required, but you can review the stages of the step in Clarity LIMS. If the run analysis is successful, the integration completes the step automatically.

Review Analysis Data

After the analysis run is complete, open the step and review the following values in the Step Details section:

  • Analysis Status - Status of the analysis run

  • Analysis Result Location - Location of the secondary analysis data. This field is empty if transfer of secondary analysis data to an external location is disabled.

  • Log - Log message during event handling by the integration.

The demultiplexing results file contains the following demultiplexing statistics for each sample:

  • Number of reads

  • Number of perfect index reads

  • Number of mismatch index reads

At this point, the entire MiSeq i100 Series Integration workflow is fully validated.

Troubleshooting

If an automation trigger does not appear to run its corresponding scripts, refer to the Troubleshooting Automation section in the Clarity LIMS (API & Database) documentation.

Validate Analysis Configuration Automation Check Fails

The Validate Analysis Configuration automation check is in the Make Bulk Pool (upon pooling) step. If a failure happens, an error message displays and the step can be aborted, or you might be prevented from continuing the step.

This check can fail for the following reasons:

  • Samples having no analysis must not be pooled with samples having analysis.

    Resolve this error as follows.

    1. Pool the samples again. Make sure that samples with analysis and those without analysis are in different pools.

  • Pooled samples assigned with the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).

    Resolve this error as follows.

    1. Pool the samples again. Make sure that samples with conflicting secondary analysis application, analysis versions or analysis settings, are in different pools.

Error When Creating Planned Runs

If you receive an error when creating a planned run, check the log message in the Load to Dry Cartridge step to identify the issue. If you cannot correct the issue, contact Illumina Support.

Incompatible Analyses in a Planned Run

Only compatible analysis versions should be combined in a single planned run. When incompatible analysis versions are combined, an error log message displays. An example of the error log message is shown below.

To resolve this error, check the ACTs that were used for the run. Create a new planned run with ACTs that have the same analysis and analysis versions.

Automated Step Starts, But Does Not Complete

If the AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v2.0) step starts, but does not complete, do as follows.

  1. Log in to the default user account and use one of the following methods to open the in progress step in Clarity LIMS:

    1. In Lab View, find the step in the Recent Activities pane.

    2. Search for the step in Clarity LIMS using the library tube strip barcode as the search term.

  2. On the Record Details screen, the Sequencing Log multiline text field contains logging information.

    If unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact Illumina Support. Provide the relevant information from the troubleshooting steps already performed.

Clarity LIMS On-Premise Planned Runs are missing from MiSeq i100 Series Control Software

For MiSeq i100 Series On-Prem Integration Package v1.1.0, planned runs configured with no analysis will be deleted from MiSeq i100 Series Control Software v1.1.0 upon restarting of the Control Software.

Recreate the planned run on instrument.

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