Configuration

The Illumina MiSeq i100 Series On-Prem Integration Package v1.0.0 supports the integration of Clarity LIMS to Illumina MiSeq i100 Series sequencer connected to the Illumina Run Manager platform on premise.

For instructions on validating and troubleshooting the Illumina MiSeq i100 Series On-Premise Integration, refer to MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

The configuration provided in this integration has been established to support MiSeq i100 Series lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the integration.

Prerequisites and Assumptions

Analysis Configuration Template

The MiSeq i100 Series On-Prem Sequencing v1.0 workflow uses analysis configuration templates (ACT) to configure secondary analysis for the planned runs. It is assumed that the required ACTs for your run has been pre-created using Illumina Run Manager UI. Please make sure that the index adapter kit (label group on Clarity) selected in the ACT has already been created on Clarity LIMS. The same label group shall be used in the library preparation step. Names of ACT must be unique. Please refer to the MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting for details on creating ACT on Illumina Run Manager User Interface (UI).

Clarity LIMS trims all leading and trailing spaces from the ACT names and ACTs created in Illumina Run Manager UI with names containing leading and/or trailing spaces may not be recognized properly on Clarity. Please ensure that the ACT do not have leading and/or trailing spaces.

Samples

It is assumed that samples that enter the MiSeq i100 Series On-Prem Sequencing v1.0 workflow have gone through library preparation and quantification processes before they are assigned to the workflow:

• Samples have been accessioned into Clarity LIMS.

• Sample and library names must contain only alphanumeric, dash, or underscore characters.

• Samples have been run through QC and library prep.

• Samples have the Molarity (nM) global field set to a valid value (required for the 'Calculate Volumes' automation in the Make Bulk Pool step).

Workflows, Protocols, and Steps

The MiSeq i100 Series On-Premise Integration Package v1.0.0 includes the following workflows:

• MiSeq i100 Series On-Prem Sequencing v1.0

• Library Prep Validation v2.3.5 (optional, but recommended for validation purposes)

The following describes the protocols and steps included in these workflows.

Library Prep Validation v2.3.5 Workflow

Protocol 1: Library Prep Validation v2.3.5

Purpose:

• Included for validation purposes only, this protocol models the library preparation steps required to advance samples to the MiSeq i100 Series On-Prem Sequencing v1.0 workflow.

  ⚠ The label group (index adapter kit) used for library preparation must be the same as the index adapter kit specified in the ACT that is being used. For more information, refer to MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

• The protocol contains a single step - Library Prep Validation v2.3.5. After this step, a routing script sends the samples to the first step of the MiSeq i100 Series On-Prem Sequencing v1.0 workflow.

Steps:

1. Library Prep Validation v2.3.5

MiSeq i100 Series On-Prem Sequencing v1.0 Workflow

Protocol 1: MiSeq i100 Series On-Prem Sequencing v1.0

Purpose:

• This protocol models the lab processes of run setup for starting a MiSeq i100 Series sequencing and secondary analysis run on premise.

Steps:

1. Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)

   • The secondary analysis of samples is configured using the ACT.

   • The labels applied to the samples are validated against the selected ACT to ensure that samples use valid labels.

2. Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)

   • Samples are pooled and resuspension buffers and reagents are added with the help of the 'Calculate Volumes' automation.

   • ACT of pooled samples are validated with Validate Analysis Configurations automation to ensure that the secondary analysis configurations remain valid after pooling.

3. Load To Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)

   • The library pools from step 2 are now ready to be loaded to the MiSeq i100 Series dry cartridge.

   • Run and analysis information is validated.

   • Samplesheet is generated and planned run is created on MiSeq i100 Illumina Run Manager.

4. AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)

   • This is a fully-automated step that is started and completed automatically once the sequencing run is started and completed on the instrument side.

   • All the sequencing run metadata e.g. run configuration, primary run metrics etc. are recorded automatically.

5. AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)

   • This is a fully-automated step that is started and completed automatically once the analysis run is started and completed on DRAGEN on premise.

   • All the analysis run metadata e.g. status and demultiplexing result files are recorded automatically.

Validation Workflow

The Library Prep Validation v2.3.5 workflow allows for validation of the system after installation is complete. This workflow can be replaced by other custom library preparation workflows. For details, refer to MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

[Optional] Configure a custom library preparation workflow. Routing of libraries from the custom workflow to the MiSeq i100 Series On-Premise Sequencing protocol can be enabled in the master step with the routing script available in the NGS package.

Step 1: Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)

In this step, secondary analyses are configured for the samples using ACTs. Each ACT contains details related to a particular secondary analysis (e.g., index adapter kit to use, reference genome setting). At this step, the user selects an ACT from a list of pre-constructed ACTs and assigns samples to it. A planned run can involve multiple ACTs and users have to repeat Step 1 for each ACT that is required for the planned run. For more information on creating ACTs and assigning samples to them, refer to MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

Validate Sample Names Automation

Automatically triggered on entry of the step, this automation validates that the sample names contain only alphanumeric, dash and underscore characters.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -exp 'submittedSamples.each { sample -> if ( !sample.name.matches( ::[A-Za-z0-9_-]+:: ) ) { fail ( ::Submitted sample :: + sample.name + :: should only have alphanumeric, dash, and underscore characters. Please rename and try again.:: ) } }' -log {compoundOutputFileLuid1}

1. Retrieve ACT List Automation

Triggered when you select '1. Retrieve ACT List' button on the Record Details screen, this automation retrieves the list of ACTS that has been created using Illumina Run Manager UI.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqi100series-onprem/automation/miseqi100series-onprem-automation.jar script:retrieve_act_list -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -n 'INSTRUMENT_USED' -t true

2. Retrieve ACT Information Automation

Triggered when you select '2. Retrieve ACT Information' button on the Record Details screen, this automation:

• Retrieves the details of the selected ACT and populates the step UDFs (e.g., Analysis Version, Library Prep Kit, Index Adapter Kit, Reference Genome)

• Saves the details into a file stored in the Analysis Configuration Metadata file placeholder, which is available for user to download.

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqi100series-onprem/automation/miseqi100series-onprem-automation.jar script:retrieve_act_info -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}' -n 'INSTRUMENT_USED'

Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation:

• Checks that the molarity of all samples are specified

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'if (!input.hasValue(::Molarity (nM)::)) { fail(::The Molarity cannot be empty.::); } else if (input.::Molarity (nM):: <= 0) { fail(::The Molarity cannot be negative or zero.::); }' -log {compoundOutputFileLuid1}

• Checks that all samples are indexed, indexes are the same as the selected ACT.

• Assign the ACT ID, ACT Name, Run Mode and Instrument ID to all the samples in the step.

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqi100series-onprem/automation/miseqi100series-onprem-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}' -n 'INSTRUMENT_USED' --derivedSampleInstrumentIdUdf 'Instrument ID' script:validate_indexes script:assign_act

• Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol - Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0).

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Master Step Fields

The following table lists the configuration details for custom fields that are defined on the Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) step.

Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Configuration

Field Name	Field Type	Options
Analysis Configuration Template	Text Dropdown	Allow Custom Entries
Application	Text	Read Only
Application Version	Text	Read Only
Index Adapter Kit	Text	Read Only
Library Prep Kit	Text	Read Only
Reference Genome	Text	Read Only
Secondary Analysis Mode	Text	Read Only

Global Fields

The following lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) step. See Global Fields section for detail configuration.

• Molarity (nM)

• ACT Name

Step 2: Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)

Samples are pooled in the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step. You can manually create working pools based on the final loading concentration required.

Validate Analysis Configurations Automation

Automatically triggered on exit of the Pooling screen, this automation performs analysis configuration validity and checks each pool for the following characteristics:

• 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).

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'output.::Instrument ID:: = input.::Instrument ID::' -log {compoundOutputFileLuid1} && \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqi100series-onprem/automation/miseqi100series-onprem-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool -n 'INPUT.Instrument ID' script:validate_analysis_config  script:validate_physical_logical_configurations

Calculate Volumes Automation

Triggered when you select 'Calculate Volumes' button on the Record Details screen, this automation completes the following actions:

• Checks to ensure all samples have molarity values assigned.

  if (!input.hasValue(::Molarity (nM)::)) { return; };

• Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.

  if (output.hasValue(::Number of Samples in Pool::)) { output.::Number of Samples in Pool:: = output.::Number of Samples in Pool:: + 1; } else { output.::Number of Samples in Pool:: = 1; }' -t true

• Calculate the Final Loading Concentration (pM)

• Copies Flowcell Type from the step inputs to the step outputs.

  output.::Required Bulk Pool Volume (ul):: = (step.::Final Loading Concentration (pM):: / 1000) * step.::Final Loading Volume (ul):: / 4 ;
      output.::MiSeq i100 Series Flowcell Type:: = step.::Flowcell Type::;

• Calculates the per sample volume required for each library to make a 2 nM intermediate library pool and sets the total sample volume to zero.

  input.::Per Sample Volume (ul):: = 4 * step.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in Pool::;
      output.::Bulk Pool Volume (ul):: = step.::Bulk Pool Volume (ul)::;
      output.::Total Sample Volume (ul):: = 0;

• Calculates the PhiX Spike-in volume if specified.

  if (step.hasValue(::% PhiX (100 pM) Spike-In::) && step.::% PhiX (100 pM) Spike-In:: > 0) { output.::PhiX Concentration (pM):: = 100; output.::PhiX Volume (ul):: = 4 * output.::Required Bulk Pool Volume (ul):: * (step.::% PhiX (100 pM) Spike-In:: / 100) / (output.::PhiX Concentration (pM):: / 1000); } else { output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; }

• Calculates the adjusted per sample volume for the pools if any of the calculated volume is lesser than the value specified in Minimum Per Sample Volume (ul) step UDF.

  script:calculate_multipool_adjusted_per_sample_volume -t

• Uses the Total Sample Volume (ul) to calculate the RSB volume (ul) required to top up the pools that are needed to create the 2 nM intermediate library pools. The RSB volume is saved to the file in the Calculation File placeholder in the Files section.

  script:evaluateDynamicExpression \
    -exp 'if (output.hasValue(::Total Sample Volume (ul)::)) { output.::Total Sample Volume (ul):: = output.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::; } else { output.::Total Sample Volume (ul):: = input.::Adjusted Per Sample Volume (ul)::; }' -t true \
    script:evaluateDynamicExpression \
    -exp 'if (output.::Total Sample Volume (ul):: > output.::Bulk Pool Volume (ul)::) { output.::RSB Volume for Intermediate Pool (ul):: = 0 } else { output.::RSB Volume for Intermediate Pool (ul):: = output.::Bulk Pool Volume (ul):: - output.::Total Sample Volume (ul):: }' -t true \
    script:evaluateDynamicExpression \
      -exp 'if (output.::Bulk Pool Volume (ul):: < output.::Required Bulk Pool Volume (ul)::) { output.::RSB Volume (ul):: = output.::Required Bulk Pool Volume (ul):: - output.::Bulk Pool Volume (ul)::; } else { output.::RSB Volume (ul):: = 0.1 * step.::Final Loading Volume (ul):: - output.::Required Bulk Pool Volume (ul)::; } ; output.::KLD Volume (ul):: = 0.9 * step.::Final Loading Volume (ul)::; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::;' -t true

• Uses the MiSeqi100Series_Bulk_Pool1.csv and MiSeqi100Series_Bulk_Pool2.csv template files to generate a single CSV file. This file contains information about the pools and the samples that they contain. The generated file is stored in the Calculation File placeholder in the Files section.

  script:driver_file_generator \
      -t /opt/gls/clarity/extensions/conf/driverfiletemplates/MiSeqi100Series_Bulk_Pool1.csv -o 1.csv \
      script:driver_file_generator \
      -t /opt/gls/clarity/extensions/conf/driverfiletemplates/MiSeqi100Series_Bulk_Pool2.csv -o 2.csv \
      script:driver_file_generator \
      -t /opt/gls/clarity/extensions/conf/driverfiletemplates/MiSeqi100Series_Bulk_Pool3.csv -o 3.csv \
      && cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csv

• Resets the Number of Samples in Pool to null before exiting the step.

  -exp 'output.::Number of Samples in Pool:: = ::::;' -t true

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation completes the following actions:

• Proceeds only if the sample has a molarity value.

• Copies the Run Mode from input to output.

• Sets the next step for samples to ADVANCE, advancing them to the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
script:evaluateDynamicExpression -exp 'if (!input.hasValue(::Molarity (nM)::)){ return; }; output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::;'"

Master Step Fields

The following table lists configuration details for the custom fields that are defined on the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step.

Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Configuration

Field Name	Field Type	Options	Additional Options and Dropdown Items
% PhiX (100 pM) Spike-In	Numeric		Range: 0 - 100
Bulk Pool Volume (ul)	Numeric	Required Field	Decimal places displayed: 2 Default: 1
Final Loading Concentration (pM)	Numeric Dropdown	Required Field Allow Custom Entries	Dropdown Items 60 70 80 90 100 110 120 130 140 150 160 170 180
Flowcell Loading Volume (ul)	Numeric	Required Field	Decimal places displayed: 2 Default: 250
Flowcell Type	Text Dropdown	Required Field	Dropdown Items 5M 25M
Minimum Per Sample Volume (ul)	Numeric	Required Field	Decimal places displayed: 2 Default: 2

Global Fields

The following lists the global custom fields that are configured to display on the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step. See Global Fields section for detail configuration.

• Final Loading Concentration (pM)

• RSB Volume (ul)

• MiSeq i100 Series Flowcell Type

Step 4: Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)

In this step, the user scans the dry cartridge barcode into the LIMS, and then manually places the working pools into the dry cartridge to be used in the MiSeq i100 Series run. In addition, this step validates the run setup and analysis information and generates samplesheet file and/or creates a planned run on instrument's Illumina Run Manager UI.

Validate Single Pool Automation

Automatically triggered on entry to the step, this automation checks only single pool is use to start the step.

script:validateSampleCount -min 1 -max 1

Validate Dry Cartridge Barcode Automation

Automatically triggered on exit of the Placement screen, this automation validates the dry cartridge barcode to ensure it conforms to the barcode mask SC[0-9]{7}-SC3.

 script:validate_output_containers -r 'MiSeq i100 Series Dry Cartridge:SC[0-9]{7}-SC3' -min 1 -max 1

Validate Run Setup and Create Planned Run Automation

Triggered when you select 'Validate Run Setup and Create Planned Run' on the Record Details screen, this automation completes the following actions:

• Validates the parameters entered on the Record Details screen. These parameters are used to set up the run, generate the sample sheet file, and create the planned run on Illumina Run Manager.

  • Run Name may only contain alphanumeric, dash, underscore or period characters. Spaces are not permitted.

    'if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes, periods and underscores. Run Name must start with alphanumeric, a dash or an underscore::); }; 

  • Run Name shall not exceed 255 characters.

    if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::); };

• Checks the Index 1 Cycles and Index 2 Cycles field values. If Index 2 Cycles is greater than 0, the Index 1 Cycles value must be greater than 0 or an error can occur.

  if (step.::Index 2 Cycles:: > 0 && step.::Index 1 Cycles:: == 0) { fail(::Index 1 Cycles cannot be zero if Index 2 Cycles is non-zero::); };

• Generates the sample sheet and creates the planned run on Illumina Run Manager. The sample sheet is attached to the step.

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqi100series-onprem/automation/miseqi100series-onprem-automation.jar  script:generate_sample_sheet -i {stepURI:v2} -u {username} -p {password} -c {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} -n 'INPUT.Instrument ID' --instrumentType '{udf:Instrument Type}' --verifyIndexLength

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation completes the following actions:

• Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Master Step Fields

The following table shows the master step fields that are configured on the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step. These fields are required for sample sheet generation and planned run creation in Illumina Run Manager.

Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Configuration

Field Name	Field Type	Options	Additional Options and Dropdown Items
Index 1 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range: 0 – 18 Presets 0 6 8
Index 2 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range: 0 – 18 Presets 0 6 8
Instrument	Text	Read Only
Planned Run ID	Text	Read Only	Hidden, use to store created planned run id
Read 1 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range: 1 – 301 Presets 101 151 301
Read 2 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range: 0 – 301 Presets 101 151 301
Run Mode	Text	Read Only	Default: Local
Run Name	Text	Required Field

Step 5: AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)

Do not add samples to the Ice Bucket, start or complete the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The integration will do this automatically.

In this step, the pooled samples in the dry cartridge are sequenced on the MiSeq i100 Series instrument.

There are no automation associated to this step.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

Master Step Field Information

Field Name	Description
Current Cycle	Current completed cycle count.
Current Read	Current completed reads count.
Dry Cartridge Barcode Serial Number	Dry Cartridge Barcode Serial Number.
Dry Cartridge Expiration Date	Flow Cell expiration data.
Flow Cell ID	Serial number of the Flow Cell.
Flow Cell Type	Flow Cell Type. Value can be 5M or 25M.
Instrument Control Software Version	Version of the instrument control software.
Instrument ID	Unique identification for the instrument.
Instrument Run ID	Unique identification for the instrument sequencing run.
Instrument Type	Type of the instrument, either MiSeqi100 or MiSeqi100Plus.
Output Folder	Output folder path for the sequencing run data.
Run End Time	Time completed for the sequencing run.
Run Name	Run name user configured for the sequencing run.
Run Start Time	Time started for the sequencing run.
Run Status	Status of the sequencing run.
Sequencing Log	Log messages by the Illumina Run Manager integration while handling the sequencing event from instrument.
Wet Cartridge Expiration Date	Wet cartridge expiration data.
Wet Cartridge Serial Number	Wet cartridge Serial Number.

All the fields above are Read-Only Text field, except Sequencing Log is a Read-Only Multiline Text.

Global Fields

The following global custom fields are used to capture the run metrics in Clarity LIMS:

• % Aligned R1

• % Aligned R2

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• % Error Rate R1

• % Error Rate R2

• % Occupied

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• % PF

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• Reads PF

• Yield (Gb) R1

• Yield (Gb) R2

At the end of the step, the pools of samples are automatically route to next step.

Step 6: AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)

Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Analysis v1.0) step. The integration does this automatically.

Data from the analysis is parsed back to Clarity LIMS. In this step, the secondary analysis configured using the Analysis Configuration Template (ACT) is performed on DRAGEN on-premise.

There are no automation associated to this step.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Information

Field Name	Description	Additional Information
Analysis Result Location	Analysis files final copy out location.
Analysis Run ID	Run ID associate with the Analysis run.	Hidden on Record Details screen by default.
Analysis Status	Status for the the analysis run.
Log	Log messages by the Illumina Run Manager integration while handling the analysis event from instrument.

All the fields above are Read-Only Text field, except Log is a Read-Only Multiline Text.

How the Integration Works

The following information summarizes how the MiSeq i100 Series On-Premise Integration works.

• After the 'Validate Run Setup and Create Planned Run' automation is triggered on the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step, the run parameters entered in the Run Details screen and the sample informations are sent to Illumina Run Manager. Illumina Run Manager validates the run and analysis configuration before the planned run is created, the automation also generates the sample sheet and attaches it to the step. In the event of incomplete information or misconfiguration on the planned run, an error will be displayed on the automation banner and details of the error will be logged in the automation log file.

• When the sequencing run starts on the instrument, the MiSeq i100 Series Control Software notifies the Illumina Run Manager integration. The events are processed and the integration service retrieves the run information from MiSeq i100 Illumina Run Manager. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

• Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics). Then, Illumina Run Manager integration retrieves the primary metrics and uses them to populate the fields in the Sample Details table (e.g., % Error Rate R1). The custom fields (e.g., Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.

• The integration tracks the analysis events and results in the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.

If a planned run with the same sample name and project name (case-insensitive) has been created previously in Illumina Run Manager, the sample sheet generated from the 'Validate Run Setup and Create Planned Run' automation can reflect the original case of the previous sample name. This can cause validation errors for analysis configurations with sample-level settings. To resolve this issue, change the sample name or the project name on Clarity LIMS and run the automation again.

Start a Sequencing Run on MiSeq i100 Series Instrument

To start a planned run, refer to information on starting a planned run in the Illumina support site..

Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes

The library preparation workflow of the samples must be configured to ensure unique derived sample names before routing the samples through the library preparation workflow.

Components Installed

The following sections describe the components (files, properties, reagent categories / label groups, reagent kits, and containers) that are installed by default as part of this integration.

Global Fields

Container Global Fields

Field Name	Field Type	Settings
% Aligned R1	Numeric	Read Only Decimal Places Displayed: 2
% Aligned R2	Numeric	Read Only Decimal Places Displayed: 2
% Bases >=Q30 R1	Numeric	Read Only Decimal Places Displayed: 2
% Bases >=Q30 R2	Numeric	Read Only Decimal Places Displayed: 2
% Error Rate R1	Numeric	Read Only Decimal Places Displayed: 2
% Error Rate R2	Numeric	Read Only Decimal Places Displayed: 2
% Occupied	Numeric	Read Only Decimal Places Displayed: 2
% PF	Numeric	Read Only Decimal Places Displayed: 2
% Phasing R1	Numeric	Read Only Decimal Places Displayed: 3
% Phasing R2	Numeric	Read Only Decimal Places Displayed: 3
% Prephasing R1	Numeric	Read Only Decimal Places Displayed: 3
% Prephasing R2	Numeric	Read Only Decimal Places Displayed: 3
Intensity Cycle 1 R1	Numeric	Read Only
Intensity Cycle 1 R2	Numeric	Read Only
Reads PF	Numeric	Read Only Decimal Places Displayed: 2
Yield (Gb) R1	Numeric	Read Only Decimal Places Displayed: 2
Yield (Gb) R2	Numeric	Read Only Decimal Places Displayed: 2

Derived Sample Global Fields

Field Name	Field Type	Settings
ACT ID	Text	Read Only
ACT Name	Text	Read Only
Adjusted Per Sample Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
Bulk Pool Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
Final Loading Concentration (pM)	Numeric Dropdown	Required Preset: 225 400 Allow custom entries
Instrument ID	Text	Read Only
KLD Volume (ul)	Numeric	Read Only Decimal Places Displayed: 1
MiSeq i100 Series Flowcell Type	Text Dropdown	Required Preset: 5M 25M
Molarity (nM)	Numeric	Decimal Places Displayed: 2
Number of Samples in Pool	Numeric
Per Sample Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
PhiX Concentration (pM)	Numeric
PhiX Volume (ul)	Numeric	Decimal Places Displayed: 2
Required Bulk Pool Volume (ul)	Numeric	Read Only Decimal Places Displayed: 1
RSB Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
RSB Volume for Intermediate Pool (ul)	Numeric	Read Only Decimal Places Displayed: 1
Run Mode	Text	Read Only
Total Sample Volume (ul)	Numeric	Read Only Decimal Places Displayed: 1

Reagent Kits

• MiSeq i100 Series Dry Cartridge

• MiSeq i100 Series Library Denature (KLD)

• MiSeq i100 Series Wet Cartridge

• Resuspension Buffer (RSB)

Container Types

• MiSeq i100 Series Dry Cartridge

Instrument Types

• MiSeq i100 Series

Rules and Constraints

• The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine the following information:

  • Which samples, and how many, can enter each step together.

  • Which samples, and how many, can be pooled together.

• All submitted samples must have an associated secondary analysis that is configured using the analysis configuration template (ACT). The ACT must be configured on MiSeq i100 Illumina Run Manager before starting the Assign Analysis Configuration Template step. The ACT names must be unique.

• Assign Analysis Configuration Template step does not support pool library as input.

• The dry cartridge barcode must be unique. There must not be multiple dry cartridge containers with the same name in the Clarity LIMS system.

• Reagent labels, or indexes, must be unique.

• One library pool can only contain one library or control with no label/index.

• The AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) and AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) steps must not be manually started or completed. These steps are fully automated and the integration service does not update samples correctly if they have been manually started.

• For the automated run to start successfully, you must click 'Validate Run Setup and Create Planned Run' in the Load to Dry Cartridge step and the automation must be ran successfully.