Illumina Preset Protocol - iSeq 100 Run Setup

Overview

The iSeq 100 Run Setup includes the following functionality.

•

Preconfigured iSeq 100 Run Setup workflow that supports the preparation of samples for use on the Illumina® iSeq™ 100 Sequencing System provides a targeted approach to next-generation sequencing (NGS).

•

Automated calculation of sample and buffer volumes.

•

Automated calculation or display of reagents at every step in the protocol.

•

Automatic step transition when required.

•

Automatic placement of samples when necessary.

•

Local Run Manager and manual sample sheet generation for use on the iSeq 100.

Protocol 1: Dilute Libraries and Run Setup (iSeq 100 Run Setup)

Protocol Type: Sequencing

Step 1: Dilute Library to 1nM (iSeq 100 Run Setup)

Master Step: Dilute Library to 1nM - iSeq 100

Step Type = Standard

Reagent Kits
Kit Name	Supplier
RSB	Illumina

Queue/Ice Bucket
Setting	Value	Specification
Well Sort Order	Column	Configured on master step

Placement
Setting	Value	Specification
Destination Containers	Low Bind 1.5 mL Tube	Configured on step

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Library Prep Kit

Text Dropdown

•

100% PhiX

•

AmpliSeq Library PLUS for Illumina

•

Nextera DNA Flex

•

Nextera Flex for Enrichment

•

Nextera XT DNA

•

TruSeq DNA Nano

•

TruSeq DNA PCR-Free

Final Molarity (nM)

Numeric

Default

•

Final Volume (uL)

Numeric

Calculated based on Library Prep Kit type:

•

100% PhiX = 12,

•

AmpliSeq Library PLUS for Illumina = 7

•

Nextera DNA Flex = 12

•

Nextera Flex for Enrichment = 10

•

Nextera XT DNA = 20

•

TruSeq DNA Nano = 20

•

TruSeq DNA PCR-Free = 12

Multiline Text Fields

Instruction Notes

Multiline Text

Select the Library Prep Kit Type and enter in the Starting Molarity before clicking on the Calculate Final & Library Volumes - iSeq button.

Step File Placeholders

File Name

Log

Auto

Table Columns

Starting Molarity (nM)

Numeric

Global field - derived sample

Required Field

User input (can be configured to pull in concentration from a previous protocol)

Library Volume (uL)

Global field - derived sample

Calculated = Final Molarity (nM) * Final Volume (uL) ) / Starting Concentration (ng/uL)

Library Prep Kit

Text

Calculated = copy from step

Final Molarity (nM)

Numeric

Calculated = copy from step

Automation
Automation Name	Setting	Value	Specification
Calculate Final & Library Volumes - iSeq	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:http} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false \` `-exp 'if (step.::Library Prep Kit:: == ::100% PhiX::) {step.::Final Volume (uL):: = 12} ; \` `if (step.::Library Prep Kit:: == ::AmpliSeq Library PLUS for Illumina::) {step.::Final Volume (uL):: = 7} ; \` `if (step.::Library Prep Kit:: == ::Nextera DNA Flex::) {step.::Final Volume (uL):: = 12} ; \` `if (step.::Library Prep Kit:: == ::Nextera Flex for Enrichment::) {step.::Final Volume (uL):: = 10} ; \` `if (step.::Library Prep Kit:: == ::TruSeq DNA Nano::) {step.::Final Volume (uL):: = 20} ; \` `if (step.::Library Prep Kit:: == ::TruSeq DNA PCR-Free::) {step.::Final Volume (uL):: = 12} ; \` `output.::Library Volume (uL):: = (step.::Final Molarity (nM):: * step.::Final Volume (uL)::) / output.::Starting Molarity (nM):: ; \` `output.::Library Prep Kit:: = step.::Library Prep Kit:: ; \` `output.::Final Molarity (nM):: = step.::Final Molarity (nM)::' \` `-log {compoundOutputFileLuid0}"`
Set Next Step - Advance	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 = ::ADVANCE::' -log {compoundOutputFileLuid0}"`

Next Step
Setting	Value
Start Next Step	Manual
Assign Next Step	Automatic
Safe Stopping Point

Step 2: Dilute Library to Loading Concentration (iSeq 100 Run Setup)

Master Step: Dilute Library to Loading Concentration - iSeq

Step Type = Standard

Reagent Kits
Kit Name	Supplier
RSB	Illumina

Queue/Ice Bucket
Setting	Value	Specification
Well Sort Order	Column	Configured on master step

Placement
Setting	Value	Specification
Destination Containers	Low Bind 1.5 mL Tube	Configured on step

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Loading Concentration (pM)

Numeric

Calculate = if

•

100% PhiX = 100

•

AmpliSeq Library PLUS for Illumina = 40

•

Nextera DNA Flex = 75

•

Nextera Flex for Enrichment = 50

•

Nextera XT DNA = 100

•

TruSeq DNA Nano = 125

•

TruSeq DNA PCR-Free = 75

Final Volume (uL)

Numeric

Default = 1

Multiline Text Fields

Loading Concentration Notes

Multiline Text

Loading concentration ranges:

•

100% PhiX = 100

•

AmpliSeq Library PLUS for Illumina = 40-60

•

Nextera DNA Flex = 75-125

•

Nextera Flex for Enrichment = 50-100

•

Nextera XT DNA = 100-200

•

TruSeq DNA Nano = 125-175

•

TruSeq DNA PCR-Free = 75-125

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Starting Molarity (nM)

Numeric

Read Only

Global field - derived sample

Required

Copy from previous step from "Final Molarity"

Library Volume (uL)

Numeric

Global field - derived sample

Calculated = (Loading Concentration (pM) * Final Volume) / (Starting Molarity (nM) * 1000)

RSB (uL)

Numeric

Calculated = Final Volume (uL) - Library Volume (uL)

Library Prep Kit

Text

Read Only

Calculated = copy from previous step

Automation
Automation Name	Setting	Value	Specification
Copy Library Prep Kit & Starting Molarity and Set Loading Concentration - iSeq	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.::Library Prep Kit:: = input.::Library Prep Kit:: ; \` `output.::Starting Molarity (nM):: = input.::Final Molarity (nM):: ; \` `if (output.::Library Prep Kit:: == ::100% PhiX::) {step.::Loading Concentration (pM):: = 100} ; \` `if (output.::Library Prep Kit:: == ::AmpliSeq Library PLUS for Illumina::) {step.::Loading Concentration (pM):: = 40} ; \` `if (output.::Library Prep Kit:: == ::Nextera DNA Flex::) {step.::Loading Concentration (pM):: = 75} ; \` `if (output.::Library Prep Kit:: == ::Nextera Flex for Enrichment::) {step.::Loading Concentration (pM):: = 50} ; \` `if (output.::Library Prep Kit:: == ::Nextera XT DNA::) {step.::Loading Concentration (pM):: = 100} ; \` `if (output.::Library Prep Kit:: == ::TruSeq DNA Nano::) {step.::Loading Concentration (pM):: = 125} ; \` `if (output.::Library Prep Kit:: == ::TruSeq DNA PCR-Free::) {step.::Loading Concentration (pM):: = 75}' \` `-log {compoundOutputFileLuid0}"`
Calculate Loading Concentration and Library & RSB Volumes - iSeq	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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \` `-exp 'output.::Library Volume (uL):: = (step.::Loading Concentration (pM):: * step.::Final Volume (uL):: ) / (output.::Starting Molarity (nM):: * 1000) ; \` `output.::RSB (uL):: = step.::Final Volume (uL):: - output.::Library Volume (uL)::' \` `-log {compoundOutputFileLuid0}"`

Next Step
Setting	Value
Start Next Step	Automatic
Assign Next Step	Manual
Must Move Forward

Step 3: Add a PhiX Control (iSeq 100)

Master Step: Add a PhiX Control

Step Type = No Outputs

Reagent Kits
Kit Name	Supplier	Cat. #
RSB	Illumina
PhiX Control v3	Illumina	FC-110-3001

Queue/Ice Bucket
Setting	Value	Specification
Well Sort Order	Column	Configured on master step

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Stock PhiX Molarity (nM)

Numeric

Default

•

Final PhiX Volume (uL)

Numeric

Default

•

Final PhiX Molarity (nM)

Numeric

Default

•

Starting PhiX Volume (uL)

Numeric

Calculated = Final PhiX Molarity (nM) * Final PhiX Volume (uL) / Stock PhiX Molarity (nM)

RSB (uL)

Numeric

Calculated = Final PhiX Volume (uL) - Starting PhiX Volume (uL)

PhiX Loading Concentration (pM)

Numeric

Default

•

100

Final Diluted PhiX Volume (uL)

Numeric

Default

•

100

PhiX Spiked In %

Numeric Dropdown

Presets

•

2 [default]

•

Multiline Text Fields

Instruction Notes

Multiline Text

Combine Starting PhiX (uL) with RSB (uL) to create your diluted PhiX solution.

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Diluted PhiX Volume (uL)

Numeric

Global field - derived sample

Calculated = (PhiX Spiked In %/100) * Final Diluted PhiX Volume (uL)

Library Kit Name

Text

Read Only

Copied from previous step

Automation
Automation Name	Setting	Value	Specification
Copy Library Prep Kit - iSeq	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.::Library Prep Kit:: = input.::Library Prep Kit::' \` `-log {compoundOutputFileLuid0}"`
Calculate Starting PhiX, RSB & Diluted PhiX Volumes	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:http} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false \` `-exp 'step.::Starting PhiX Volume (uL):: = (step.::Final PhiX Molarity (nM):: * step.::Final PhiX Volume (uL)::) / step.::Stock PhiX Molarity (nM):: ; \` `step.::RSB (uL):: = step.::Final PhiX Volume (uL):: - step.::Starting PhiX Volume (uL):: ; \` `input.::Diluted PhiX Volume (uL):: = (step.::PhiX Spiked In %:: / 100) * step.::Final Diluted PhiX Volume (uL)::' \` `-log {compoundOutputFileLuid0}"`
Set Next Step - Advance	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 = ::ADVANCE::' -log {compoundOutputFileLuid0}"`

Next Step
Setting	Value
Start Next Step	Automatic
Assign Next Step	Automatic
Must move forward

Step 4: Run Setup (iSeq 100)

Master Step: Run Setup - iSeq 100

Step Type = Standard

Reagent Kits
Kit Name	Supplier	Cat. #
iSeq 100 i1 Reagent v2	Illumina	300 cycle single - 20031371; 300 cycle four-pack - 20031374; 300 cycle eight-pack - 20040760

Queue/Ice Bucket
Setting	Value	Specification
Well Sort Order	Column	Configured on master step

Placement
Setting	Value	Specification
Destination Containers	iSeq Reagent Cartridge	Configured on step

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Local Run Manager Analysis Id

Text

Experiment Name

Text

Required Field

Module

Text

Workflow

Text

Default

•

GenerateFASTQ

Library Prep Kit

Copied from previous step

Chemistry

Read 1 Cycles

Numeric

Required Field

Range = 26 - 151

Read 2 Cycles

Numeric

Required Field

Range = 26 - 151

Read Type

Text Dropdown

Required Field

Presets

•

Single Read

•

Paired End

IndexStrategy

Text Dropdown

Required Field

Presets

•

NoIndex

•

SingleOnly

•

DualOnly

•

SingleAndDual

•

NoAndSingle

•

NoAndDual

Adapter

Text

Default

•

CTGTCTCTTATACACATCT

BaseSpace Sequence Hub

Text Dropdown

Presets

•

Run Monitoring Only

•

Run Monitoring and Storage

Output Folder Location

Text

Step File Placeholders

File Name

Log

Auto

LRM Sample Sheet

Auto

Script

Manual Sample Sheet

Auto

Script

Automation
Automation Name	Setting	Value	Specification
Copy Library Kit Type - iSeq	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.::Library Prep Kit:: = input.::Library Prep Kit::' \` `-log {compoundOutputFileLuid0}"`
iSeq LRM sample sheet	Trigger Location	Record Details
	Trigger Style	Button
			Command line `bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {processURI:v2} -u {username} -p {password} \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/iSeq_Samplesheet.csv \` `-o {compoundOutputFileLuid1}.csv \` `-q true \` `-destLIMSID {compoundOutputFileLuid1} \` `-l {compoundOutputFileLuid0}"`
iSeq manual sample sheet	Trigger Location	Record Details
	Trigger Style	Button
			Command line `bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {processURI:v2} -u {username} -p {password} \` `script:driver_file_generator \` `-t /opt/gls/clarity/extensions/conf/driverfiletemplates/iSeqManual_Samplesheet.csv \` `-o {compoundOutputFileLuid2}.csv \` `-q true \` `-destLIMSID {compoundOutputFileLuid2} \` `-l {compoundOutputFileLuid0}"`

End of Workflow