Library Preparation v1.1

Overview

The Library Prep AmpliSeq for Illumina Comprehensive Cancer Panel is part of the AmpliSeq for Illumina Comprehensive Cancer Panel protocols that include the following functionality:

• Preconfigured AmpliSeq for Illumina Comprehensive Cancer Panel protocol that supports the preparation of up to 96 uniquely indexed libraries of genomic DNA using the AmpliSeq for Illumina workflow.

• 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.

• Automated assignment of QC Pass/Fail, based on user-selected threshold values. A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

Protocol 1: Library Prep (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Dilute DNA (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

• Master Step Name = Dilute DNA (AmpliSeq for Illumina v1.1)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Copy Concentration and Set Sample Volume

• 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.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Sample Volume (uL):: = 5' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t true \
        -h false \
        -exp 'output.::Total Volume (uL):: = (output.::Concentration::/step.::Desired Concentration (ng/uL)::) * output.::Sample Volume (uL):: ; \
output.::Low TE (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \
output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Total 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 = 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
  Desired Concentration (ng/uL)	Numeric		Range = 20 To 50 Decimal Places Displayed = 0
  Directions	Multiline Text	Read Only	Default = If enough DNA is available, dilute to intermediate concentration of ~20-50 ng/uL.

• 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	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Numeric	Required Field
    Derived Sample	Input Amount (ng)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Low TE (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Volume (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Total Volume (uL)	Numeric		Decimal Places Displayed = 2
    Project	Project Name	Built-in

Step 2: Qubit (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

• Master Step Name = Qubit (AmpliSeq for Illumina v1.1)

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Assign QC flags (Qubit QC)

• 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 {processURI:v2} -u {username} -p {password} script:assignQC -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}"

Set Next Step and Copy Concentration

• 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:: ; input.::Concentration:: = output.::Concentration:: ; input.::Conc. Units:: = output.::Conc. Units::' -log {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
  Criteria 1 - Operator	Text		Default = >=
  Criteria 1 - Source Data Field	Text		Default = Concentration
  Criteria 1 - Threshold Value	Numeric
  Criteria 2 - Operator	Text		Default = <=
  Criteria 2 - Source Data Field	Text		Default = Concentration
  Criteria 2 - Threshold Value	Numeric

• Step File Placeholders

  • Log - Automatically attached

  • QC Log File - Automatically attached

  • QC Result File - Automatically attached

  • Upload File - Manually uploaded

• Sample Table

  • Enable QC Flags = Yes

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • File Column Options

    • File Column Display = Hide

    • File Attachment Method = Auto

  • 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	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Sample Name	Built-in
    Measurement	Concentration	Numeric		Decimal Places Displayed = 2
    Measurement	Conc. Units	Text
    Project	Project Name	Built-in

Step 3: Dilute DNA to Final Concentration (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

• Master Step Name = Dilute DNA (AmpliSeq for Illumina v1.1)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Copy Concentration and Set Sample Volume

• 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.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Sample Volume (uL):: = 5' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t true \
        -h false \
        -exp 'output.::Total Volume (uL):: = (output.::Concentration::/step.::Desired Concentration (ng/uL)::) * output.::Sample Volume (uL):: ; \
output.::Low TE (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \
output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Total 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 = 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
  Desired Concentration (ng/uL)	Numeric		Range = 20 To 50 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	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Numeric	Required Field
    Derived Sample	Input Amount (ng)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Low TE (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Volume (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Total Volume (uL)	Numeric		Decimal Places Displayed = 2
    Project	Project Name	Built-in

Step 4: Amplify Targets Replicates (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

• Master Step Name = Amplify Targets Replicates (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • AmpliSeq Comprehensive Cancer Panel for Illumina

    • Supplier = Illumina

    • Catalog Number = 20019160

    • Website = www.illumina.com

  • AmpliSeq Library PLUS for Illumina

    • Supplier = Illumina

    • Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103

    • Website = www.illumina.com

Automations

Copy Concentration & Input Amount - CCP

• 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.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Total Volume (uL):: = input.::Total Volume (uL):: ; output.::Input Amount (ng):: = input.::Input Amount (ng):: ; output.::5X AmpliSeq HiFi Mix (uL):: = 9.0'\
        -log {compoundOutputFileLuid0}"

Calculate Prep Input

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t false \
        -h false \
        -exp 'output.::Prep Input Volume (uL):: = output.::Prep Input Amount (ng):: / output.::Concentration:: ; \
if (output.::Prep Input Volume (uL):: < 13.5) {output.::Nuclease-free water (uL):: = 13.5 - output.::Prep Input 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 = 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

Group of Defaults

CCP

• Recommended Prep Input (ng) = This protocol supports 1-100 ng per pool. The recommended input is 10 ng high-quality DNA per pool.

• Thermal Cycler Program = AMP_CCP

• Step Data

  • Group of Defaults = CCP

  • Master Step Fields

    Field Name	Field Type	Options	Additional Options and Dropdown Items
    20x AmpliSeq Sample ID Panel For Illumina	Toggle Switch		Default = None Set
    20x AmpliSeq Sample ID Panel For Illumina Lot#	Text
    Instructions	Multiline Text		Default = Add 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 1 (blue cap) to the first well, and 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 2 (blue cap) to the second.Add 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 3 (blue cap) to the third well, and 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 4 (blue cap) to the fourth.
    PCR Cycles	Numeric		Decimal Places Displayed = 0
    Recommended Prep Input (ng)	Multiline Text	Read Only	Default = This protocol supports 1-100 ng per pool. The recommended input is 10 ng high-quality DNA per pool.
    Thermal Cycler Program	Text Dropdown	Custom Entries	Presets AMP_CCP

• 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	5X AmpliSeq HiFi Mix (uL)	Numeric		Decimal Places Displayed = 1
    Derived Sample	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Input Amount (ng)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Nuclease-free water (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Prep Input Amount (ng)	Numeric	Required Field	Range = 2 To 200 Decimal Places Displayed = 0
    Derived Sample	Prep Input Volume (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Total Volume (uL)	Numeric		Decimal Places Displayed = 2
    Project	Project Name	Built-in

Step 5: Partially Digested Amplicons with Pooling (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

• Master Step Name = Partially Digested Amplicons with Pooling (AmpliSeq for Illumina v1.1)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

• Reagent Kits

  • AmpliSeq Library PLUS for Illumina

    • Supplier = Illumina

    • Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103

    • Website = www.illumina.com

Automations

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}"

Route AmpliSeq RNA Samples for Plate Transfer - Comp. Panel

• Trigger Location = Not Used

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 {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Queue for Plate Transfer' \
--FIELD_VALUE 'Transfer Amplicons' \
--WORKFLOW 'DNA Library Prep AmpliSeq for Illumina Comprehensive Panel v3 v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina Comprehensive Panel v3 v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' "

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

Pooling

• Label Uniqueness = Off

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

Placement = Enabled

• Defaults

  • Well Sort Order = Row

  • Placement Pattern = Column

• Destination Containers

  • 96 well plate

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Thermal Cycler Program	Text		Default = FUPA

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

Step 6: Ligate Indexes (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)

• Master Step Name = Ligate Indexes (AmpliSeq for Illumina CCP v1.1)

• Step Type = Add Labels

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}-{AppliedReagentLabels}

• Reagent Kits

  • AmpliSeq Library PLUS for Illumina

    • Supplier = Illumina

    • Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103

    • Website = www.illumina.com

Automations

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}"

Route AmpliSeq Samples - CCP

• Trigger Location = Step

• 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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Comprehensive Cancer Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Comprehensive Cancer Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Comprehensive Cancer Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The workflow version and step version for the routing script may vary depending on the version of the IPP.

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

Add Labels

• Label Groups

  • AmpliSeq CD Indexes Set A for Illumina

  • AmpliSeq CD Indexes Set B for Illumina

  • AmpliSeq CD Indexes Set C for Illumina

  • AmpliSeq CD Indexes Set D for Illumina

  • AmpliSeq UD Indexes for Illumina (24 Indexes)

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Thermal Cycler Program	Text		Default = LIGATE

• 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	AmpliSeq Workflow	Text Dropdown	Required Field	Preset Standard Workflow Equalizer Workflow
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in