⚠️ 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 (global or master step) — could break the process.
Prerequisites and Assumptions
The Illumina NextSeq 1000/2000 Integration Package v2.4.0 is compatible with Illumina cloud hosted deployments only.
It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.3 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.
The NextSeq 1000/2000 Integration Package v2.4.0 includes the following workflows:
[Optional] Library Prep Validation v2.3.1 (recommended for validation purposes).
NextSeq 1000/2000 Sequencing v2.3
ℹ️ Samples are routed to this protocol after they have gone through Library Prep Validation v2.3.1 (or any other library prep workflow). Samples are pooled, diluted to final loading concentration, and added to a reagent cartridge in preparation for the sequencing run.
ℹ️ The workflow configuration includes a global field named Illumina Universal Sample Identifier. This text field is reserved for Clarity LIMS Product Analytics support. The field is optional and not required for this integration.
Library Prep Validation v2.3.1 Workflow
Protocol 1: Library Prep Validation v2.3.1
Purpose:
Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NextSeq 1000/2000 Sequencing v2.3 workflow.
The protocol contains a single step—Library Prep Validation v2.3.1. At the end of this step, a routing script sends the samples to the first step of the NextSeq 1000/2000 Sequencing v2.3 workflow. The first step is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3).
Steps:
Library Prep Validation v2.3.1
NextSeq 1000/2000 Sequencing v2.3 Workflow
Protocol 1: NextSeq 1000/2000 Sequencing v2.3
Purpose:
This protocol models the lab processes of starting a NextSeq 1000/2000 sequencing run.
Steps:
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
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 v2.3)
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 v2.3)
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 (E.g., run configuration, primary run metrics, etc.) of the sequencing run are recorded automatically.
GenerateFastQ secondary analysis is planned by default for the run. Samples continues to the next step.
⚠️ Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
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.
Register Step Started¹
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.
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 (µl) 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 (µl):: = 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 (24µl in volume).
-exp 'input.::Per Sample Volume (µl):: = (48/ input.::Molarity (nM)::); step.::RSB Volume for Pool (µl):: -= input.::Per Sample Volume (µl)::'
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 (µl).
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 v2.3):
nextStep = ::ADVANCE::
Register Pools & Register Step Completed¹
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 Clarity LIMS Product Analytics support only.
Master Step Fields
The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Master Step Field Configuration
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
RSB Volume for Pool (ul)
Numeric
Read Only
Hidden
Global Fields
The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Global Field Configuration
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 (µl)
Numeric
Read Only
Decimal places displayed = 2
Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)
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 on Illumina Connected Analytics, depending on the Run Mode selected.
ℹ️ 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.
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 v2.3 workflow.
script:validateSampleCount -min 1 -max 1
Registers the start time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This script is only used for Clarity LIMS Product Analytics 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-Za-z]{2}[0-9]{7}-[A-Za-z0-9]{4}:
if (!output.container.name.matches(::[A-Za-z]{2}[0-9]{7}-[A-Za-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 completes the following actions:
Fetches the available analysis workflow versions from Illumina Connected Analytics.
Updates the preset values of both Local Analysis Workflow Versions & Cloud Analysis Workflow Versions fields.
⚠️ Do not modify or disable the Retrieve Analysis Workflow Versions automation script. Modifying or disabling the script breaks the integration.
Validate Run Setup and Create Planned Run 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 and/or create the planned run on Illumina Connected Analytics.
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.
if (step.::Run Mode:: == ::Cloud::){
step.::Local Analysis Workflow Versions:: = ::None::;
if (step.::Cloud Analysis Workflow Versions:: == ::None::){
fail(::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 limitation, 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 on Illumina Connected Analytics.
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol—AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3). 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 Clarity LIMS Product Analytics through Illumina Connected Analytics. This script is only used for Clarity LIMS Product Analytics support.
¹ These automations are required for Clarity LIMS Product Analytics support only.
² These automations are required for the NextSeq 1000/2000 Sequencing v2.3 workflows and contain additional logic needed for Clarity LIMS Product Analytics support. If you would like to remove Clarity LIMS Product Analytics support, contact Illumina Support.
Master Step Fields
There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step. These fields are required for sample sheet generation and planned run creation on Illumina Connected Analytics (ICA).
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 Dropdown
Required Field
Presets
Local
Hybrid
Cloud
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
301
151
101
51
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
301
151
101
51
Index Reads
Text Dropdown
Required Field
Presets
No Index
Single Index
Dual Index
Index Read 1
Numeric Dropdown
Required Field
Custom Entries
Presets
0
6
8
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Presets
0
6
8
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
2
Barcode Mismatches Index 2
Numeric Dropdown
Presets
0
1
2
Override Cycles
Text
Cloud Run ID
(Used in creating planned run in ICA)
Text
Read Only
Hidden
Use Custom Index Read 1 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Use Custom Index Read 2 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Use Custom Read 1 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Use Custom Read 2 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Instructions
Multiline Text
Read Only
Default
If GenerateFASTQ analysis is required, please select the DRAGEN version in Cloud Analysis Workflow Versions (for Cloud run mode) or Local Analysis Workflow Versions (for Local/Hybrid run mode) custom fields above.
Local Analysis Workflow Versions
Text Dropdown
Default
None
Cloud Analysis Workflow Versions
Text Dropdown
Default
None
FASTQ Compression Format
Text Dropdown
Required Field
Presets
gzip
DRAGEN
None
Default
gzip
Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
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.
⚠️ Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
Register Step Started & Register NextSeq1k2k Run & Register NextSeq1k2k Run Association & Register NextSeq1k2k RunStarted¹
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 Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics 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 v2.3) step.
Registers the completion time of the step.
The automation completes these actions by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support.
¹ These automations are required for Clarity LIMS Product Analytics support only.
Master Step Fields
There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) 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.
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
Instrument Control Software Version
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
Flow Cell Mode
FlowCellMode
Hidden
Instrument Run ID
Derived from OutputFolder
Hidden
Run End Time
RunEndTime
Hidden
Run Start Time
RunStartTime
Hidden
Secondary Analysis Mode
SecondaryAnalysisMode
Hidden
Secondary Analysis Platform Version
SecondaryAnalysisPlatformVersion
Hidden
SkipObdd
SkipObdd
Hidden
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.
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 from Instrument Type step field from previous Load to Reagent Cartridge step.
Visible
Run Status
One of the following options:
RunStarted
RunCompletedSuccessfully
RunAbortedByUser
RunErroredOut
Set by the integration service¹
Visible
Sequencing Log
Filled in by the integration service¹ as the sequencing run proceeds
Visible
BaseSpace Run ID
Received as part of the event payload¹
Hidden
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 v2.3) step.
⚠️ Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
⚠️ For the demultiplexing information in the Sample Details table to display correctly, the following conditions must be met:
The DRAGEN version must be earlier than v3.9.3.
The samples in the run must not repeat.
The run must be for the single lane flow cell (P1 or P2).
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 Automation
Automatically triggered at the beginning of the step, this automation registers the start time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics 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.
Automatically triggered on exit of the step, this automation performs the following actions:
Registers the NextSeq 1000/2000 analysis status and result.
Registers the completion time of the step.
These actions are done by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support.
The following table lists the 12 master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) master step.
Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) Step
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
# of >= Q30 Bases (PF)
Criteria 2 - Source Data Field
Text Dropdown
Custom Entries
Presets
# Reads
# Perfect Index Reads
# One Mismatch Index Reads
# of >= Q30 Bases (PF)
Criteria 1 - Operator
Text Dropdown
Custom Entries
Presets
>=
<=
=
!=
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 Completed Time
Text
Read Only
Hidden
Analysis Started Time
Text
Read Only
Hidden
Analysis Status
Text
Read Only
Hidden
BSSH Analysis ID
Text
Read Only
Hidden
Reagent Cartridge ID
(Used for parsing demultiplexing results)
Text
Read Only
Hidden
Global Fields
The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) 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 Sequencing v2.3) Step
Field Name
Field Type
Field Constraints/Options
# of >= Q30 Bases (PF)
Numeric
Read Only
# One Mismatch Index Reads
Numeric
Read Only
# Perfect Index Reads
Numeric
Read Only
# Reads
Numeric
Read Only
Mean Quality Score (PF)
Numeric
Read Only
How the NextSeq 1000/2000 Integration Works
Sample Sheet Generation and Planned Run Creation
On the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) 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 sent to Illumina Connected Analytics (ICA). ICA validates the run and analysis configuration, and sends an error message to Clarity LIMS if the validation fails. If the validation passes, ICA generates the sample sheet (and sends back to Clarity LIMS) and/or creates a planned run depending on the Run Mode selected. NextSeq 1000/2000 Integration supports local, hybrid and cloud run mode.
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 (BSSH) that the sequencing run has started.
BSSH then does the following actions:
Local run mode: Creates a run on the BSSH database with the run configuration information received from NCS.
Hybrid/Cloud run mode: Creates a run on the BSSH database by copying the run configuration information from the planned run created in ICA, using a special Cloud Run ID received from NCS.
After the run is created on BSSH, a run update event message that carries the RunStarted equivalent message is created on BSSH and sent to the integration service which processes the message.
Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) 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 then updates all the other step fields (eg, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BSSH 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.
Run Status
Shown in Step
Details
RunStarted
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunStarted.
All other step fields (except Run End Time) are updated based on RunParameters.xml detail obtained from BSSH, the SQS event message, and the step field value from the Load to Reagent Cartridge (NextSeq 1000/2000 v2.3) step.
Reagent cartridge lot information received from BSSH is compared against the details of the pre-accessioned reagent cartridge on Clarity LIMS.
If the expiration date is different, it is updated to the date received from BSSH.
If reagent cartridge is not found, it is created and set to Active.
If reagent cartridge is not in Active status, it is set to Active.
Multiline Sequencing Log field is updated.
RunCompletedSuccessfully
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunCompletedSuccessfully
Current Read and Current Cycle fields are updated to the final cycle number and read number based on RunParameters.xml detail obtained from BSSH.
Run End Time field is updated based on RunParameters.xml detail obtained from BSSH.
Sequencing run result (primary metrics) is downloaded from BSSH and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values.
Multiline Sequencing Log field is updated.
The integration automatically completes the step and routes the samples in the reagent cartridge container to the next step (e.g., Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)) step.
RunErroredOut
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunErroredOut.
Current Read and Current Cycle fields are updated to the final cycle number and read number that the run stopped based on RunParameters.xml detail obtained from BSSH.
Run End Time field is updated based on RunParameters.xml detail obtained from BSSH.
Sequencing run result (primary metrics) is downloaded from BSSH and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values.
Multiline Sequencing Log field is updated.
The integration does not automatically complete the step. Complete the step manually.
RunAbortedByUser
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunAbortedByUser.
Current Read and Current Cycle fields are updated to the final cycle number and read number that was aborted based on RunParameters.xml details obtained from BSSH.
Run End Time field is updated based on RunParameters.xml detail obtained from BSSH.
Sequencing run result (primary metrics) is downloaded from BSSH and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values.
Multiline Sequencing Log field is updated.
The integration does not automatically complete the step. Complete the step manually.
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.
Components Installed
This section describes the various components that are installed by default as part of this integration.
This integration supports the NextSeq 1000/2000 reagent cartridge with barcode provided in the format [A-Za-z]{2}[0-9]{7}-[A-Za-z0-9]{4} (eg, EC1234567-EC03).
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 (E.g., appending LIMS ID to the default naming convention, like OutputItemLIMSID).
Select Save.
Rules and Constraints
⚠️ 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.
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 v2.3) 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 v2.3) 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 NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after it has been successfully validated. Modifying the barcode would result in Clarity failing to update subsequent steps and sample details properly.
