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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

    • 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

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