iSeq 100 Run Setup v1.0

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 v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Dilute Library to 1nM (iSeq 100 v1.0)

• Master Step Name = Dilute Library to 1nM - iSeq 100 v1.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Resuspension Buffer (RSB)

Automations

Calculate Final & Library Volumes - iSeq

• 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: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

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}"

Queue/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	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Molarity (nM)	Numeric		Default = 1 Decimal Places Displayed = 0
  Final Volume (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = Select the Library Kit Type and enter in the Starting Molarity before clicking on the Calculate Final & Library Volumes - iSeq button.
  Library Prep Kit	Text Dropdown	Required Field Custom Entries	Presets 100% PhiX AmpliSeq Library PLUS for Illumina Nextera DNA Flex Nextera Flex for Enrichment Nextera XT DNA TruSeq DNA Nano TruSeq DNA PCR-Free

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • 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	Final Molarity (nM)	Numeric
    Derived Sample	Library Prep Kit	Text
    Derived Sample	Library Volume (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	Starting Molarity (nM)	Numeric		Decimal Places Displayed = 0
    Project	Project Name	Built-in

Step 2: Dilute Library to Loading Concentration (iSeq 100 v1.0)

• Master Step Name = Dilute Library to Loading Concentration - iSeq 100 v1.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Resuspension Buffer (RSB)

Automations

Copy Library Prep Kit & Starting Molarity and Set Loading Concentration - iSeq

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

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 Library & RSB Volumes - iSeq

• 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: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}"

Set Next Step - Advance

• 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:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/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	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Volume (uL)	Numeric		Default = 100 Decimal Places Displayed = 0
  Loading Concentration (pM)	Numeric		Decimal Places Displayed = 0
  Loading Concentration Notes	Multiline Text	Read Only	Default = 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

  • Log - Manually uploaded

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • 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	Library Prep Kit	Text
    Derived Sample	Library Volume (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	Starting Molarity (nM)	Numeric		Decimal Places Displayed = 0
    Project	Project Name	Built-in

Step 3: Add a PhiX Control (iSeq 100 v1.0)

• Master Step Name = Add a PhiX Control (iSeq 100 v1.0)

• Step Type = No Outputs

• Reagent Kits

  • PhiX Control v3

    • Supplier = Illumina

    • Catalog Number = FC-110-3001

    • Website = www.illumina.com

  • Resuspension Buffer (RSB)

Automations

Copy Library Prep Kit- iSeq

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

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

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

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}"

Queue/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	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Diluted PhiX Volume (uL)	Numeric		Default = 100 Decimal Places Displayed = 0
  Final PhiX Molarity (nM)	Numeric		Default = 1 Decimal Places Displayed = 0
  Final PhiX Volume (uL)	Numeric		Default = 50 Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = Combine Starting PhiX Volume (uL) with RSB (uL) to create your diluted PhiX solution.
  PhiX Loading Concentration (pM)	Numeric		Default = 100 Decimal Places Displayed = 0
  PhiX Spiked In %	Numeric Dropdown	Custom Entries	Presets 2 10 Default = 2
  RSB (uL)	Numeric		Decimal Places Displayed = 0
  Starting PhiX Volume (uL)	Numeric		Decimal Places Displayed = 0
  Stock PhiX Molarity (nM)	Numeric		Default = 10 Decimal Places Displayed = 0

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • 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	Diluted PhiX Volume (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Library Prep Kit	Text
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 4: Run Setup (iSeq 100 v1.0)

• Master Step Name = Run Setup - iSeq 100 v1.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • iSeq 100 i1 Reagent v2

    • Supplier = Illumina

    • Catalog Number = 300 cycle single - 20031371; 300 cycle four-pack - 20031374; 300 cycle eight-pack - 20040760

    • Website = www.illumina.com

Automations

Copy Library Prep Kit- iSeq

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

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}"

1. iseq LRM sample sheet

• 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/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}"

2. iseq manual sample sheet

• 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/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}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table (Column Headers)

  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	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Adapter	Text		Default = CTGTCTCTTATACACATCT
  BaseSpace Sequence Hub	Text Dropdown		Presets Run Monitoring Only Run Monitoring and Storage
  Chemistry	Text
  Experiment Name	Text	Required Field
  IndexStrategy	Text Dropdown	Required Field	Presets NoIndex SingleOnly DualOnly SingleAndDual NoAndSingle NoAndDual
  Library Prep Kit	Text
  Local Run Manager Analysis Id	Numeric
  Manifest File Name	Text
  Module	Text
  Output Folder Location	Text
  Read 1 Cycles	Numeric	Required Field	Range = 26 To 151
  Read 2 Cycles	Numeric	Required Field
  Read Type	Text Dropdown	Required Field Custom Entries	Presets Single Read Paired End
  Workflow	Text		Default = GenerateFASTQ

• Step File Placeholders

  • Log - Automatically attached

  • LRM Sample Sheet - Automatically attached

  • Manual Sample Sheet - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Row

  • 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	Sample Name	Built-in
    Project	Project Name	Built-in