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IGA NovaSeq Sequencing v2.1

PreviousIGA Library Prep Automated v2.1 NextViral Pathogen Protocols

Last updated 2 months ago

Overview

IGA NovaSeq Sequencing v2.1 contains preset protocols that support the setup of previously prepared libraries used with the NovaSeq instrument. These protocols can be used with the IGA Library Prep Automated v2.1 workflow. They also provide the following functions:

File-based integration with the Hamilton robots that are used for liquid handling steps.
Automated calculation of sample and buffer volumes.
Automated calculation or display of reagents.
When required, automatic step transition.

Protocol 1: Run Format (IGA)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Define Run Format (IGA)

Master Step Name = Define Run Format (IGA v2.1)
Step Type = No Outputs

Automations

Trigger Location = Step
Trigger Style = Automatic upon entry

bash -l -c "/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 {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/register_sample.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 {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_started.groovy'"

Define Run Format - Assign Workflow and Flowcell Type to Samples & Set Next Steps to Remove from Workflow (IGA v2.1)

Trigger Location = Record Details
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::REMOVE:: ; input.::Minimum Molarity (nM):: = (5 * input.::Final Loading Concentration (pM)::)/1000 ; if (!input.hasValue(::Normalized Molarity (nM)::)) { fail(::The Normalized Molarity cannot be empty.::) ; } else if (input.::Normalized Molarity (nM):: < input.::Minimum Molarity (nM)::) { input.::Warning:: = ::The Normalized Molarity is too low.:: ; input.::Loading Workflow Type:: = ::[Remove from workflow]:: ; } else { input.::Warning:: = ::n/a:: }' -log {compoundOutputFileLuid0}"

Define Run Format - Routing Script (IGA v2.1)

Trigger Location = Step
Trigger Style = Automatic upon exit

bash -c "/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 {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Standard' \
--WORKFLOW 'Illumina Genomics Architecture - NovaSeq Sequencing v2.1' \
--STEP 'Make Bulk Pool for NovaSeq Standard (IGA v2.1)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Xp' \
--WORKFLOW 'Illumina Genomics Architecture - NovaSeq Sequencing v2.1' \
--STEP 'Make Bulk Pool for NovaSeq Xp (IGA v2.1)' \
--INPUTS_OR_OUTPUTS 'INPUTS'"

Queue

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Column
Sample Table
- Column Headers
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  Container Name
  Built-in
  Container
  Well
  Built-in
  Derived Sample
  Reagent Name
  Built-in
  Derived Sample
  Sample Name
  Built-in
  Derived Sample
  Waiting
  Built-in
- Expanded View Fields
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  LIMS ID (Container)
  Built-in
  Project
  Project Name
  Built-in

Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Column
Sample Table
- Column Headers
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  Container Name
  Built-in
  Container
  Well
  Built-in
  Derived Sample
  Reagent Name
  Built-in
  Derived Sample
  Sample Name
  Built-in
  Derived Sample
  Waiting
  Built-in
- Expanded View Fields
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  LIMS ID (Container)
  Built-in
  Project
  Project Name
  Built-in

Record Details

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
IGA WF Build Version
Text
Read Only
Default = 2.1.0.1
Quick reference checklist: Define Run Format.
Multiline Text
Read Only
Defaults = User tasks for this page: - Below, assign 'Loading Workflow Type', 'Flowcell Type', and the NovaSeq 'Final Loading Concentration (pM)'. - Click 'Next Steps'.
Step File Placeholders
- Log file - Automatically attached
Sample Table (Table Columns - Global Fields)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Container
Well
Built-in
Derived Sample
Adjusted Per Sample Volume (ul)
Numeric
Read Only
Decimal Places Displayed = 2
Derived Sample
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal Places Displayed = 0
Derived Sample
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Derived Sample
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Derived Sample
Minimum Molarity (nM)
Numeric
Decimal Places Displayed = 2
Derived Sample
Normalized Molarity (nM)
Numeric
Decimal Places Displayed = 2
Derived Sample
Per Sample Volume (ul)
Numeric
Read Only
Decimal Places Displayed = 2
Derived Sample
Sample Name
Built-in
Derived Sample
Warning
Text
Read Only
Project
Project Name
Built-in

Protocol 2: NovaSeq Standard

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Make Bulk Pool for NovaSeq Standard

Master Step Name = Make Bulk Pool for NovaSeq Standard (IGA v2.1)
Step Type = Pooling
Aliquot Generation = Fixed, 1
Naming Convention = {PoolName}
Control Types
- PhiX v3

Automations

Trigger Location = Step
Trigger Style = Automatic upon entry

bash -l -c "/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 {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/register_sample.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 {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_started.groovy'"

Pooling NovaSeq Standard - Show Warning Message (IGA v2.1)

Trigger Location = Pooling
Trigger Style = Automatic upon entry

bash -l -c "PYTHONPATH=/opt/gls/clarity/extensions/IGA_2.1 python3 \
/opt/gls/clarity/extensions/IGA_2.1/iga_novaseq/NovaSeq_pooling_warning.py \
-s {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid0}"

Validate Inputs Flowcell Type and Number of Pools (IGA v2.1)

Trigger Location = Pooling
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:validate_flowcell_for_input_pools -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -validateSingleOutput false -poolType bulk \
&& PYTHONPATH=/opt/gls/clarity/extensions/IGA_2.1 python3 /opt/gls/clarity/extensions/IGA_2.1/iga_novaseq/validate_flowcell_pools.py -s {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0}"

NovaSeq Make Bulk Pool - Output Plate BPP Barcode Check (Duplicates Allowed) (IGA v2.1)

Trigger Location = Placement
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -exp 'if ( !output.container.name.matches( ::LP[0-9]{7}-BPP:: ) ) {fail ( ::Invalid BPP Plate Barcode. This plate ID must be in the format LPxxxxxxx-BPP, where 'x' is a digit 0-9, e.g. LP1234567-BPP. Please verify and try again.:: ) }' -log {compoundOutputFileLuid0} \
&& PYTHONPATH=/opt/gls/clarity/extensions/IGA_2.1 python3 /opt/gls/clarity/extensions/IGA_2.1/iga_novaseq/validate_multiple_destination_plates.py -s {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0}"

Make Bulk Pool NovaSeq Standard - Calculate Volumes and Generate Hamilton Input File (IGA v2.1)

Trigger Location = Record Details
Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -t true -log {compoundOutputFileLuid0} \
    script:evaluateDynamicExpression \
        -h false \
        -exp 'step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: + 1' \
    script:evaluateDynamicExpression \
        -h false \
        -exp 'if ( input.::Flowcell Type:: == ::SP:: ) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100 + 30 } ; if ( input.::Flowcell Type:: == ::S1:: ) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100 + 30 } ; if ( input.::Flowcell Type:: == ::S2:: ) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 150 + 30 } ; if ( input.::Flowcell Type:: == ::S4:: ) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 310 + 30 } ; input.::Per Sample Volume (ul):: = 0' \
&& PYTHONPATH=/opt/gls/clarity/extensions/IGA_2.1 python3 \
/opt/gls/clarity/extensions/IGA_2.1/make_bpp/calculate_sample_volume.py \
-s {stepURI:v2} \
-u {username} \
-p {password} \
&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -t true -log {compoundOutputFileLuid0} \
    script:calculate_adjusted_per_sample_volume \
    script:evaluateDynamicExpression \
        -h false \
        -exp 'step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::' \
    script:evaluateDynamicExpression \
        -h false \
        -exp 'if (step.::Total Sample Volume (ul):: >= step.::Bulk Pool Volume (ul)::) {output.::RSB Volume (ul):: = 0} else {output.::RSB Volume (ul):: = step.::Bulk Pool Volume (ul):: - step.::Total Sample Volume (ul)::} ; output.::Flowcell Type:: = input.::Flowcell Type:: ; output.::Loading Workflow Type:: = input.::Loading Workflow Type:: ; if ( input.::Flowcell Type:: == ::SP:: ) { output.::Volume of Pool to Denature (ul):: = 100 ; output.::NaOH Volume (ul):: = 25 ; output.::Tris-HCl Volume (ul):: = 25 } ;  if ( input.::Flowcell Type:: == ::S1:: ) { output.::Volume of Pool to Denature (ul):: = 100 ; output.::NaOH Volume (ul):: = 25 ; output.::Tris-HCl Volume (ul):: = 25 } ; if ( input.::Flowcell Type:: == ::S2:: ) {output.::Volume of Pool to Denature (ul):: = 150 ; output.::NaOH Volume (ul):: = 37.50 ; output.::Tris-HCl Volume (ul):: = 37.50 } ; ; if ( input.::Flowcell Type:: == ::S4:: ) { output.::Volume of Pool to Denature (ul):: = 310 ; output.::NaOH Volume (ul):: = 77.50 ; output.::Tris-HCl Volume (ul):: = 77.50 }' \
&& \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
-i {stepURI:v2} -u {username} -p {password} -t true -log {compoundOutputFileLuid0} \
script:evaluateDynamicExpression \
-h false \
-exp 'step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: - 1; step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: - input.::Adjusted Per Sample Volume (ul)::' \
&& \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar \
script:driver_file_generator \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-q true \
-destLIMSID {compoundOutputFileLuid1} \
-t /opt/gls/clarity/extensions/IGA_2.1/hamilton_fast_prep/templatefiles/MakeBPFile_NovaSeq_template.csv \
-o Output_plate_name.csv \
-l {compoundOutputFileLuid2} && \
PYTHONPATH=/opt/gls/clarity/extensions/IGA_2.1 python3 \
/opt/gls/clarity/extensions/IGA_2.1/make_bpp/apply_multiple_bulk_pools.py \
-s {stepURI:v2} \
-u {username} \
-p {password} \
--min-volume {udf:Minimum Per Sample Volume (ul)} \
--selected-cf {udf:Apply Correction Factors} \
--hamilton-limsid {compoundOutputFileLuid1} \
--custom-cf-limsid {compoundOutputFileLuid4} \
-l {compoundOutputFileLuid5} "

Make Bulk Pool NovaSeq Standard - Validate Hamilton Output File Name if Present, Parse Instrument ID, Set Next Steps to Advance (IGA v2.1)

Trigger Location = Record Details
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'step.::Liquid Handling Instrument ID:: = ::::' -log {compoundOutputFileLuid0} \
&& \
/usr/bin/env python /opt/gls/clarity/extensions/IGA_2.1/iga_novaseq/01_NovaSeqStd_confirmPlateIDAndStoreInstrumentID_OptionalFile.py -u {username} -p {password} -s {stepURI} -f {compoundOutputFileLuid3} -i 'Liquid Handling Instrument ID' \
&& \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'if (!step.hasValue(::Liquid Handling Instrument ID::)) {step.::Liquid Handling Instrument ID:: = ::NO HAMILTON OUTPUT FILE UPLOADED:: } ; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid0}"

Trigger Location = Step
Trigger Style = Automatic upon exit

bash -l -c "/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 {compoundOutputFileLuid0} -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 {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/pooling.groovy'"

Validate Unique Indexes (IGA v2.1)

Trigger Location = Not Used

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:validateUniqueIndexes"

Queue

Defaults
- Sample Grouping = Group by Containers