Illumina Genomics Architecture - NovaSeq Sequencing v1.0

Overview

The Illumina Genomics Architecture - NovaSeq Sequencing v1.0 includes the following functionality:

•

Preset Illumina Genomics Architecture - NovaSeq Sequencing v1.0 protocols that support the setup of previously prepared libraries for sequencing on the NovaSeq platform.

•

Can be used with the Illumina Genomics Architecture - Library Prep Automated v1.0 workflow.

•

Tight file-based integration with Hamilton robots for performing various steps of liquid handling.

•

Automated calculation of sample and buffer volumes.

•

Automated calculation or display of reagents.

•

Automatic step transition when required.

Protocol 1: Run Format (IGA v1.0)

Step 1: Define Run Format (IGA v1.0)

Master Step 1: Define Run Format (IGA v1.0)

Step Type = No Outputs

Input = Indexed or Pooled/Captured 96-well plates

Output = Indexed or Pooled/Captured 96-well plates

Queue/Ice Bucket

Section

Subsection

Value

Sample Table

Column Headers

•

Sample Name

•

Reagent Name

•

Well

•

Container Name

•

Waiting

Expanded View Fields

•

Project Name

•

LIMS ID (Container)

Defaults

Sample Grouping

Group by Containers

Well Sort Order

Column

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Quick Reference Checklist: Define Run Format

Multiline Text

Read Only

Default

User tasks for this page:

•

Assign Loading Workflow Type, Flowcell Type, and the NovaSeq Final Loading Concentration (pM)

•

Select Next Steps

Field Order: Vertical

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Heading

Sample Table

Enable QC Flags

Sample Display Default

Expand

Well Sort Order

Column

Table Columns

Sample Name

Warning

Text Dropdown

•

Global Field = Derived Sample

•

Required field = No

•

Read Only = Yes

•

Custom Entries = Yes

Presets

•

The Normalized Molarity (nM) is too low

•

N/A

Loading Workflow Type

Text Dropdown

•

Global Field = Derived Sample

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = No

•

Requires user input

Presets

•

NovaSeq Standard

•

NovaSeq Xp

•

(Remove from workflow)

Flowcell Type

Text Dropdown

•

Global Field = Derived Sample

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = No

•

Requires user input

Presets

•

Final Loading Concentration (pM)

Numeric Dropdown

•

Global Field = Derived Sample

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = Yes

•

Requires user input

Presets

•

225

•

400

Normalized Molarity (nM)

Numeric

•

Global field - measurement and derived sample

•

Decimal Places Displayed = 2

•

Required Field = No

•

Read Only = No

Container Name

Well

LIMS ID (Container)

Project Name

Per Sample Volume (µl)

Numeric

•

Global Field = Derived Sample

•

Decimal Places Displayed = 2

•

Required Field = No

•

Read Only = Yes

Adjusted Per Sample Volume (µl)

Numeric

•

Global Field = Derived Sample

•

Decimal Places Displayed = 2

•

Required Field = No

•

Read Only = Yes

Minimum Molarity (nM)

Numeric

•

Global Field = Derived Sample

•

Decimal Places Displayed = 2

•

Required Field = No

•

Read Only = No

Automation - Script Description
Automation Name	Setting	Value	Specification
Define Run Format - Assign Workflow and Flowcell Type to Samples & Set Next Steps to Remove from Workflow (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Automatic upon exit
			Command line 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 v1.0)	Trigger Location	Step
	Trigger Style	Automatic upon exit
			Command line `bash -c "/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 v1.0' \` `--STEP 'Make Bulk Pool for NovaSeq Standard (IGA v1.0)' \` `--INPUTS_OR_OUTPUTS 'INPUTS' \` `\` `--FIELD_NAME 'Loading Workflow Type' \` `--FIELD_VALUE 'NovaSeq Xp' \` `--WORKFLOW 'Illumina Genomics Architecture - NovaSeq Sequencing v1.0' \` `--STEP 'Make Bulk Pool for NovaSeq Xp (IGA v1.0)' \` `--INPUTS_OR_OUTPUTS 'INPUTS'"`

Next Step
Setting	Value
Make Bulk Pool for NovaSeq Standard (IGA v1.0)
Make Bulk Pool for NovaSeq Xp (IGA v1.0)
Assign Next Step	Automatic
Safe Stopping Point

Protocol 2: NovaSeq Standard (IGA v1.0)

Step 1: Make Bulk Pool for NovaSeq Standard (IGA v1.0)

Master Step 1: Make Bulk Pool for NovaSeq Standard (IGA v1.0)

Step Type = Pooling

Input = Indexed or Pool/Captured 96-well plates

Output = Pooled libraries in a Bulk Pool Plate.

Queue/Ice Bucket

Section

Subsection

Value

Sample Table

Column Headers

•

Sample Name

•

Flowcell Type

•

Normalized Molarity (nM)

•

Well

•

Container Name

•

Waiting

Expanded View Fields

•

Project Name

•

LIMS ID (Container)

Defaults

Sample Grouping

Group by Containers

Well Sort Order

Column

Pooling
Section	Subsection	Value
Pool Settings	Label Uniqueness	On
Defaults	Sample Grouping	Group by Containers
	Well Sort Order	Column

Placement
Setting	Value	Specification
Destination Containers	96 Well Plate
Defaults	Well Sort Order	Column
	Placement Pattern	Column
	Skip Alternating Rows	No
	Skip Alternating Columns	No

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Number of Flowcells to Sequence

Numeric

Required Field = Yes

Read Only = No

Default = 1

Range = 1 to 10

Decimal Places Displayed = 0

Minimum Per Sample Volume (µl)

Numeric

Required Field = Yes

Read Only = No

Default = 2

Decimal Places Displayed = 2

Liquid Handling Instrument ID

Text

Required Field = Yes

Read Only = No

Multiline Text Fields

Quick Reference Checklist: Make Bulk Pool for NovaSeq Standard. i) pre Liquid Handler

Multiline Text

Required Field = No

Read Only = Yes

Default

User tasks for this page:

•

Select Number Of Flowcells and Min Per Sample Volume (µl).

•

Run the Make Bulk Pool automation, and download the resulting Hamilton Make Bulk Pool Input File.

Quick Reference Checklist: Make Bulk Pool for NovaSeq Standard. i) pre Liquid Handler

Multiline Text

Required Field = No

Read Only = Yes

Default

User tasks for this page:

•

After running MakeBPP on the liquid handler, upload resulting Hamilton Output File

•

Select Next Steps.

Step File Placeholders

Calculation

File

Auto

Calculation

Log

Auto

Hamilton Make Bulk Pool Input

File

Auto

Hamilton

Log

Auto

Hamilton Output

File

Manual

Sample Table

Table Heading

Sample Table

Enable QC Flags

Sample Display Default

Expand

Well Sort Order

Column

Table Columns

Sample Name

RSB Volume (µl)

Numeric

•

Global Field = Derived Sample

•

Required field = No

•

Read Only = Yes

•

Decimal Places Displayed = 2

Volume of Pool to Denature (µl)

Numeric

•

Global Field = Derived Sample

•

Required field = No

•

Read Only = Yes

•

Decimal Places Displayed = 2

NaOH Volme (µl)

Numeric

•

Global Field = Derived Sample

•

Required field = No

•

Read Only = Yes

•

Decimal Places Displayed = 2

Tris-HCl Volume (µl)

Numeric

•

Global Field = Derived Sample

•

Required field = No

•

Read Only = Yes

•

Decimal Places Displayed = 2

Flowcell Type

Text Dropdown

•

Global Field = Derived Sample

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = No

Presets

•

Loading Workflow Type

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

NovaSeq Standard

•

NovaSeq Xp

•

(Remove from workflow)

Container Name

Well

LIMS ID (Container)

Project Name

Automation - Script Description
Automation Name	Setting	Value	Specification
Validate Inputs Flowcell Type and Single Pool (IGA v1.0)	Trigger Location	Pooling
	Trigger Style	Automatic upon exit
			Command line `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 {compoundOutputFileLuid1} -validateSingleOutput true -poolType bulk"`
NovaSeq Make Bulk Pool - Output Plate BPP Barcode Check (Duplicates Allowed) (IGA v1.0)	Trigger Location	Placement
	Trigger Style	Automatic upon exit
			Command line `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 {compoundOutputFileLuid1}"`
Make Bulk Pool NovaSeq Standard - Calculate Volumes and Generate Hamilton Input File (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Manual button
			Command line `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 {compoundOutputFileLuid1} \` `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 (µl):: = step.::Number of Flowcells to Sequence:: * 100 + 30 } ; if ( input.::Flowcell Type:: == ::S1:: ) { step.::Bulk Pool Volume (µl):: = step.::Number of Flowcells to Sequence:: * 100 + 30 } ; if ( input.::Flowcell Type:: == ::S2:: ) { step.::Bulk Pool Volume (µl):: = step.::Number of Flowcells to Sequence:: * 150 + 30 } ; if ( input.::Flowcell Type:: == ::S4:: ) { step.::Bulk Pool Volume (µl):: = step.::Number of Flowcells to Sequence:: * 310 + 30 } ; input.::Per Sample Volume (µl):: = ( ( (input.::Final Loading Concentration (pM):: * 5 / 1000) / input.::Normalized Molarity (nM):: ) * step.::Bulk Pool Volume (µl):: ) / step.::Number of Samples in Pool::' \ `script:calculate_adjusted_per_sample_volume \` `script:evaluateDynamicExpression \` `-h false \` `-exp 'step.::Total Sample Volume (µl):: = step.::Total Sample Volume (µl):: + input.::Adjusted Per Sample Volume (µl)::' \` `script:evaluateDynamicExpression \` `-h false \` -exp 'if (step.::Total Sample Volume (µl):: >= step.::Bulk Pool Volume (µl)::) {output.::RSB Volume (µl):: = 0} else {output.::RSB Volume (µl):: = step.::Bulk Pool Volume (µl):: - step.::Total Sample Volume (µl)::} ; 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 (µl):: = 100 ; output.::NaOH Volume (µl):: = 25 ; output.::Tris-HCl Volume (µl):: = 25 } ; if ( input.::Flowcell Type:: == ::S1:: ) { output.::Volume of Pool to Denature (µl):: = 100 ; output.::NaOH Volume (µl):: = 25 ; output.::Tris-HCl Volume (µl):: = 25 } ; if ( input.::Flowcell Type:: == ::S2:: ) {output.::Volume of Pool to Denature (µl):: = 150 ; output.::NaOH Volume (µl):: = 37.50 ; output.::Tris-HCl Volume (µl):: = 37.50 } ; ; if ( input.::Flowcell Type:: == ::S4:: ) { output.::Volume of Pool to Denature (µl):: = 310 ; output.::NaOH Volume (µl):: = 77.50 ; output.::Tris-HCl Volume (µl):: = 77.50 }' \ `&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Standard_Bulk_Pool1.csv \` `-o 1.csv \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Standard_Bulk_Pool2.csv \` `-o 2.csv \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Standard_Bulk_Pool3.csv \` `-o 3.csv \` `&& cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csv \` `&& \` `/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 {compoundOutputFileLuid1} \` `script:evaluateDynamicExpression \` `-h false \` `-exp 'step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: - 1; step.::Total Sample Volume (µl):: = step.::Total Sample Volume (µl):: - input.::Adjusted Per Sample Volume (µl)::' \` `&& \` `/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 {compoundOutputFileLuid2} \` `-t /opt/gls/clarity/extensions/hamilton_fast_prep/templatefiles/MakeBPFile_NovaSeq_template.csv \` `-o Output_plate_name.csv \` `-l {compoundOutputFileLuid3}"`
Make Bulk Pool NovaSeq Standard - Validate Hamilton Output File Name if Present, Parse Instrument ID, Set Next Steps to Advance (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Automatic upon exit
			Command line `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 {compoundOutputFileLuid1} \` `&& \` `/usr/bin/env python /opt/gls/clarity/extensions/iga_novaseq/01_NovaSeqStd_confirmPlateIDAndStoreInstrumentID_OptionalFile.py -u {username} -p {password} -s {stepURI} -f {compoundOutputFileLuid4} -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 {compoundOutputFileLuid1}"`

Next Step
Setting	Value
Dilute and Denature (IGA v1.0)
Start Next Step	Manual
Assign Next Step	Manual
Safe Stopping Point

Step 2: Dilute and Denature (IGA v1.0)

Master Step 2: Dilute and Denature (IGA v1.0)

Step Type = STANDARD

Input = Bulk Pooled Libraries

Output = Bulk pooled libraries prepared in a Library Tube for sequencing.

Reagent Kits
Kit Name	Supplier
NaOH
Resuspension Buffer (RSB)
Tris-HCl

Queue/Ice Bucket

Section

Subsection

Value

Sample Table

Column Headers

•

Sample Name

•

Flowcell Type

•

Normalized Molarity (nM)

•

Well

•

Container Name

•

Waiting

Expanded View Fields

•

Project Name

•

LIMS ID (Container)

Defaults

Sample Grouping

Group by Containers

Well Sort Order

Column

Placement
Setting	Value	Specification
Destination Containers	Library Tube
Defaults	Sample Grouping	Group by Containers
	Well Sort Order	Column
	Placement Pattern	Column
	Skip Alternating Rows	No
	Skip Alternating Columns	No

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Experiment Name

Text

Required Field = Yes

Read Only = No

BaseSpace Sequence Hub Configuration

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

Run Monitoring and Storage

•

Run Monitoring Only

•

Not Used

Workflow

Text

Required Field = No

Read Only = Yes

Default = GenerateFASTQ

Workflow Type

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

•

Dual Index

•

Single Index

•

No Index

•

Custom

Index Read 1

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 0 to 20

Decimal Places Displayed = 0

Presets:

•

Index Read 2

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 0 to 20

Decimal Places Displayed = 0

Presets:

•

Paired End

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

True

•

False

Read Cycles 1

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 1 to 301

Decimal Places Displayed = 0

Presets:

•

151

•

251

•

101

•

Read Cycles 2

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 0 to 301

Decimal Places Displayed = 0

Presets:

•

151

•

251

•

101

•

Use Custom Read 1 Primer

Toggle Switch

Required Field = Yes

Read Only = No

Default = No

Use Custom Read 2 Primer

Toggle Switch

Required Field = Yes

Read Only = No

Default = No

Use Custom Index Read 1 Primer

Toggle Switch

Required Field = No

Read Only = No

Default = No

Output Folder

Text

Required Field = Yes

Read Only = No

Use Custom Recipe

Toggle Switch

Required Field = Yes

Read Only = No

Default = No

Custom Recipe Path

Text

Required Field = No

Read Only = No

Adapter

Text

Required Field = Yes

Read Only = No

Default = CTGTCTCTTATACACATCT

Adapter Read 2

Text

Required Field = Yes

Read Only = No

Default = CTGTCTCTTATACACATCT

BaseSpace Sequence Hub Analysis Workflow

Text

Required Field = Yes

Read Only = No

Default = TSS Connect v1.1

BaseSpace Sequence Hub Project Name

Text

Required Field = Yes

Read Only = No

Enable Secondary Analysis

Toggle Switch

Required Field = Yes

Read Only = No

Default = Yes

Liquid Handling Instrument ID

Text

Required Field = No

Read Only = Yes

Required Yield Gbp

Numeric

Required Field = Yes

Read Only = No

Default = 90

SampleSheet Version

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

V2 (Set as Default = Yes)

•

Multiline Text Fields

Quick Reference Checklist: Dilute and Denature

Multiline Text

Required Field = No

Read Only = Yes

Default

User tasks for this page:

•

Select NaOH/TrisHCl/RSB lots, and input run configuration.

•

Run Dilute and Denature automation, download resulting input Hamilton Load to Library tube file.

•

Select Next Steps and then Finish Step to move library tube through to AUTOMATED - NovaSeq Run.

Step File Placeholders

Sample Sheet

File

Auto

Sample Sheet

Log

Auto

Hamilton Load to Library tube

File

Auto

Hamilton

Log

Auto

Biosample Manifest

File

Auto

Sample Table

Table Heading

Sample Table

Enable QC Flags

Sample Display Default

Expand

Well Sort Order

Column

Table Columns

Sample Name

Flowcell Type

Text Dropdown

•

Global Field = Derived Sample

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = No

Presets

•

Loading Workflow Type

Text Dropdown

Global Field = Derived Sample

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

NovaSeq Standard

•

NovaSeq Xp

•

(Remove from workflow)

Container Name

Well

LIMS ID (Container)

Project Name

Automation - Script Description
Automation Name	Setting	Value	Specification
Validate Single Input (IGA v1.0)	Trigger Location	Step
	Trigger Style	Automatic upon entry
			Command line `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:validateSampleCount -min 1 -max 1"`
Validate Library Tube Barcode (IGA v1.0)	Trigger Location	Placement
	Trigger Style	Automatic upon exit
			Command line 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 -exp 'if (!output.container.name.matches( ::[A-Za-z]{2}[0-9]{7}-[A-Za-z]{3}:: ) ) {fail ( ::Invalid Library Tube Barcode. This must be in the format CCxxxxxxx-CCC, where 'x' is a digit 0-9 and 'C' is an uppercase letter, e.g. LP1234567-ABC. Please verify and try again.:: ) } ; output.::Flowcell Type:: = input.::Flowcell Type:: ; output.::Loading Workflow Type:: = input.::Loading Workflow Type::' -log {compoundOutputFileLuid1}"
Dilute and Denature NovaSeq Standard - Generate Hamilton Library Tube Input File, Validate Run Setup, and Generate SampleSheet (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Manual Button
			Command line `bash -l -c "/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 {compoundOutputFileLuid2} \` `-t /opt/gls/clarity/extensions/hamilton_fast_prep/templatefiles/LoadToLibTubeFile_NovaSeqStd_template.csv \` `-o Output_plate_name.csv \` `-l {compoundOutputFileLuid3} && \` `/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 \` -exp 'step.::Run Mode:: = input.::Flowcell Type:: ; if (!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) { fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::) }; if (step.::Workflow Type:: == ::No Index::) { if (step.::Index Read 1:: != 0) { fail(::Index Read 1 must be 0 if the Workflow Type is No Index.::) } } else { if (step.::Index Read 1:: == 0) { fail(::Index Read 1 must be greater than 0 if the Workflow Type is :: + step.::Workflow Type:: + ::.::) } }; if (step.::Paired End::.toBoolean() && step.::Read 2 Cycles:: == 0) { fail(::Read 2 Cycles must not be zero if it is Paired End read.::) }; if (step.::Workflow Type:: == ::Dual Index:: && step.::Index Read 2:: == 0) { fail(::Index Read 2 must not be zero if Workflow Type is Dual Index.::) }; if (step.::Workflow Type:: == ::No Index:: \|\| step.::Workflow Type:: == ::Single Index::) { step.::Index Read 2:: = 0 }; if (!step.::Paired End::.toBoolean()) { step.::Read 2 Cycles:: = 0 }; if (input.::Flowcell Type::!=::SP:: && step.::Read 1 Cycles:: > 151) { fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell::) };if (input.::Flowcell Type::!=::SP:: && step.::Read 2 Cycles:: > 151) { fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell::) }; nextStep = ::REMOVE::' \ `-log {compoundOutputFileLuid1} \` `-t true \` `&& if [[ '{udf:SampleSheet Version}' == 'V2' && '{udf:Enable Secondary Analysis}' == 'false' ]]; then \` `templateFilename='/opt/gls/clarity/extensions/ss_converter_templates/NovaSeq_BCL2FASTQ_Samplesheet_Std_V2.csv'; \` `else \` `templateFilename='/opt/gls/clarity/extensions/ss_converter_templates/NovaSeq_BCL2FASTQ_Samplesheet_Std_V1.csv'; \` `fi \` `&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \` `script:driver_file_generator \` `-t $templateFilename \` `-o {compoundOutputFileLuid0}.csv \` `-q true \` `-destLIMSID {compoundOutputFileLuid0} \` `-l {compoundOutputFileLuid1} \` `&& if [[ '{udf:Enable Secondary Analysis}' == 'true' ]]; then \` `PYTHONPATH=/opt/gls/clarity/extensions python3 /opt/gls/clarity/extensions/ss_converter \` `--input {compoundOutputFileLuid0}.csv \` `--stepURI {stepURI:v2} -u {username} -p {password} -q \` `--destLIMSID {compoundOutputFileLuid0} 2>&1; \` `fi && \` `/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar \` `-i {stepURI:v2} -u {username} -p {password} \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/iga_novaseq/Biosample_Manifest.csv \` `-o {compoundOutputFileLuid4}.csv \` `-q true \` `-destLIMSID {compoundOutputFileLuid4} \` `-l {compoundOutputFileLuid1}"`
Dilute and Denature NovaSeq Standard - Set Next Steps to Remove from Workflow (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Automatic upon exit
			Command line `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 {compoundOutputFileLuid1} \` `&& \` `/usr/bin/env python /opt/gls/clarity/extensions/iga_novaseq/02_NovaSeqStd_confirmPlateIDAndStoreInstrumentID_OptionalFile_DiluteDenature.py -u {username} -p {password} -s {stepURI} -f {compoundOutputFileLuid4} -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 = ::REMOVE::' -log {compoundOutputFileLuid1}"`
Dilute and Denature NovaSeq Standard - Routing Script (IGA v1.0)	Trigger Location	Step
	Trigger Style	Automatic upon exit
			Command line `bash -l -c "/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 {compoundOutputFileLuid1} script:changeWorkflow \` `\` `--FIELD_NAME 'N/A' \` `--FIELD_VALUE 'N/A' \` `--WORKFLOW 'Illumina Genomics Architecture - NovaSeq Sequencing v1.0' \` `--STEP 'AUTOMATED - NovaSeq Run (IGA v1.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS'"`

Next Step
Setting	Value
AUTOMATED - NovaSeq Run (IGA v1.0)
Assign Next Step	Automatic
Safe Stopping Point

Protocol 3: NovaSeq Xp (IGA v1.0)

Step 1: Make Bulk Pool for NovaSeq Xp (IGA v1.0)

Master Step 1: Make Bulk Pool for NovaSeq Xp (IGA v1.0)

Step Type = Pooling

Aliquot Generation = Fixed 1

Input = Indexed or Pool/Captured 96-well plates

Output = Indexed or Pool/Captured 96-well plates (ready to be pooled into a Bulk Pool Plate in the Next Step)

Queue/Ice Bucket

Section

Subsection

Value

Sample Table

Column Headers

•

Sample Name

•

Flowcell Type

•

Normalized Molarity (nM)

•

Loading Workflow Type

•

Well

•

Container Name

•

Waiting

Expanded View Fields

•

Project Name

•

LIMS ID (Container)

Defaults

Sample Grouping

Group by Containers

Well Sort Order

Row

Pooling
Section	Subsection	Value
Pool Settings	Label Uniqueness	On
Defaults	Sample Grouping	Group by Containers
	Well Sort Order	Row

Placement
Setting	Value	Specification
Destination Containers	96 Well Plate
Defaults	Well Sort Order	Row
	Placement Pattern	Column
	Skip Alternating Rows	No
	Skip Alternating Columns	No

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Number of Lanes to Sequence

Numeric

Required Field = Yes

Read Only = No

Decimal Places Displayed = 0

Minimum Per Sample Volume (µl)

Numeric

Required Field = Yes

Read Only = No

Default = 2

Decimal Places Displayed = 2

Multiline Text Fields

Quick Reference Checklist: Make Bulk Pool for NovaSeq Xp. i) pre Liquid Handler

Multiline Text

Required Field = No

Read Only = Yes

Default

User Tasks for the page:

•

Select Number Of Lanes and Min Per Sample Volume (µl).

•

Run the Make Bulk Pool automation.

•

Select Next Steps and Finish Steps. To add another Bulk Pool to this BPP, repeat Make Bulk Pool for NovaSeq Xp. After your BPP contains the pools you need, continue on to Dilute and Denature to get the collated MakeBPP Liquid Handler input file.

Step File Placeholders

Calculation

File

Auto

Log

Auto

Hamilton Make Bulk Pool Input

File

Auto

Hamilton Input File Generation

Log

Auto

Sample Table

Table Heading

Sample Table

Enable QC Flags

Sample Display Default

Expand

Well Sort Order

Row

Table Columns

Sample Name

RSB Volume (µl)

Numeric

•

Global Field = Derived Sample

•

Required Field = No

•

Read Only = Yes

•

Decimal Places Displayed = 2

Flowcell Type

Text Dropdown

•

Global Field = Derived Sample

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = No

Presets:

•

Loading Workflow Type

Text Dropdown

•

Global Field = Derived Sample

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = No

Presets:

•

NovaSeq Standard

•

NovaSeq Xp

•

(Remove from workflow)

Container Name

Well

LIMS ID (Container)

Project Name

Automation - Script Description
Automation Name	Setting	Value	Specification
Validate Inputs Flowcell Type and Single Pool (IGA v1.0)	Trigger Location	Pooling
	Trigger Style	Automatic upon exit
			Command line `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 {compoundOutputFileLuid1} -validateSingleOutput true -poolType bulk"`
NovaSeq Make Bulk Pool - Output Plate BPP Barcode Check (Duplicates Allowed) (IGA v1.0)	Trigger Location	Placement
	Trigger Style	Automatic upon exit
			Command line `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 {compoundOutputFileLuid1}"`
Make Bulk Pool NovaSeq Xp - Reset Loading Workflow Type and Flowcell Type (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Automatic upon entry
			Command line `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 'output.::Loading Workflow Type:: = input.::Loading Workflow Type::; output.::Flowcell Type:: = input.::Flowcell Type::' -log {compoundOutputFileLuid1}"`
Make Bulk Pool NovaSeq Xp - Calculate Volumes and Generate Hamilton Input File (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Manual Button
			Command line `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 {compoundOutputFileLuid1} \` `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 (µl):: = step.::Number of Lanes to Sequence:: * 18+ 30 } ; if ( input.::Flowcell Type:: == ::S1:: ) { step.::Bulk Pool Volume (µl):: = step.::Number of Lanes to Sequence:: * 18+ 30 } ; if ( input.::Flowcell Type:: == ::S2:: ) { step.::Bulk Pool Volume (µl):: = step.::Number of Lanes to Sequence:: * 22 + 30 } ; if ( input.::Flowcell Type:: == ::S4:: ) { step.::Bulk Pool Volume (µl):: = step.::Number of Lanes to Sequence:: * 30 + 30 } ; input.::Per Sample Volume (µl):: = ( ( (input.::Final Loading Concentration (pM):: * 5 / 1000) / input.::Normalized Molarity (nM):: ) * step.::Bulk Pool Volume (µl):: ) / step.::Number of Samples in Pool::' \ `script:calculate_adjusted_per_sample_volume \` `script:evaluateDynamicExpression \` `-h false \` `-exp 'step.::Total Sample Volume (µl):: = step.::Total Sample Volume (µl):: + input.::Adjusted Per Sample Volume (µl)::' \` `script:evaluateDynamicExpression \` `-h false \` `-exp 'if (step.::Total Sample Volume (µl):: >= step.::Bulk Pool Volume (µl)::) {output.::RSB Volume (µl):: = 0} else {output.::RSB Volume (µl):: = step.::Bulk Pool Volume (µl):: - step.::Total Sample Volume (µl)::} ; output.::Flowcell Type:: = input.::Flowcell Type:: ; output.::Loading Workflow Type:: = input.::Loading Workflow Type::' \` `&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Bulk_Pool.csv \` `-o {compoundOutputFileLuid0}.csv \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Bulk_Pool2.csv \` `-o append.csv \` `&& cat append.csv >> {compoundOutputFileLuid0}.csv \` `&& \` `/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 {compoundOutputFileLuid1} \` `script:evaluateDynamicExpression \` `-h false \` `-exp 'step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: - 1; step.::Total Sample Volume (µl):: = step.::Total Sample Volume (µl):: - input.::Adjusted Per Sample Volume (µl)::' \` `&& \` `/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 {compoundOutputFileLuid2} \` `-t /opt/gls/clarity/extensions/hamilton_fast_prep/templatefiles/MakeBPFile_NovaSeq_template.csv \` `-o Output_plate_name.csv \` `-l {compoundOutputFileLuid3}"`
Set Flowcell Type and Next Step (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Automatic upon exit
			Command line `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 'output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}"`

Next Step
Setting	Value
Dilute, Denature & ExAmp (IGA v1.0)
Start Next Step	Manual
Assign Next Step	Manual
Safe Stopping Point

Step 2: Dilute, Denature & ExAmp (IGA v1.0)

Step Type = Standard

Input = Indexed or Pool/Captured 96-well plates

Output = Bulk pooled libraries ready to load to a flowcell.

Reagent Kits
Kit Name	Supplier	Cat. #
DPX1	Illumina	20019916
DPX2	Illumina	20019917
DPX3	Illumina	20019918
NaOH
Resuspension Buffer (RSB)
Tris-HCl

Queue/Ice Bucket

Section

Subsection

Value

Sample Table

Column Headers

•

Sample Name

•

Flowcell Type

•

Loading Workflow Type

•

Well

•

Container Name

•

Waiting

Expanded View Fields

•

Project Name

•

LIMS ID (Container)

Defaults

Sample Grouping

Group by Containers

Well Sort Order

Row

Step Setup

Section

Subsection

Value

File Placeholders

Hamilton Consolidated MakeBPP Bulk Pool Input File

•

The file contains consolidated Bulk Pool information to run the Hamilton MakeBPP method. It refers to previous Xp Make Bulk Pool steps in Clarity that all use the same BPP output plate name. Run this file with the Hamilton MakeBPP method before continuing with this step.

•

Auto

Upload Hamilton MakeBPP Output File

•

Upload the Hamilton output file from the MakeBPP run.

•

Manual

Placement
Setting	Value	Specification
Destination Containers	96 Well Plate
Defaults	Sample Grouping	Group by Containers
	Well Sort Order	Row
	Placement Pattern	Column
	Skip Alternating Rows	No
	Skip Alternating Columns	No

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

DPX1 Volume (µl)

Numeric

Required Field = No

Read Only = Yes

Decimal Places Displayed = 0

DPX2 Volume (µl)

Numeric

Required Field = No

Read Only = Yes

Decimal Places Displayed = 0

DPX3 Volume (µl)

Numeric

Required Field = No

Read Only = Yes

Decimal Places Displayed = 0

Liquid Handling Instrument ID

Text

Required Field = No

Read Only = Yes

Multiline Text Fields

Quick Reference Checklist: Dilute, Denature and ExAm

Multiline Text

Required Field = No

Read Only = Yes

Default

User Tasks for the page:

•

Input DPX/NaOH/RSB/TrisHCl lots.

•

Select Next Steps.

Step File Placeholders

Calculation

File

Auto

Log

Auto

Incomplete WPP Hamilton Files

(Do not use. Required for next step)

File

Auto

Sample Table

Table Heading

Sample Table

Enable QC Flags

Sample Display Default

Expand

Well Sort Order

Row

Table Columns

Sample Name

BP Aliquot Volume (µl)

Numeric

Global Field = Derived Sample

Required Field = No

Read Only = Yes

Decimal Places Displayed = 0

Mastermix per Lane (µl)

Numeric

Global Field = Derived Sample

Required Field = No

Read Only = Yes

Decimal Places Displayed = 0

NaOH Volume (µl)

Numeric

Global Field = Derived Sample

Required Field = No

Read Only = Yes

Decimal Places Displayed = 2

Tris-HCl Volume (µl)

Numeric

Global Field = Derived Sample

Required Field = No

Read Only = Yes

Decimal Places Displayed = 2

Flowcell Type

Text Dropdown

Global Field = Derived Sample

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

Loading Workflow Type

Text Dropdown

Global Field = Derived Sample

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

NovaSeq Standard

•

NovaSeq Xp

•

(Remove from workflow)

Container Name

Well

LIMS ID (Container)

Project Name

Automation - Script Description
Automation Name	Setting	Value	Specification
Dil/Den/ExAmp NovaSeq Xp - Generate Hamilton Consolidated Bulk Pool MakeBPP Input File and Validate Input Flowcell Type (IGA v1.0)	Trigger Location	Step
	Trigger Style	Automatic upon entry
			Command line `bash-l -c "/usr/bin/env python /opt/gls/clarity/extensions/iga_novaseq/03_NovaSeqXp_consolidateBulkPoolFiles.py \` `-u {username} -p {password} -s {stepURI} -o consolidated.csv --destLIMSID {compoundOutputFileLuid2} && \` `/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 {compoundOutputFileLuid1}"`
Dil/Den/ExAmp NovaSeq Xp - Validate Hamilton BPP Output File Name & Parse Instrument ID (IGA v1.0)	Trigger Location	Placement
	Trigger Style	Automatic upon entry
			Command line `bash -l -c "/usr/bin/env python /opt/gls/clarity/extensions/iga_novaseq/03_NovaSeqXp_confirmBPPPlateAndStoreInstrumentID.py -u {username} -p {password} -s {stepURI} -f {compoundOutputFileLuid3} -i 'Liquid Handling Instrument ID'"`
Dil/Den/ExAmp NovaSeq Xp - Output Plate WPP Barcode Uniqueness Check (IGA v1.0)	Trigger Location	Placement
	Trigger Style	Automatic upon exit
			Command line 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}-WPP:: ) ) {fail ( ::Invalid WPP Plate Barcode. This plate ID must be in the format LPxxxxxxx-WPP, where 'x' is a digit 0-9, e.g. LP1234567-WPP. Please verify and try again.:: ) }' -log {compoundOutputFileLuid1} && /usr/bin/env python /opt/gls/clarity/extensions/hamilton_fast_prep/01_A_CFP_Batching_defaultContainerCheck.py -u {username} -p {password} -s {stepURI}"
Dil/Den/ExAmp NovaSeq Xp - Calculate Volumes and Generate Incomplete WPP Driver File for Next Step (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Automatic upon entry
			Command line `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 true \` `-h false \` -exp 'if ( input.::Flowcell Type:: == ::SP:: ) { output.::BP Aliquot Volume (ul):: = 18 ; output.::NaOH Volume (ul):: = 4.50 ; output.::Tris-HCl Volume (ul):: = 4.50 ; step.::DPX1 Volume (ul):: = 126 ; step.::DPX2 Volume (ul):: = 18 ; step.::DPX3 Volume (ul):: = 66 ; output.::Mastermix per Lane (ul):: = 63} ; if ( input.::Flowcell Type:: == ::S1:: ) { output.::BP Aliquot Volume (ul):: = 18 ; output.::NaOH Volume (ul):: = 4.50 ; output.::Tris-HCl Volume (ul):: = 4.50 ; step.::DPX1 Volume (ul):: = 126 ; step.::DPX2 Volume (ul):: = 18 ; step.::DPX3 Volume (ul):: = 66 ; output.::Mastermix per Lane (ul):: = 63} ; if ( input.::Flowcell Type:: == ::S2:: ) { output.::BP Aliquot Volume (ul):: = 22 ; output.::NaOH Volume (ul):: = 5.50 ; output.::Tris-HCl Volume (ul):: = 5.50 ; step.::DPX1 Volume (ul):: = 126 ; step.::DPX2 Volume (ul):: = 18 ; step.::DPX3 Volume (ul):: = 66 ; output.::Mastermix per Lane (ul):: = 77} ; if ( input.::Flowcell Type:: == ::S4:: ) { output.::BP Aliquot Volume (ul):: = 30 ; output.::NaOH Volume (ul):: = 7.50 ; output.::Tris-HCl Volume (ul):: = 7.50 ; step.::DPX1 Volume (ul):: = 315 ; step.::DPX2 Volume (ul):: = 45 ; step.::DPX3 Volume (ul):: = 165 ; output.::Mastermix per Lane (ul):: = 105} ; output.::Flowcell Type:: = input.::Flowcell Type:: ; output.::Loading Workflow Type:: = input.::Loading Workflow Type::' \ `-log {compoundOutputFileLuid1} \` `&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Working_Pool.csv \` `-o {compoundOutputFileLuid0}.csv \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Working_Pool2.csv \` `-o append.csv \` `&& cat append.csv >> {compoundOutputFileLuid0}.csv \` `&& \` `/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 {compoundOutputFileLuid4} \` `-t /opt/gls/clarity/extensions/hamilton_fast_prep/templatefiles/LoadToFlowcell_NovaSeqXp_Incomplete_template.csv \` `-o Output_plate_name.csv \` `-l {compoundOutputFileLuid1}"`
Dil/Den/ExAmp NovaSeq Xp - Set Next Steps to Advance (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Automatic upon exit
			Command line `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 'output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}"`

Next Step
Setting	Value
Load to Flowcell (IGA v1.0)
Start Next Step	Manual
Assign Next Step	Manual
Safe Stopping Point

Step 3: Load to Flowcell (IGA v1.0)

Master Step 3: Load to Flowcell (IGA v1.0)

Step Type = Standard

Input = Diluted and denatured Working Pool Plate

Output = Loaded flowcell ready for sequencing

Queue/Ice Bucket

Section

Subsection

Value

Sample Table

Column Headers

•

Sample Name

•

Flowcell Type

•

Well

•

Container Name

•

Waiting

Expanded View Fields

•

Project Name

•

LIMS ID (Container)

Defaults

Sample Grouping

Group by Containers

Well Sort Order

Row

Placement
Setting	Value	Specification
Destination Containers	SP
	S1
	S2
	S4
Defaults	Sample Grouping	Group by Containers
	Well Sort Order	Row
	Placement Pattern	Column
	Skip Alternating Rows	No
	Skip Alternating Columns	No

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Experiment Name

Text

Required Field = Yes

Read Only = No

BaseSpace Sequence Hub Configuration

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

Run Monitoring and Storage (Set as Default = Yes)

•

Run Monitoring Only

•

Not Used

Workflow

Text

Required Field = No

Read Only = Yes

Default = GenerateFASTQ

Workflow Type

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

•

Dual Index (Set as Default = Yes)

•

Single Index

•

No Index

•

Custom

Index Read 1

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 0 to 20

Decimal Places Displayed = 0

Presets:

•

10 (Set as Default = Yes)

•

Index Read 2

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 0 to 20

Decimal Places Displayed = 0

Presets:

•

10 (Set as Default = Yes)

•

Paired End

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

True (Set as Default = Yes)

•

False

Read Cycles 1

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 1 to 301

Decimal Places Displayed = 0

Presets:

•

151 (Set as Default = Yes)

•

251

•

101

•

Read Cycles 2

Numeric Dropdown

Required Field = Yes

Read Only = No

Custom Entries = Yes

Range = 0 to 301

Decimal Places Displayed = 0

Presets:

•

151 (Set as Default = Yes)

•

251

•

101

•

Use Custom Read 1 Primer

Toggle Switch

Required Field = Yes

Read Only = No

Default = No

Use Custom Read 2 Primer

Toggle Switch

Required Field = Yes

Read Only = No

Default = No

Use Custom Index Read 1 Primer

Toggle Switch

Required Field = Yes

Read Only = No

Default = No

Output Folder

Text

Required Field = Yes

Read Only = No

Use Custom Recipe

Toggle Switch

Required Field = Yes

Read Only = No

Default = No

Custom Recipe Path

Text

Required Field = No

Read Only = No

Library Tube Barcode

Text

Required Field = Yes

Read Only = No

Adapter

Text

Required Field = Yes

Read Only = No

Default = CTGTCTCTTATACACATCT

Adapter Read 2

Text

Required Field = Yes

Read Only = No

Default = CTGTCTCTTATACACATCT

BaseSpace Sequence Hub Analysis Workflow

Text

Required Field = Yes

Read Only = No

Default = TSS Connect v1.1

BaseSpace Sequence Hub Project Name

Text

Required Field = Yes

Read Only = No

Enable Secondary Analysis

Toggle Switch

Required Field = Yes

Read Only = No

Default = Yes

Required Yield Gbp

Numeric

Required Field = No

Read Only = No

Default = 90

SampleSheet Version

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

V2 (Set as Default = Yes)

•

Multiline Text Fields

Quick Reference Checklist: Load to Flowcell

Multiline Text

Required Field = No

Read Only = Yes

Default

User tasks for this page:

•

Populate run configuration settings.

•

Run Load To Flowcell automation, download resulting Hamilton Input File (BPP to WPP to Flow Cell) file.

•

Run Validate Run Setup and Generate Sample Sheet (IGA v1.0) automation.

•

Select Next Steps and then Finish Step to move flowcell through to AUTOMATED - NovaSeq Run.

Step File Placeholders

Sample Sheet

File

Auto

Log

Auto

Hamilton Input File (BPP to WPP to Flow Cell)

File

Auto

Biosample Manifest

File

Auto

Sample Table

Table Heading

Sample Table

Enable QC Flags

Sample Display Default

Expand

Well Sort Order

Row

Table Columns

Sample Name

Flowcell Type

Text Dropdown

•

Required Field = Yes

•

Read Only = No

•

Custom Entries = No

Presets

•

Loading Workflow Type

Text Dropdown

Required Field = Yes

Read Only = No

Custom Entries = No

Presets:

•

NovaSeq Standard

•

NovaSeq Xp

•

(Remove from workflow)

Container Name

Well

LIMS ID (Container)

Project Name

Automation - Script Description
Automation Name	Setting	Value	Specification
Validate Inputs and Selected Container (IGA v1.0)	Trigger Location	Step
	Trigger Style	Automatic upon entry
			Command line `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 {compoundOutputFileLuid1} -validateSelectedContainer true"`
Validate Flowcell Barcode with New Mask Fixes (IGA v1.0)	Trigger Location	Placement
	Trigger Style	Automatic upon exit
			Command line `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 \` -exp 'if (input.::Flowcell Type:: == ::SP:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DRX[XY2357]:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; if (input.::Flowcell Type:: == ::S1:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DRX[XY2357]:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; if (input.::Flowcell Type:: == ::S2:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DMX[XY2357]:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; if (input.::Flowcell Type:: == ::S4:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DSX[XY2357]:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; output.::Flowcell Type:: = input.::Flowcell Type:: ; output.::Loading Workflow Type:: = input.::Loading Workflow Type::' \ `-log {compoundOutputFileLuid1} && \` `/usr/bin/env python /opt/gls/clarity/extensions/iga_novaseq/validate_floorcell_container.py -u {username} -p {password} -s {stepURI}"`
Load to Flowcell NovaSeq Xp - Generate Hamilton WPP Input File (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Manual Button
			Command line `bash -l -c "/usr/bin/env python /opt/gls/clarity/extensions/iga_novaseq/04_NovaSeqXp_completeAndConsolidateWPPZipFile.py -u {username} -p {password} -s {stepURI} -o wpp_output --destLIMSID {compoundOutputFileLuid2}"`
Validate Run Setup and Generate SampleSheet (IGA v1.0)	Trigger Location	Record Details
	Trigger Style	Manual Button
			Command line `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 \` -exp 'step.::Run Mode:: = input.::Flowcell Type:: ; if (!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) { fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::) }; if (step.::Workflow Type:: == ::No Index::) { if (step.::Index Read 1:: != 0) { fail(::Index Read 1 must be 0 if the Workflow Type is No Index.::) } } else { if (step.::Index Read 1:: == 0) { fail(::Index Read 1 must be greater than 0 if the Workflow Type is :: + step.::Workflow Type:: + ::.::) } }; if (step.::Paired End::.toBoolean() && step.::Read 2 Cycles:: == 0) { fail(::Read 2 Cycles must not be zero if it is Paired End read.::) }; if (step.::Workflow Type:: == ::Dual Index:: && step.::Index Read 2:: == 0) { fail(::Index Read 2 must not be zero if Workflow Type is Dual Index.::) }; if (step.::Workflow Type:: == ::No Index:: \|\| step.::Workflow Type:: == ::Single Index::) { step.::Index Read 2:: = 0 }; if (!step.::Paired End::.toBoolean()) { step.::Read 2 Cycles:: = 0 }; if (input.::Flowcell Type::!=::SP:: && step.::Read 1 Cycles:: > 151) { fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell::) };if (input.::Flowcell Type::!=::SP:: && step.::Read 2 Cycles:: > 151) { fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell::) }; nextStep = ::ADVANCE::' \ `-log {compoundOutputFileLuid1} \` `-t true \` `&& if [[ '{udf:SampleSheet Version}' == 'V2' && '{udf:Enable Secondary Analysis}' == 'false' ]]; then \` `templateFilename='/opt/gls/clarity/extensions/ss_converter_templates/NovaSeq_BCL2FASTQ_Samplesheet_XP_V2.csv'; \` `else \` `templateFilename='/opt/gls/clarity/extensions/ss_converter_templates/NovaSeq_BCL2FASTQ_Samplesheet_XP_V1.csv'; \` `fi \` `&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \` `script:driver_file_generator \` `-t $templateFilename \` `-o {compoundOutputFileLuid0}.csv \` `-q true \` `-destLIMSID {compoundOutputFileLuid0} \` `-l {compoundOutputFileLuid1} \` `&& if [[ '{udf:Enable Secondary Analysis}' == 'true' ]]; then \` `PYTHONPATH=/opt/gls/clarity/extensions python3 /opt/gls/clarity/extensions/ss_converter \` `--input {compoundOutputFileLuid0}.csv \` `--stepURI {stepURI:v2} -u {username} -p {password} -q \` `--destLIMSID {compoundOutputFileLuid0} 2>&1; \` `fi && \` `/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar \` `-i {stepURI:v2} -u {username} -p {password} \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/iga_novaseq/Biosample_Manifest.csv \` `-o {compoundOutputFileLuid3}.csv \` `-q true \` `-destLIMSID {compoundOutputFileLuid3} \` `-l {compoundOutputFileLuid1}"`

Next Step
Setting	Value
AUTOMATED - NovaSeq Run (IGA v1.0)
Assign Next Step	Manual
Must move forward (30 minutes to start sequencing)

Protocol 4: AUTOMATED - NovaSeq Run (IGA v1.0)

Step 1: AUTOMATED = NovaSeq Run (IGA v1.0)

Master Step 1: AUTOMATED - NovaSeq Run (IGA v1.0)

Do not add samples to the Ice Bucket, start, or complete the AUTOMATED - NovaSeq Run (IGA v1.0) step. The integration does this automatically.

Step Type = Analysis

Input = Library Tube or Flowcell ready for sequencing

Output = None

Reagent Kits
Kit Name	Supplier
Buffer Cartridge	Illumina
Cluster Cartridge	Illumina
SBS Cartridge	Illumina

Queue/Ice Bucket

Section

Subsection

Value

Sample Table

Column Headers

•

Sample Name

•

Well

•

Container Name

•

Waiting

Expanded View Fields

•

Project Name

•

LIMS ID (Container)

Defaults

Sample Grouping

Group by Containers

Well Sort Order

Row

Record Details
Section	Subsection	Setting	Value	Specification
Step Data	Master Step Fields	Run ID	Text	Required Field = No Read Only = Yes
		Current Cycle	Numeric	Required Field = Yes Read Only = No Decimal Places Displayed = 0
		Flow Cell ID	Text	Required Field = No Read Only = Yes
		Flow Cell Part Number	Text	Required Field = No Read Only = Yes
		Instrument ID	Text	Required Field = No Read Only = Yes
		Firmware Version	Text	Required Field = No Read Only = Yes
		Run Status	Text	Required Field = No Read Only = Yes
		Current Read	Numeric	Required Field = No Read Only = Yes Decimal Places Displayed = 0
		Flow Cell Side	Text	Required Field = No Read Only = Yes
		Flow Cell Lot Number	Text	Required Field = No Read Only = Yes
		Instrument Type	Text	Required Field = No Read Only = Yes
		RTA Version	Text	Required Field = No Read Only = Yes
		Output Folder	Text	Required Field = No Read Only = Yes
		Loading Workflow Type	Text	Required Field = No Read Only = Yes
		Flow Cell Mode	Text	Required Field = No Read Only = Yes
		Flow Cell Expiration Date	Date	Required Field = No Read Only = Yes
		Instrument Control Software Version	Text	Required Field = No Read Only = Yes
	Multiline Text Fields	Sequencing Log	Multiline Text	Required Field = No Read Only = Yes
Step File Placeholders		None
Sample Table		Table Heading	Sample Table
		Enable QC Flags	No
		Sample Display Default	Collapse
		Well Sort Order	Row
	Table Columns
		Sample Name
		Container Name
		Yield PF (Gb) R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		Yield PF (Gb) R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		Reads PF (M) R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		Reads PF (M) R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		Cluster Density (K/mm^2) R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 0
		Cluster Density (K/mm^2) R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		%PF R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		%PF R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		% Bases >=Q30 R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		% Bases >=Q30 R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 0
		Intensity Cycle 1 R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 0
		Intensity Cycle 1 R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 0
		% Phasing R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 3
		% Phasing R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 3
		% Prephasing R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 3
		% Prephasing R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 3
		% Aligned R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		% Aligned R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		% Error Rate R1	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		% Error Rate R2	Numeric	Global field - Measurement & Container Required Field = No Read Only = Yes Decimal Places Displayed = 2
		Well
		LIMS ID (Container)
		Project Name

Next Step
Setting	Value
None - End of Workflow
Assign Next Step	Manual