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.::)
}
}
}
