The Illumina NextSeq 1000/2000 Integration Package v2.5.0 is compatible with Illumina cloud hosted deployments only.
It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.4 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 Integration Package v2.5.0 includes the following workflows:
NextSeq 1000/2000 Sequencing v2.4
[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 Sequencing v2.4 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 Sequencing v2.4 workflow. The first step is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4).
Steps:
Library Prep Validation v2.3.3
NextSeq 1000/2000 Sequencing v2.4 Workflow
Protocol 1: NextSeq 1000/2000 Sequencing v2.4
Purpose:
This protocol models the lab processes of starting a NextSeq 1000/2000 sequencing run.
Steps:
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)
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 Sequencing v2.4)
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 Sequencing v2.4)
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 and primary run metrics) of the sequencing run are recorded automatically.
GenerateFastQ secondary analysis is planned by default for the run. Samples continues to the next step.
Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)
In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.
Register Step Started Automation¹
This automation registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics. This automation is automatically triggered on entry to the step.
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).
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.
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 Sequencing v2.4):
This automation is automatically triggered on exit of the step and performs the following tasks:
Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library preparation.
Registers the completion time of the step by publishing messages to CLPA through Illumina Connected Analytics.
¹ These automations are required for CLPA support only.
Master Step Fields
The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) 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 Sequencing v2.4) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) Global Field Configuration
Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)
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 and/or creates a planned run depending on the Run Mode selected.
Validate Single Input & Register Step Started Automation²
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. This script is required for the NextSeq 1000/2000 Sequencing v2.4 workflow.
script:validateSampleCount -min 1 -max 1
Registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics. This script is only used for CLPA support.
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.::)
}
Retrieve Analysis Workflow Versions Automation
Automatically triggered upon entry of the Record Details screen, this automation fetches the available analysis workflow versions and updates the preset values of both of the Local Analysis Workflow Versions and Cloud Analysis Workflow Versions custom fields.
Validate Run Setup and Create Planned Run Automation
Automatically triggered when a button on the Record Details screen is selected, this automation validates the parameters entered on the Record Details screen. These parameters are used to set up the run and generate the sample sheet file and/or create the planned run.
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.
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.::)
}
When the Run Mode is Cloud, the script does the following actions:
Sets Local Analysis Workflow Versions to None.
If the Cloud Analysis Workflow Versions is None, the script displays the following error: Invalid option selected for Cloud Analysis Workflow Versions field.
Due to automation script limitation, a validation rule resides in the backend service to perform the following action: If Cloud Analysis Workflow Versions is 3.7.4 or later, set FASTQ Compression Format to gzip.
When the Run Mode is Local or Hybrid, the script does the following actions:
Sets Cloud Analysis Workflow Versions to None.
If Local Analysis Workflow Versions is None, the script displays the following error message: Invalid option selected for Local Analysis Workflow Versions field.
if (step.::Run Mode:: == ::Local:: || step.::Run Mode:: == ::Hybrid::) {
step.::Cloud Analysis Workflow Versions:: = ::None::;
if (step.::Local Analysis Workflow Versions:: == ::None::) {
fail(::Invalid option selected for Local Analysis Workflow Versions field.::);
}
};
Due to automation script limitations, a validation rule resides in the backend service. If Local Analysis Workflow Versions is 3.7.4 or later, an error occurs if FASTQ Compression Format is None.
Generates the sample sheet and/or creates the planned run.
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol—AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4). The automation is automatically triggered on exit of the Record Details screen.
nextStep = ::ADVANCE::
Register Step Completed¹
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through Illumina Connected Analytics. This script is only used for CLPA support.
¹ These automations are required for CLPA support only.
² These automations are required for the NextSeq 1000/2000 Sequencing v2.4 workflows and contain additional logic needed for CLPA support. If you would like to remove CLPA support, contact Illumina Support.
Master Step Fields
There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step. These fields are required for sample sheet generation and planned run creation.
Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)
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.
Register Step Started & Register NextSeq1k2k Run & Register NextSeq1k2k Run Association & Register NextSeq1k2k RunStarted Automation¹
Automatically triggered at the beginning of the step, this automation completes the following actions:
Registers the start time of the step.
Registers the NextSeq 1000/2000 sequencing run configuration.
Registers the association of the sequencing run with the actual samples by linking the Instrument Run ID with the IDs for the samples.
Registers the time that the sequencing run starts.
The automation completes these actions by publishing messages to CLPA through Illumina Connected Analytics. This automation is only used for CLPA support.
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the sequencing run completion time, run status, and run metrics. This script requires the Cloud Run ID field from the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step.
Registers the completion time of the step.
The automation completes these actions by publishing messages to CLPA through Illumina Connected Analytics. This automation is only used for CLPA support.
¹ These automations are required for CLPA support only.
Master Step Fields
There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) 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.
¹ For information on how the integration works, refer to Run Status, Primary Metrics, and Analysis Results Parsing and Recording in How the NextSeq 1000/2000 Integration Works.
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 Sequencing v2.4) 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.
Register Step Started Automation¹
Automatically triggered at the beginning of the step, this automation registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics. This automation is only used for CLPA support.
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.
¹ These automations are required for CLPA support only.
Master Step Fields
The following table lists the master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) master step.
Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step
Global Fields
The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool.
The demultiplexing statistics are aggregated for multi-lane flow cells. For each demultiplexing field (for example, # Reads) displayed in the Sample Details table for the multi-lane flow cell (for example, P3), the displayed value is the sum of the statistics across all lanes.
Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step
How the NextSeq 1000/2000 Integration Works
Sample Sheet Generation and Planned Run Creation
On the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step, when the Validate Run Setup and Create Planned Run automation is triggered, the run and analysis parameters entered in the Run Details screen are used to create a planned run. Then, the run and analysis configuration is validated. If the validation fails, an error message is sent to Clarity LIMS. If the validation passes, the sample sheet is generated (and sent back to Clarity LIMS) and/or a planned run is created depending on the Run Mode selected.
Local run mode: Only the sample sheet is generated and stored on Clarity LIMS. The sample sheet contains all the run and analysis configuration required to start the run on the instrument.
Hybrid/Cloud run mode: A planned run is created. This planned run contains all the run and analysis configuration required to start the run on the instrument. The sample sheet is also generated and stored on Clarity LIMS for reference purposes.
If a planned run with the same sample and project name has been created previously, the original case with the previous sample name can be reflected in the sample sheet generated from the Validate Run Setup and Create Planned Run automation. To resolve this issue, change the sample or project name in Clarity LIMS and run the automation again.
Run Status, Primary Metrics, and Analysis Results Parsing and Recording
After the sequencing run is started on the instrument, NextSeq 1000/2000 Control Software (NCS) notifies BaseSpace Sequence Hub that the sequencing run has started.
BaseSpace Sequence Hub then does the following actions:
Local run mode: Creates a run on the BaseSpace Sequence Hub database with the run configuration information received from NCS.
Hybrid/Cloud run mode: Creates a run on the BaseSpace Sequence Hub database by copying the run configuration information from the planned run using a special Cloud Run ID received from NCS.
After the run is created on BaseSpace Sequence Hub, a run update event message that carries the RunStarted equivalent message is created on BaseSpace Sequence Hub and delivered to Clarity LIMS SQS queue service. The message is captured and processed by the integration.
Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration service looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) step for a container with the same reagent cartridge barcode. When identified, the step is started automatically and the Run Status field on Record Details screen is updated to RunStarted. The integration service then updates all the other step fields (for example, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BaseSpace Sequence Hub by NCS.
The NextSeq 1000/2000 Control Software (NCS) continues to notify BaseSpace Sequence Hub (BSSH) of any run status change throughout the course of the sequencing run. The run update event delivery process continues as described in the previous section.
The following table lists the run status details and how the integration service handles each status.
Start A Sequencing Run On Instrument With Different Run Modes
Configuring a run with the Proactive, Run Monitory and Storage option. Local runs are done in Local mode, and cloud or hybrid runs are done in Cloud or Hybrid mode.
Starting a local, cloud, or hybrid run.
Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes
Clarity LIMS requires unique library names when samples are re-queued to the workflow in the NextSeq 1000/2000 integration. Assign unique names to libraries after going through library preparation.
The following steps are used to configure the library preparation workflow correctly before routing the samples for re-queuing:
From Configuration, select the Lab Work tab.
Search for the library preparation workflow used for the re-queued samples.
In the Master Step of the library preparation workflow, modify the naming convention under Step Type to generate unique library names (for example, appending LIMS ID to the default naming convention, like OutputItemLIMSID).
Select Save.
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:
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 should 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 Sequencing v2.4) 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 Sequencing v2.4) step. This step is semi-automated, and the integration service does not update the demultiplexing result correctly if they have been manually started.
For the automated run to start successfully, select Validate Run Setup and Create Planned Run in the Load to Reagent Cartridge step.
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.
This integration requires a Personal, Professional, or Enterprise BaseSpace Sequence Hub subscription.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
Create one pool per step. The Calculate Volumes automation supports one pool only.
Use alphanumeric, dash, or underscore characters only in the submitted sample names. Any other characters can cause sample sheet validation failure in the Load to Reagent Cartridge step. The NextSeq 1000/2000 Sequencing workflow handles these characters in the Load to Reagent Cartridge step by replacing them with an underscore in the submitted sample name. The Sample Details table in the Demultiplexing step reflects the modified submitted sample name.
The NextSeq 1000/2000 reagent cartridges support different read cycle numbers. Make sure that the read cycle values configured for the planned run are within the maximum allowable reads for the cartridge type.
Do not modify or disable the Retrieve Analysis Workflow Versions automation script. Modifying or disabling the script breaks the integration.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
Do not modify the master step field names. This action causes the integration to break.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
The NextSeq 1000/2000 Reagent Cartridge barcode should 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.
