Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)
This step is a semi-automated step.
The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. The lab scientist reviews the demultiplexing result parsed into Clarity LIMS, assigns QC flags, and completes the step.
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). |
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
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 the LINK service. This automation is only used for Clarity LIMS Product Analytics support.
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'
Assign Demultiplexing QC Flags Automation
Automatically triggered when you select a button on the Record Details screen, this automation assigns QC flags based on the criteria set in the step fields.
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} script:assignQCForDemuxStep -i {processURI:v2} -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation sets the next step to Advance and the samples to complete the protocol.
nextStep = ::ADVANCE::
Register Analysis Completed & Register Step Completed Automation
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 the LINK service. This automation is only used for Clarity LIMS Product Analytics support.
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_analysisrun_completed.groovy'
Master Step Fields
The following table lists the 12 master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) master 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
|
||||||||||||
|
Criteria 2 - Source Data Field |
Text Dropdown |
Custom Entries |
Presets
|
||||||||||||
|
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 Do not delete, rename, or modify. These actions will break the integration.
|
||||||||||||
|
Analysis Started Time |
Text |
Read Only |
Hidden Do not delete, rename, or modify. These actions will break the integration. |
||||||||||||
|
Analysis Status |
Text |
Read Only |
Hidden Do not delete, rename, or modify. These actions will break the integration. |
||||||||||||
|
BSSH Analysis ID |
Text |
Read Only |
Hidden Do not delete, rename, or modify. These actions will break the integration. |
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|
Reagent Cartridge ID |
Text |
Read Only |
Hidden Used for parsing demultiplexing results Do not delete, rename, or modify. These actions will break the integration. |
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.
|
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 |
User Interaction
Refer to NextSeq 1000/2000 Integration v2.4 Validation and Troubleshooting for details.
