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Configuration

The Illumina NextSeq 1000/2000 On-Prem Integration Package v1.0.0 supports the on-premise integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems.

For instructions on validating and troubleshooting the NextSeq 1000/2000 Integration, refer to NextSeq 1000/2000 On-Prem Integration v1.0.0 User Interaction, Validation and Troubleshooting.

The configuration provided in this integration has been established to support NextSeq 1000/2000 lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the process.

When the Library Prep Validation v2.3.3 workflow is imported, it includes the Illumina Universal Sample Identifier global field. This field is a text field that is reserved for CLPA support and is optional. The value of this field is not required for this integration.

Prerequisites and Assumptions

It is assumed that samples entering the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow have gone through library preparation and quantification processes. Before they are assigned to the workflow, samples have completed the following actions:

Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to some value.
The Calculate Volumes automation in the Library Pooling and Dilution step requires a value in the Molarity (nM) global field.

For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Workflows, Protocols, and Steps

The NextSeq 1000/2000 On-Prem Integration Package v1.0.0 includes the following workflows:

NextSeq 1000/2000 On-Prem Sequencing v1.0
[Optional] Library Prep Validation v2.3.3 (recommended for validation purposes)

The following protocols and steps are included in these workflows.

Library Prep Validation v2.3.3 Workflow

Protocol 1: Library Prep Validation v2.3.3

Purpose:

Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow.
The protocol contains a single step—Library Prep Validation v2.3.3. At the end of this step, a routing script sends the samples to the first step of the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow. The first step is Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0).

Steps:

Library Prep Validation v2.3.3

NextSeq 1000/2000 On-Prem Sequencing v1.0 Workflow

Protocol 1: NextSeq 1000/2000 On-Prem Sequencing v1.0

Purpose:

This protocol models the lab processes of starting a NextSeq 1000/2000 sequencing run.

Steps:

Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- Samples are pooled and diluted to the final loading concentration with the help of the Calculate Volume script.
Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- The library pool from step 1 is then ready to be loaded to the NextSeq 1000/2000 reagent cartridge.
- Run and analysis information is validated.
- Sample sheet is generated and/or planned run is created.
AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- This step is a fully automated step that is started and completed automatically after the sequencing run is started and completed on the instrument side.
- All the metadata (for example, run configuration, primary run metrics) of the sequencing run is recorded automatically.
- GenerateFastQ secondary analysis is planned by default for the run. Samples continue to the next step.
Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- This step is a semi-automated step that is started automatically after the GenerateFastQ secondary analysis has started.
- Demultiplexing result is parsed and recorded automatically.
- User is required to assign QC label (Pass/Fail) to the individual library in the pool and complete the step.

Validation Workflow

The Library Prep Validation v2.3.3 workflow allows for validation of the system after installation is complete. For details, refer to NextSeq 1000/2000 On-Prem Integration v1.0.0 User Interaction, Validation and Troubleshooting.

Step 1: Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)

In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.

Calculate Volumes Automation

This automation is triggered when you select Calculate Volumes on the Record Details screen. The automation completes the following actions:

Sets RSB Volume for Pool (ul) field value to 24 for calculation purpose of the 2 nM intermediate library pool.

Copies the Final Loading Concentration (pM) from step inputs to step outputs.

-exp 'step.::RSB Volume for Pool (ul):: = 24; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::'

Calculate per sample volume required for each library to make the 2 nM intermediate library pool (24 µl in volume).

-exp 'input.::Per Sample Volume (ul):: = ( 48/ input.::Molarity (nM):: ); step.::RSB Volume for Pool (ul):: -= input.::Per Sample Volume (ul)::'

Calculate the volume required of the 2 nM library pool to be diluted further to the Final Loading Concentration (pM) with the Final Loading Volume (ul).

-exp 'step.::Library Pool Volume (ul):: = (step.::Final Loading Concentration (pM):: * step.::Final Loading Volume (ul)::/2000); output.::RSB Volume (ul):: = step.::Final Loading Volume (ul):: - step.::Library Pool Volume (ul)::'

Uses the NextSeq1K2K_Pool1.csv, NextSeq1K2K_Pool2.csv, and NextSeq1K2K_Pool3.csv template files to generate a single CSV file containing information about the pool and the samples it contains. The generated file is stored in the Calculation File placeholder, in the Files section, for download.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool1.csv \
-o 1.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool2.csv \
-o 2.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool3.csv \
-o 3.csv \
&& cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csv

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load to Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0):

nextStep = ::ADVANCE::

Master Step Fields

The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.

Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) Master Step Field Configuration

Global Fields

The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.

Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) Global Field Configuration

Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0)

In this step, scan the reagent cartridge barcode into Clarity LIMS, then manually place the working pool into the reagent cartridge for the NextSeq 1000/2000 run. This step validates the run setup and analysis information and generates the sample sheet file.

Validate Single Input Automation

Automatically triggered at the beginning of the step, this automation does the following actions:

Checks that there is only one container input to the step.

script:validateSampleCount -min 1 -max 1

Validate Reagent Cartridge Barcode Automation

Automatically triggered on exit of the Placement screen, the following automation validates the reagent cartridge barcode to make sure it conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z0-9]{4}:

if (!output.container.name.matches( ::[A-Z]{2}[0-9]{7}-[A-Z0-9]{4}:: ) )
{
    fail ( ::Invalid Reagent Cartridge Barcode. Please verify and try again.:: ) 
}

Validate Run Setup and Create Sample Sheet Automation

Automatically triggered when a button on the Record Details screen is selected, this automation does the following actions:

Validates the parameters entered on the Record Details screen. These parameters are used to set up the run and generate the sample sheet file.
- Run Name can only contain alphanumeric, dash, underscore, or period characters. Spaces are not permitted.
  if (!step.::Run Name::.matches(::[a-zA-Z0-9-_.]+::)){ fail(::Run Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, _, and .::) }
- Run Name must not exceed 255 characters.
  if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::) };

Checks the Index Reads, Index Read 1, and Index Read 2 field values.

If Index Reads is No Index, Index Read 1 and Index Read 2 values must be 0 (error results if it is otherwise).
If Index Reads is Single Index, Index Read 1 value must be greater than 0, and Index Read 2 values must be 0 (error results if it is otherwise).
If Index Reads is Dual Index, Index Read 1 and Index Read 2 values must be greater than 0 (error results if it is otherwise).

if (step.::Index Reads:: == ::No Index::){ 
    if (step.::Index Read 1:: != 0 || step.::Index Read 2:: != 0) { 
        fail(::Index Read 1 and Index Read 2 must be 0 if the Index Reads is No Index.::) 
    } 
}
else{ 
    if (step.::Index Reads:: == ::Single Index::){ 
        if (step.::Index Read 1:: == 0 || step.::Index Read 2:: != 0) { 
            fail(::Index Read 1 must be greater than 0 and Index Read 2 must be 0 if the Index Reads is Single Index.::) 
        } 
    }
    else{ 
        if (step.::Index Read 1:: == 0 || step.::Index Read 2:: == 0) { 
            fail(::Index Read 1 and Index Read 2 must be greater than 0 if the Index Reads is Dual Index.::) 
        } 
    }
}

Checks the Paired End, Read 2 Cycles, and Index Read 2 field values.

If Paired End is set to True and Read 2 Cycles value is 0, an error is generated.
If Paired End is set to False and Read 2 Cycles or Index Read 2 values are greater than 0, an error is generated.

if (step.::Paired End::.toBoolean()){ 
    if (step.::Read 2 Cycles:: == 0) { 
        fail(::Read 2 Cycles must not be zero if it is Paired End read.::) 
    }
}
else{
    if (step.::Read 2 Cycles:: != 0 || step.::Index Read 2:: != 0) {
        fail(::Read 2 Cycles and Index 2 Cycles must be 0 if it is not Paired End Read.:: )
    }
}

Checks the Adapter Sequence Read 1 and Adapter Sequence Read 2 field values.

Adapter Sequence Read 1 and Adapter Sequence Read 2 can only contain ACTG+ characters.

if (step.hasValue(::Adapter Sequence Read 1::) && !step.::Adapter Sequence Read 1::.matches(::[ACTG+]+::)) { 
    fail(::Adapter Sequence Read 1 contains prohibited characters. Allowed characters are: ACTG+::) 
};
if (step.hasValue(::Adapter Sequence Read 2::) && !step.::Adapter Sequence Read 2::.matches(::[ACTG+]+::)) { 
    fail(::Adapter Sequence Read 2 contains prohibited characters. Allowed characters are: ACTG+::) 
}

Checks Override Cycles field value.

Override Cycles can only contain Y, N, I, U, 0–9, and semicolon characters.

if (step.hasValue(::Override Cycles::) && !step.::Override Cycles::.matches(::[YNIU0-9;]+::)){ 
    fail(::Override Cycles contains prohibited characters. Allowed characters are: Y, N, I, U, 0-9 and ;. Example: N1Y150;I8;I7N1;Y141U10.::) 
}

Checks Local Analysis Workflow Versions field value.

Local Analysis Workflow Versions can only contain 0–9 and period characters. This field must start and end with numbers that are separated by a period (for example, 3.8.4).

if (!step.::Local Analysis Workflow Version::.matches(::^\\d+\\.\\d+\\.\\d+$::)) { 
    fail(::Local Analysis Workflow Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::) 
};

Generates the sample sheet.

Sample sheet is attached to the step and is also copied to the sample sheet directory that is configured during installation.
Refer to NextSeq 1000/2000 On-Prem Integration v1.0.0 User Interaction, Validation and Troubleshooting for submitted sample name character restrictions.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_OnPrem_Samplesheet.csv \
-o {compoundOutputFileLuid0}.csv \
-q true \
-destLIMSID {compoundOutputFileLuid0} \
-l {compoundOutputFileLuid2} \
&& /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:download_attachments \
-b 'prop:integration.nextseq1k2k-onprem.v1.sampleSheetDir' \
-s 'CONTAINER_NAME' \
-f {compoundOutputFileLuid0} \
-l {compoundOutputFileLuid1}"

Set Next Step Automation

Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol—AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0). The automation is automatically triggered on exit of the Record Details screen.

nextStep = ::ADVANCE::

Master Step Fields

The following fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0) step are required for sample sheet generation.

Load to Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0) Master Step Fields Configuration

Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0)

This step is fully automated.

The integration starts and completes the step automatically. Data from the run is parsed back to Clarity LIMS. No user interaction is required. In this step, the pooled samples in the reagent cartridge are sequenced on the NextSeq 1000/2000 instrument.

Master Step Fields

The following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. These fields are used to display the run status and sequencing run and analysis configuration parsed from the RunParameters.xml file of the sequencing run.

Run Parameters and Corresponding Clarity LIMS Step Fields

The following table shows how some of the step fields map to the fields on the RunParameters.xml file, and whether the field is visible on the Record Details screen.

Additional Master Step Fields and Values

The following table shows how the other step fields derive their values, and whether the step field is visible on the Record Details screen.

Global Fields

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

% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2

At the end of this step, the pool of samples is automatically advanced to (and queued for) the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.

Step 4: Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0)

This step is a semi-automated step.

The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. The lab scientist reviews the demultiplexing result parsed into Clarity LIMS, assigns QC flags, and completes the step.

Assign Demultiplexing QC Flags Automation

Automatically triggered when you select a button on the Record Details screen, this automation assigns QC flags based on the criteria set in the step fields.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} script:assignQCForDemuxStep -i {processURI:v2} -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation sets the next step to Advance and the samples to complete the protocol.

nextStep = ::ADVANCE::

Master Step Fields

The following fields are configured on the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) master step.

Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) Step

Global Fields

The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool.

Global Field Configuration for Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) Step

How the NextSeq 1000/2000 On-Premise Integration Works

The following diagram shows how the integration works between the NextSeq 1000/2000 and Clarity LIMS.

Sample Sheet Generation

On the Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0) step, the run and analysis parameters entered in the Run Details screen are validated using the scripts in this step. This validation occurs when the Validate Run Setup and Create Sample Sheet automation is triggered. If the validation passes, Clarity LIMS generates the sample sheet using the driver file generator. The sample sheet contains all the run and analysis configuration required to start the run on the instrument.

For more information on how to start a local run, refer to instructions for initiating a sequence run in the NextSeq 1000/2000 Product Documentation.

Run Status, Primary Metrics, and Analysis Results Parsing and Recording

After the sequencing run is started on the instrument, the instrument generates the files as the sequencing and analysis run progresses. The integration monitors these files to track the run.

The following table shows the run statuses and how the integration service handles them.

Run Status

The integration immediately starts the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step after completing the AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. In the Demultiplexing step, the integration does the following actions:

Searches for the CopyComplete.txt file produced by the instrument as a sign that the analysis is complete.
- After analysis is completed, the integration downloads the Demultiplex_Stats.csv file that contains the demultiplexing results. Then, the integration zips the run metric files (Adapter_Metrics.csv, Demultiplex_Stats.csv, Index_Hopping_Counts.csv, Quality_Metrics.csv, and Top_Unknown_Barcodes.csv) and attaches the Run_Metrics.zip file to the step. After detecting the Demultiplex_Stats.csv file, the Analysis Status field is updated to Completed. Otherwise, this field is set to Failed to signal that the BCL Convert analysis has failed.
- The Demultiplex_Stats.csv file is parsed and details are recorded in the Sample Details table for each library in the library pool. For multi-lane flow cells, the demultiplex details (for example, number of reads) are aggregated as a sum.
Updates the Multiline Sequencing Log field.

The integration does not automatically complete the step. You must assign the QC flag to the individual library before manually completing the step.

Start A Sequencing Run On Instrument

For more information on how to start a local run, refer to instructions for initiating a standard SBS or XLEAP-SBS sequencing run in the NextSeq 1000/2000 Product Documentation.

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 various components that are installed by default as part of this integration. These components include the following items:

Reagent categories/label groups
Reagent kits
Control types
Containers

Information on installed workflows, protocols, steps, and automation points is provided in the Workflows, Protocols, and Steps section of NextSeq 1000/2000 On-Prem Integration v1.0.0 User Interacion, Validation and Troubleshooting.

Reagent Categories/Label Groups

TruSeq HT adapters v2 (D7-D5)

Reagent Kits

Resuspension Buffer (RSB)
NextSeq 1000/2000 reagent cartridge

Container Types

Tube
96-well plate
NextSeq 1000/2000 reagent cartridge

Control Types

PhiX v3

This integration supports the NextSeq 1000/2000 reagent cartridge with barcode provided in the format [A-Z]{2}[0-9]{7}-[A-Z0-9]{4} (for example, EC1234567-EC03).

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:
- Which samples, and how many, can enter each step together.
- Which samples, and how many, can be pooled together.
The NextSeq 1000/2000 reagent cartridge barcode must be unique. There must not be multiple NextSeq 1000/2000 reagent cartridge containers in the system with the same name.
Reagent labels (indexes) must be unique.
One library pool can only contain one library or control with no label (index).
Do not manually start or complete the AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. This step is a fully automated step, and the integration service does not update samples correctly if they have been manually started.
Do not manually start the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. This step is semi-automated, and the SIS integration service does not update the demultiplexing results correctly if they have been manually started.

Configuration

The Illumina NextSeq 1000/2000 On-Prem Integration Package v1.0.0 supports the on-premise integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems.

For instructions on validating and troubleshooting the NextSeq 1000/2000 Integration, refer to NextSeq 1000/2000 On-Prem Integration v1.0.0 User Interaction, Validation and Troubleshooting.

The configuration provided in this integration has been established to support NextSeq 1000/2000 lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the process.

When the Library Prep Validation v2.3.3 workflow is imported, it includes the Illumina Universal Sample Identifier global field. This field is a text field that is reserved for CLPA support and is optional. The value of this field is not required for this integration.

Prerequisites and Assumptions

Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to some value.
The Calculate Volumes automation in the Library Pooling and Dilution step requires a value in the Molarity (nM) global field.

Workflows, Protocols, and Steps

The NextSeq 1000/2000 On-Prem Integration Package v1.0.0 includes the following workflows:

NextSeq 1000/2000 On-Prem Sequencing v1.0
[Optional] Library Prep Validation v2.3.3 (recommended for validation purposes)

The following protocols and steps are included in these workflows.

Library Prep Validation v2.3.3 Workflow

Protocol 1: Library Prep Validation v2.3.3

Purpose:

Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow.
The protocol contains a single step—Library Prep Validation v2.3.3. At the end of this step, a routing script sends the samples to the first step of the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow. The first step is Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0).

Steps:

Library Prep Validation v2.3.3

NextSeq 1000/2000 On-Prem Sequencing v1.0 Workflow

Protocol 1: NextSeq 1000/2000 On-Prem Sequencing v1.0

Purpose:

This protocol models the lab processes of starting a NextSeq 1000/2000 sequencing run.

Steps:

Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- Samples are pooled and diluted to the final loading concentration with the help of the Calculate Volume script.
Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- The library pool from step 1 is then ready to be loaded to the NextSeq 1000/2000 reagent cartridge.
- Run and analysis information is validated.
- Sample sheet is generated and/or planned run is created.
AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- This step is a fully automated step that is started and completed automatically after the sequencing run is started and completed on the instrument side.
- All the metadata (for example, run configuration, primary run metrics) of the sequencing run is recorded automatically.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
- GenerateFastQ secondary analysis is planned by default for the run. Samples continue to the next step.
Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0)
- This step is a semi-automated step that is started automatically after the GenerateFastQ secondary analysis has started.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
- Demultiplexing result is parsed and recorded automatically.
- User is required to assign QC label (Pass/Fail) to the individual library in the pool and complete the step.

Validation Workflow

Step 1: Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)

In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.

Create one pool per step. The Calculate Volumes automation supports one pool only.

Calculate Volumes Automation

This automation is triggered when you select Calculate Volumes on the Record Details screen. The automation completes the following actions:

Sets RSB Volume for Pool (ul) field value to 24 for calculation purpose of the 2 nM intermediate library pool.

Copies the Final Loading Concentration (pM) from step inputs to step outputs.

-exp 'step.::RSB Volume for Pool (ul):: = 24; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::'

Calculate per sample volume required for each library to make the 2 nM intermediate library pool (24 µl in volume).

-exp 'input.::Per Sample Volume (ul):: = ( 48/ input.::Molarity (nM):: ); step.::RSB Volume for Pool (ul):: -= input.::Per Sample Volume (ul)::'

Calculate the volume required of the 2 nM library pool to be diluted further to the Final Loading Concentration (pM) with the Final Loading Volume (ul).

-exp 'step.::Library Pool Volume (ul):: = (step.::Final Loading Concentration (pM):: * step.::Final Loading Volume (ul)::/2000); output.::RSB Volume (ul):: = step.::Final Loading Volume (ul):: - step.::Library Pool Volume (ul)::'

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool1.csv \
-o 1.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool2.csv \
-o 2.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool3.csv \
-o 3.csv \
&& cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csv

Set Next Step Automation

nextStep = ::ADVANCE::

Master Step Fields

The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.

Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) Master Step Field Configuration

Global Fields

The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.

Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) Global Field Configuration

Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0)

The NextSeq 1000/2000 reagent cartridges support different read cycle numbers. Make sure that the read cycle values configured are within the maximum allowable reads for the cartridge type.

Validate Single Input Automation

Automatically triggered at the beginning of the step, this automation does the following actions:

Checks that there is only one container input to the step.

script:validateSampleCount -min 1 -max 1

Validate Reagent Cartridge Barcode Automation

Automatically triggered on exit of the Placement screen, the following automation validates the reagent cartridge barcode to make sure it conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z0-9]{4}:

if (!output.container.name.matches( ::[A-Z]{2}[0-9]{7}-[A-Z0-9]{4}:: ) )
{
    fail ( ::Invalid Reagent Cartridge Barcode. Please verify and try again.:: ) 
}

Validate Run Setup and Create Sample Sheet Automation

Automatically triggered when a button on the Record Details screen is selected, this automation does the following actions:

Validates the parameters entered on the Record Details screen. These parameters are used to set up the run and generate the sample sheet file.
- Run Name can only contain alphanumeric, dash, underscore, or period characters. Spaces are not permitted.
  if (!step.::Run Name::.matches(::[a-zA-Z0-9-_.]+::)){ fail(::Run Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, _, and .::) }
- Run Name must not exceed 255 characters.
  if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::) };

Checks the Index Reads, Index Read 1, and Index Read 2 field values.

If Index Reads is No Index, Index Read 1 and Index Read 2 values must be 0 (error results if it is otherwise).
If Index Reads is Single Index, Index Read 1 value must be greater than 0, and Index Read 2 values must be 0 (error results if it is otherwise).
If Index Reads is Dual Index, Index Read 1 and Index Read 2 values must be greater than 0 (error results if it is otherwise).

if (step.::Index Reads:: == ::No Index::){ 
    if (step.::Index Read 1:: != 0 || step.::Index Read 2:: != 0) { 
        fail(::Index Read 1 and Index Read 2 must be 0 if the Index Reads is No Index.::) 
    } 
}
else{ 
    if (step.::Index Reads:: == ::Single Index::){ 
        if (step.::Index Read 1:: == 0 || step.::Index Read 2:: != 0) { 
            fail(::Index Read 1 must be greater than 0 and Index Read 2 must be 0 if the Index Reads is Single Index.::) 
        } 
    }
    else{ 
        if (step.::Index Read 1:: == 0 || step.::Index Read 2:: == 0) { 
            fail(::Index Read 1 and Index Read 2 must be greater than 0 if the Index Reads is Dual Index.::) 
        } 
    }
}

Checks the Paired End, Read 2 Cycles, and Index Read 2 field values.

If Paired End is set to True and Read 2 Cycles value is 0, an error is generated.
If Paired End is set to False and Read 2 Cycles or Index Read 2 values are greater than 0, an error is generated.

if (step.::Paired End::.toBoolean()){ 
    if (step.::Read 2 Cycles:: == 0) { 
        fail(::Read 2 Cycles must not be zero if it is Paired End read.::) 
    }
}
else{
    if (step.::Read 2 Cycles:: != 0 || step.::Index Read 2:: != 0) {
        fail(::Read 2 Cycles and Index 2 Cycles must be 0 if it is not Paired End Read.:: )
    }
}

Checks the Adapter Sequence Read 1 and Adapter Sequence Read 2 field values.

Adapter Sequence Read 1 and Adapter Sequence Read 2 can only contain ACTG+ characters.

if (step.hasValue(::Adapter Sequence Read 1::) && !step.::Adapter Sequence Read 1::.matches(::[ACTG+]+::)) { 
    fail(::Adapter Sequence Read 1 contains prohibited characters. Allowed characters are: ACTG+::) 
};
if (step.hasValue(::Adapter Sequence Read 2::) && !step.::Adapter Sequence Read 2::.matches(::[ACTG+]+::)) { 
    fail(::Adapter Sequence Read 2 contains prohibited characters. Allowed characters are: ACTG+::) 
}

Checks Override Cycles field value.

Override Cycles can only contain Y, N, I, U, 0–9, and semicolon characters.

if (step.hasValue(::Override Cycles::) && !step.::Override Cycles::.matches(::[YNIU0-9;]+::)){ 
    fail(::Override Cycles contains prohibited characters. Allowed characters are: Y, N, I, U, 0-9 and ;. Example: N1Y150;I8;I7N1;Y141U10.::) 
}

Checks Local Analysis Workflow Versions field value.

Local Analysis Workflow Versions can only contain 0–9 and period characters. This field must start and end with numbers that are separated by a period (for example, 3.8.4).

if (!step.::Local Analysis Workflow Version::.matches(::^\\d+\\.\\d+\\.\\d+$::)) { 
    fail(::Local Analysis Workflow Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::) 
};

Generates the sample sheet.

Sample sheet is attached to the step and is also copied to the sample sheet directory that is configured during installation.
Refer to NextSeq 1000/2000 On-Prem Integration v1.0.0 User Interaction, Validation and Troubleshooting for submitted sample name character restrictions.

Sample sheet validation in the Load to Reagent Cartridge step must only have alphanumeric, dash, and underscore characters in the submitted sample name. Any other characters are replaced with an underscore. The Sample Details table in the Demultiplexing step reflects the modified submitted sample name.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_OnPrem_Samplesheet.csv \
-o {compoundOutputFileLuid0}.csv \
-q true \
-destLIMSID {compoundOutputFileLuid0} \
-l {compoundOutputFileLuid2} \
&& /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:download_attachments \
-b 'prop:integration.nextseq1k2k-onprem.v1.sampleSheetDir' \
-s 'CONTAINER_NAME' \
-f {compoundOutputFileLuid0} \
-l {compoundOutputFileLuid1}"

Set Next Step Automation

nextStep = ::ADVANCE::

Master Step Fields

The following fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0) step are required for sample sheet generation.

Load to Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0) Master Step Fields Configuration

Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0)

This step is fully automated.

Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.

Master Step Fields

Run Parameters and Corresponding Clarity LIMS Step Fields

The following table shows how some of the step fields map to the fields on the RunParameters.xml file, and whether the field is visible on the Record Details screen.

Additional Master Step Fields and Values

The following table shows how the other step fields derive their values, and whether the step field is visible on the Record Details screen.

Global Fields

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

% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2

At the end of this step, the pool of samples is automatically advanced to (and queued for) the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.

Step 4: Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0)

This step is a semi-automated step.

Do not add samples to the Ice Bucket or start the AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. The integration completes these actions automatically.

Assign Demultiplexing QC Flags Automation

Automatically triggered when you select a button on the Record Details screen, this automation assigns QC flags based on the criteria set in the step fields.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} script:assignQCForDemuxStep -i {processURI:v2} -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation sets the next step to Advance and the samples to complete the protocol.

nextStep = ::ADVANCE::

Master Step Fields

The following fields are configured on the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) master step.

Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) Step

Global Fields

Global Field Configuration for Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) Step

How the NextSeq 1000/2000 On-Premise Integration Works

The following diagram shows how the integration works between the NextSeq 1000/2000 and Clarity LIMS.

Sample Sheet Generation

For more information on how to start a local run, refer to instructions for initiating a sequence run in the NextSeq 1000/2000 Product Documentation.

Run Status, Primary Metrics, and Analysis Results Parsing and Recording

After the sequencing run is started on the instrument, the instrument generates the files as the sequencing and analysis run progresses. The integration monitors these files to track the run.

The following table shows the run statuses and how the integration service handles them.

Run Status

Searches for the CopyComplete.txt file produced by the instrument as a sign that the analysis is complete.
- After analysis is completed, the integration downloads the Demultiplex_Stats.csv file that contains the demultiplexing results. Then, the integration zips the run metric files (Adapter_Metrics.csv, Demultiplex_Stats.csv, Index_Hopping_Counts.csv, Quality_Metrics.csv, and Top_Unknown_Barcodes.csv) and attaches the Run_Metrics.zip file to the step. After detecting the Demultiplex_Stats.csv file, the Analysis Status field is updated to Completed. Otherwise, this field is set to Failed to signal that the BCL Convert analysis has failed.
- The Demultiplex_Stats.csv file is parsed and details are recorded in the Sample Details table for each library in the library pool. For multi-lane flow cells, the demultiplex details (for example, number of reads) are aggregated as a sum.
Updates the Multiline Sequencing Log field.

The integration does not automatically complete the step. You must assign the QC flag to the individual library before manually completing the step.

Start A Sequencing Run On Instrument

For more information on how to start a local run, refer to instructions for initiating a standard SBS or XLEAP-SBS sequencing run in the NextSeq 1000/2000 Product Documentation.

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 various components that are installed by default as part of this integration. These components include the following items:

Reagent categories/label groups
Reagent kits
Control types
Containers

Reagent Categories/Label Groups

TruSeq HT adapters v2 (D7-D5)

Reagent Kits

Resuspension Buffer (RSB)
NextSeq 1000/2000 reagent cartridge

Container Types

Tube
96-well plate
NextSeq 1000/2000 reagent cartridge

Control Types

PhiX v3

This integration supports the NextSeq 1000/2000 reagent cartridge with barcode provided in the format [A-Z]{2}[0-9]{7}-[A-Z0-9]{4} (for example, EC1234567-EC03).

Rules and Constraints

The NextSeq 1000/2000 Reagent Cartridge barcode must not be modified after a successful validation. Modifications can cause issues when Clarity LIMS tries to update the status and sample details of subsequent steps.

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:
- Which samples, and how many, can enter each step together.
- Which samples, and how many, can be pooled together.
The NextSeq 1000/2000 reagent cartridge barcode must be unique. There must not be multiple NextSeq 1000/2000 reagent cartridge containers in the system with the same name.
Reagent labels (indexes) must be unique.
One library pool can only contain one library or control with no label (index).
Do not manually start or complete the AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. This step is a fully automated step, and the integration service does not update samples correctly if they have been manually started.
Do not manually start the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. This step is semi-automated, and the SIS integration service does not update the demultiplexing results correctly if they have been manually started.