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Library Preparation v1.1

PreviousCancer HotSpot Panel v2 NextEqualizer v1.1

Last updated 16 days ago

Overview

The Library Prep AmpliSeq for Illumina Cancer HotSpot Panel V2 is part of the AmpliSeq for Illumina Cancer HotSpot Panel V2 protocols that include the following functionality:

Preconfigured AmpliSeq for Illumina Cancer HotSpot 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.

Protocol 1: Library Prep (AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Dilute DNA (AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1)

Master Step Name = Dilute DNA (AmpliSeq for Illumina v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- AmpliSeq Library PLUS for Illumina
  - Supplier = Illumina
  - Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
  - Website = www.illumina.com

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
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
  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

Step 2: Qubit (AmpliSeq for Illumina Cancer HotSpot Panel V2 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
  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

Step 3: Dilute DNA to Final Concentration (AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1)

Master Step Name = Dilute DNA (AmpliSeq for Illumina v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- AmpliSeq Library PLUS for Illumina
  - Supplier = Illumina
  - Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
  - Website = www.illumina.com

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
  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

Step 4: Amplify Targets (AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1)

Master Step Name = Amplify Targets (AmpliSeq for Cancer Hotspot Illumina v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- AmpliSeq Cancer HotSpot Panel V2 for Illumina
  - Supplier = Illumina
  - Catalog Number = 20019161
- AmpliSeq Library PLUS for Illumina
  - Supplier = Illumina
  - Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
  - Website = www.illumina.com

Automations

Concentration,Total Volume and Input Amount - Cancer HotSpot v2

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):: = 4.0 ; output.::5X AmpliSeq Cancer HotSpot v2 Panel (uL):: = 4.0'\
        -log {compoundOutputFileLuid0}"

Calculate Prep Input - CH v2

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):: < 12) {output.::Nuclease-free water (uL):: = 12 - 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

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
PCR Cycles
Numeric
Decimal Places Displayed = 0
Recommended Prep Input (ng)
Multiline Text
Read Only
Default = This protocol supports 1-100 ng. The recommended input is 10 ng high-quality DNA.
Thermal Cycler Program
Text
Default = AMP
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 Cancer HotSpot v2 Panel (uL)
  Numeric
  Decimal Places Displayed = 1
  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