These release notes describe the key changes to software components for the Clarity LIMS NextSeq 1000/2000 On-Prem Integration Package v1.0.0.
Compatibility
Refer to under Instruments & Integrations.
New Features
The new on-premise integration for NextSeq 1000/2000 supports the following functions:
Automated generation of a sample sheet for use with the BCL Convert analysis software.
Automated tracking of the sequencing run and parsing of run statistics into Clarity LIMS.
Defect Repairs
None
Known Issues
Sequencing run error triggered by disk space full on instrument causes RunParameters.xml to be empty and the run event cannot be processed on Clarity LIMS. This error is unlikely to occur as pre-run checks on the instrument check for sufficient disk space before run is started.
Limitations
Does not support analysis requeue.
Revision History
Automated tracking of the local DRAGEN BCL Convert analysis (BCL Convert v4.2.7 and earlier) and parsing of demultiplexing metrics into Clarity LIMS. The metrics are aggregated for multi-lane flow cells (for example, P3).
Version
Changes
2
Updated Compatibility section to reference Compatibility matrix table.
The documents in this section support the Illumina NextSeq 1000/2000 Integrations. These integrations are compatible with Illumina cloud hosted deployments as well as on-premise integrations without internet connections.
NextSeq 1000/2000 On-Prem v1.0.0
The NextSeq 1000/2000 On-Prem Integration Package v1.0.0 enables run planning through the v2 sample sheet that is generated or imported. The integration also tracks both sequencing run and GenerateFASTQ analysis run events without a connection to BaseSpace Sequence Hub or ICA.
The integration includes:
The preconfigured NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow that maps to lab protocols, instrument runs, and GenerateFASTQ analysis.
The following preconfigured steps:
Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)
Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0)
AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0)
Automated generation of the v2 sample sheet using Driver File Generator to set up Local Run Mode sequencing and GenerateFASTQ analysis runs.
Automated copying of the generated v2 sample sheet to a network drive location so that the instrument can import it.
Automated tracking of the sequencing run and parsing of run statistics into Clarity LIMS, including:
Progress and metrics of sequencing run
Sequencing run parameters
Automated tracking of local secondary analysis using DRAGEN (up to BCL Convert only) and parsing of demultiplexing metrics into Clarity LIMS. The local BCL Convert is supported up to v4.2.7.
[Optional] Preconfigured Library Prep Validation v2.3.3 protocol used for validation purposes only. This workflow contains a single-step protocol that models the library prep required to produce libraries tagged with index sequences that are ready for the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow. For more information, refer to .
Reagent lot information
Sequencing run data directory location
Instrument software version
Real-Time Analysis (RTA) version and other run specific information
The Illumina NextSeq 1000/2000 On-Prem Integration Package v1.0.0 supports the integration of Clarity LIMS to NextSeq 1000/2000 instruments.
Prerequisites
NextSeq 1000/2000 On-Prem Integration v1.0.0 has the following prerequisites:
Illumina Preset Protocols (IPP) v2.8 or later, which contains the NextSeq 1000/2000 On-Prem Sequencing workflow
NGS Package v5.25.0 or later, which contains automation scripts used by the workflow
Clarity LIMS v6.2 or later with NAS shares and mount points
Prerequisite 1: IPP v2.8 and NGS v5.25.0 Installation
The NextSeq 1000/2000 On-Prem Integration v1.0.0 includes the workflows, protocols, and steps provided in IPP v2.8. The following workflows can be installed with IPP v2.8:
Prerequisite 2: Clarity LIMS with NAS Share and Mount Points
âš The mount points and the on-premise Clarity instance are recommended to be co-located for optimal performance.
The following NAS shares and mount points are required to enable files to be transmitted between Clarity LIMS (v6.2 or later) and the NextSeq 1000/2000 instrument.
Sample sheet directory
Installation
The NextSeq 1000/2000 On-Prem Integration Package v1.0.0 is distributed as the ClarityLIMS-NextSeq1k2k-OnPremise RPM package. This package must be installed on the Clarity LIMS server.
The RPM package installs the following files:
Use the following instructions to install the integration package. Install RPM Package
Log in using the admin credentials.
Run the following command to install the ClarityLIMS-NextSeq1k2k-OnPremise RPM package from the repository:
Run Configuration Scripts
Make sure that you are using the glsjboss user credentials.
Run the following script to set properties in the Clarity LIMS database:
After running this script, the following prompts display:
For more information about the properties set by this script and how to modify them, refer to .
Start the Sequencing Service
Log in as the root or glsjboss user.
Run the following command to start the sequencing service:
Run the following command to check the status of the integration service:
After the sequencing service starts, the following files are created at /opt/gls/clarity/extensions/nextseq1k2k-onprem:
During the initial run of the integration service, it can take some time to scan the files in the run folders within the configured Run parent directory before events are processed.
Configuration Properties
Refer to for the properties installed for this integration. Depending on the modified property, the integration service may need to be restarted for the changes to take effect.
This section explains how to validate the installation of the Illumina NextSeq 1000/2000 On-Prem Integration Package v1.0.0.
The validation process involves the following items:
Running samples through the Library Prep Validation v2.3.3 workflow.
The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow.
Running the libraries through the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow. This process validates the following information:
Successful sequential step advancement of samples in the following steps:
Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)
Before running the validation steps below, these steps assume that the NextSeq 1000/2000 On-Prem Integration Package v1.0.0 is installed, and that you have imported the default Clarity LIMS configuration.
For information on how the integration works, refer to .
Activate Workflow, Create Project, Add and Assign Samples
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.3 and NextSeq 1000/2000 On-Prem Sequencing v1.0 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.3 and NextSeq 1000/2000 On-Prem Sequencing v1.0 workflows.
For instructions, refer to the information on configuring workflows in the .
On the Projects and Samples screen, create a project and add samples to it.
âš Use only alphanumeric, dash, and underscore characters in the submitted sample names. Failing to do so causes sample sheet validation failure in Load To Reagent Cartridge step.
In Lab View, locate the NextSeq 1000/2000 On-Prem Sequencing v1.0 protocol. The samples are queued for the Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
At the end of this step, the pool of samples automatically advances to Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.
In Lab View, locate the NextSeq 1000/2000 On-Prem Sequencing v1.0 protocol.
The pool of samples is queued for the Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
At the end of this step, the pool of samples automatically advances to and is queued for the AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0) step. The sequencing run is ready to be started. For details on how to start the sequencing run for the local run mode, refer to .
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
The integration starts the step automatically and data from the run is parsed back to Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS. Do not perform any action when reviewing the data.
Read summary metrics are recorded for the library pool in the Step Details section and the Sample Details table.
Values are populated in the following master step fields:
Run Name
Current Read
Flow Cell ID
Reagent Cartridge Lot Number
The summary metrics (per run level) populate in the following global fields.
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
At the end of this step, the pool of samples automatically advances to (and queues for) the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. Review the data parsed and assign QC, depending on the criteria set, and complete the step.
In the Record Details screen, perform the following actions:
Review demultiplexing data.
Demultiplexing metrics are recorded for the library pool in the Sample Details table. For multi-lane flow cells (for example, P3), the metrics are aggregated by summing them across all lanes. For example, the # Reads displayed in the Sample Details table for a P3 flow cell (two lanes) is the summed value of the number of reads in Lane 1 and Lane 2 of Demultiplex_Stats.csv.
At this point, the whole NextSeq 1000/2000 On-Prem Integration workflow is fully validated.
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, refer to Troubleshooting Automation in the .
If the AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0) step starts but does not finish, complete the following steps:
Log in to Clarity LIMS using the default user account and use one of the following methods to open the step in Clarity LIMS:
Method 1: In Lab View, find the step in the Recent Activities pane.
Method 2: Search for the step in Clarity LIMS using reagent cartridge barcode as the search term.
If you cannot reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting steps already performed.
Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0)
AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0)
Automatic validation of run setup information before sample sheet generation.
Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.
Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics to Clarity LIMS.
Automated tracking of the local secondary analysis using DRAGEN (up to BCL Convert only).
Parsing of demultiplexing metrics into Clarity LIMS.
Assign the samples to the Library Prep Validation v2.3.3 workflow.
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 1000/2000 On-Prem Sequencing v1.0 workflow, which is Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.
On the Pooling screen, perform the following actions:
Create a pool by dragging samples into the Pool Creator.
âš Do not create more than one pool for this step. The Calculate Volumes automation in this step supports one pool only.
Type a name for the pool or accept the default name (Pool #1).
Select Record Details.
On the Record Details screen, the Reagent Lot Tracking section tracks the Resuspension Buffer (RSB) lot information used in the step.
In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.
The Step Details area contains two required fields:
Final Loading Concentration (pM) — The value entered in this field is the recommended final loading concentration specified in the Illumina NextSeq 1000/2000 Sequencing System Guide. The value depends on the library type. The default drop-down list contains the values 650, 750, 1000, and 2000. A custom value is acceptable.
Final Loading Volume (ul) — The value in this field is the final loading volume of the pool into the reagent cartridge. The field is prepopulated with the configured default value, 24 µl, specified in the . The value is editable when more volume is necessary.
Select Calculate Volumes.
This selection triggers the Calculate Volumes automation. This automation calculates the volume required for each library to form a pool that has the concentration and volume specified in the step details fields.
The automation also generates the Calculation File (CSV) and attaches it to the step. This file contains volume information of each of the samples and RSB buffer to add to the pool. Select the file to download it, then open it in Excel.
In the Sample Details table, select the pool icon to view details on the pool composition.
Select Next Steps.
This selection triggers the Set Next Step automation, which 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).
On the Assign Next Steps screen, the next step for samples is already set to the next step in the workflow. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 On-Prem Sequencing v1.0).
Select Finish Step.
The Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.
On the Placement screen, perform the following actions:
Drag the pool into the NextSeq 1000/2000 Reagent Cartridge field in the Placed Samples area.
Scan or type the barcode of the reagent cartridge into the NextSeq 1000/2000 Reagent Cartridge field.
Select Record Details.
On exit of the Placement screen, the Validate Reagent Cartridge Barcode automation checks that the reagent cartridge barcode conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z0-9]{4}. If not, an error message displays.
âš 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.
On the Record Details screen, the Reagent Lot Tracking section tracks the NextSeq 1000/2000 Reagent Cartridge lot information used in the step. Follow the steps in Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0) step if you must add a lot.
In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.
The fields displayed on the Record Details screen are used to create planned run and generate the sample sheet file.
Run Name — Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
Instrument Type — Select from preset options (NextSeq 1000 or NextSeq 2000).
Run Mode — Read-only. This field is set to Local.
Paired End — Select from preset options (True or False).
Read 1 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.
Read 2 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.
Index Reads — Select from preset options (No Index, Single Index, Dual Index).
Index Read 1 — Select from preset options (0, 6, 8) or type a custom value.
Index Read 2 — Select from preset options (0, 6, 8) or type a custom value.
Analysis Workflow — Read only. This field is set to GenerateFASTQ.
Local Analysis Workflow Versions — This field is required. Enter the DRAGEN version of the GenerateFASTQ application installed on the instrument.
FASTQ Compression Format — Select gzip (default) or DRAGEN.
[Optional] Adapter Sequence Read 1 — Enter the Read 1 adapter sequence of the index adapter kit.
[Optional] Adapter Sequence Read 2 — Enter the Read 2 adapter sequence of the index adapter kit.
[Optional] Barcode Mismatches Index 1 — Select from preset options (0, 1, 2). Leave it blank if you are unsure.
[Optional] Barcode Mismatches Index 2 — Select from preset options (0, 1, 2). Leave it blank if you are unsure.
[Optional] Override Cycles — String used to specify UMI cycles and mask out cycles of a read (e.g., N1Y150;I8;I7N1;Y141U10). Leave it blank if you are unsure.
On the Record Details screen, select Validate Run Setup and Create Sample Sheet.
This selection triggers the automation script, which does the following actions:
Validates the parameters entered on the Record Details screen.
Generates the sample sheet and attaches it to the placeholder in the Files area of the Record Details screen. A copy of the sample sheet is copied to the sample sheet directory configuring during installation of the integration.
Select Next Steps.
On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow. The next step is AUTOMATED - Sequencing Run (NextSeq 1000/2000 On-Prem Sequencing v1.0).
Select Finish Step.
Instrument ID
Run Status
Current Cycle
Flow Cell Lot Number
Instrument Platform
Instrument Control Software Version
Output Folder
Secondary Analysis Workflow
Reagent Cartridge ID
Instrument Type
RTA Version
Sequencing Log
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
The metric files that are generated by the DRAGEN onboard are stored under the Files section as Run_Metrics.zip.
Assign QC flags to the individual samples. There are two ways of doing this step.
Manually assign QC flags through the QC column in Sample Details table.
Automatically assign QC flag by running Assign QC flags automation. This option is for scenarios where a huge number of libraries are involved.
In the Step Details section, the following step fields are visible. (N is the number of criteria. You can use one or more criteria.)
Criteria N - Source Data Field — Select from preset options (for example, # Reads).
Criteria N - Operator — Select from preset options (for example, >= (greater than or equal to)).
Criteria N - Threshold Value — Enter the desired threshold value.
After filling up the criteria fields, select Assign QC flags. This selection triggers the automation script, which loops through each library in the pool and apply QC flag base on the criteria set previously.
This automation also generates an AssignQC Result file under Files section.
Select Next Steps.
On the Assign Next Steps screen, the Next Step field for all samples is prepopulated with Mark protocol as complete.
Select Finish Step.
On the Record Details screen, locate the Sequencing Log field.
The multiline text field contains logging information.
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 .
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 .
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.
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.
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):
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
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.
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.
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}:
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.
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.
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.
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.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
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
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.
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.
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.
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.
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 .
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 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 .
Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes
The 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 .
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 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
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.
âš 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.
This step is a semi-automated step that is started automatically after the GenerateFastQ secondary analysis has started.
âš 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.
Copies the Final Loading Concentration (pM) from step inputs to step outputs.
Calculate per sample volume required for each library to make the 2 nM intermediate library pool (24 µl in volume).
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.
RSB Volume for Pool (ul)
Numeric
Read Only
Hidden
ℹ This field is used by the Calculate Volumes automation.
Run Name can only contain alphanumeric, dash, underscore, or period characters. Spaces are not permitted.
Run Name must 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).
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.
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.
Checks Override Cycles field value.
Override Cycles can only contain Y, N, I, U, 0–9, and semicolon characters.
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).
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.
ℹ 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.
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
301
151
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
301
151
Index Reads
Text Dropdown
Required Field
Presets
No Index
Single Index
Index Read 1
Numeric Dropdown
Required Field
Custom Entries
Presets
0
6
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Presets
0
6
Analysis Workflow
Text
Required Field
Read Only
Default
GenerateFASTQ
Adapter Sequence Read 1
Text
Adapter Sequence Read 2
Text
Barcode Mismatches Index 1
Numeric Dropdown
Presets
0
1
Barcode Mismatches Index 2
Numeric Dropdown
Presets
0
1
Override Cycles
Text
Local Analysis Workflow Versions
Text
Required Field
FASTQ Compression Format
Text Dropdown
Required Field
Presets
gzip
DRAGEN
ApplicationVersion
Visible
Instrument ID
InstrumentSerialNumber
Visible
Output Folder
OutputFolder
Visible
Reagent Cartridge ID
CartridgeSerialNumber
Visible
Reagent Cartridge Lot Number
CartridgeLotNumber
Visible
RTA Version
RtaVersion
Visible
Run Name
ExperimentName
Visible
Secondary Analysis Workflow
SecondaryAnalysisWorkflow
Visible
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
Criteria 2 - Operator
Text Dropdown
Custom Entries
Presets
>=
<=
=
Criteria 1 - Threshold Value
Numeric
Valid integer value
Criteria 2 - Threshold Value
Numeric
Valid integer value
Log
Multiline Text
Read Only
Analysis Status
Text
Read Only
Reagent Cartridge ID
Text
Read Only
Hidden
ℹ Used for parsing demultiplexing results
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 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.
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Presets
650
750
1000
2000
Final Loading Volume (ul)
Numeric
Required Field
Default
24
Library Pool Volume (ul)
Numeric
Read Only
Hidden
ℹ This field is used by the Calculate Volumes automation.
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Decimal places displayed = 0
Presets
225
400
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Run Name
Text
Required Field
Instrument Type
Text Dropdown
Required Field
Presets
NextSeq1000
NextSeq2000
Run Mode
Text
Required Field
Read Only
Presets
Local
Master Step Field
RunParameters.xml Field
On Record Details Screen
Current Cycle
Calculated based on CompletedCycles Field
Visible
Current Read
Calculated based on CompletedCycles field against PlannedCycles
Visible
Flow Cell ID
FlowCellSerialNumber
Visible
Flow Cell Lot Number
FlowCellLotNumber
Visible
Master Step Field
RunParameters.xml Field
On Record Details Screen
Instrument Platform
NextSeq 1000/2000
Constant value
Visible
Instrument Type
One of the following options:
NextSeq1000
NextSeq2000
Determined by the integration and run event
Visible
Run Status
One of the following options:
RunStarted
RunCompletedSuccessfully
RunAbortedByUser
RunErroredOut
Visible
Sequencing Log
Generated by the integration service as the sequencing run proceeds
Visible
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Criteria 1 - Source Data Field
Text Dropdown
Custom Entries
Presets
# Reads
# Perfect Index Reads
# One Mismatch Index Reads
Criteria 2 - Source Data Field
Text Dropdown
Custom Entries
Presets
# Reads
# Perfect Index Reads
# One Mismatch Index Reads
Criteria 1 - Operator
Text Dropdown
Custom Entries
Presets
>=
<=
=
!=
Field Name
Field Type
Field Constraints/Options
# One Mismatch Index Reads
Numeric
Read Only
# Perfect Index Reads
Numeric
Read Only
# Reads
Numeric
Read Only
Status
Description
RunStarted
The Run Status field is updated to RunStarted after detecting the RunParameters.xml file.
All step custom fields are updated based on the RunParameters.xml file and the step field value of the Load to Reagent Cartridge step.
The Multiline Sequencing Log field is updated.
RunCompletedSuccessfully
RunErroredOut
RunAbortedByUser
After detecting the RunCompletionStatus.xml file, the Run Status field is updated with the run status from the XML file.
The Current Read and Current Cycle fields are updated to the final cycle and read numbers based on the RunCompletionStatus.xml file.
The sequencing run result (primary metrics) is parsed from InterOp.
The Multiline Sequencing Log field is updated.
The integration service automatically completes the step and routes the samples in the reagent cartridge container to the Demultiplexing (NextSeq 1000/2000 On-Prem Sequencing v1.0) step.
if (step.::Run Name::.length() > 255) {
fail(::Run Name shall not exceed 255 characters.::)
};
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.:: )
}
}
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.::)
}
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::)
};
nextStep = ::ADVANCE::
script:validateSampleCount -min 1 -max 1
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.:: )
}
-exp 'step.::RSB Volume for Pool (ul):: = 24; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::'
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 .::)
}
101
51
Range: 1–351
Decimal places displayed: 0
101
51
Range: 0–351
Decimal places displayed: 0
Dual Index
8
Range: 0–20
Decimal places displayed: 0
8
Range: 0–20
Decimal places displayed: 0
2
2
Default
gzip
!=
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.::)
}
}
}
