Illumina DNA PCR-Free Library Prep Manual v1.1

Overview

The Illumina DNA PCR-Free Library Prep Manual workflow includes the following functionality.

• Preset Illumina DNA PCR-Free Library Prep Manual protocols that support the preparation of up to 96 dual-indexed paired-end single-stranded libraries from DNA using the Illumina DNA PCR-Free Library Prep 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: Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Sort Sample (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Sort Sample (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = No Outputs

ℹ️ The version of Sort Sample master step name may be different depending on the version of IPP installed.

Automations

Set Next Step - DNA PCR-Free Lib Prep

• 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 'if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::gDNA:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Qubit - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::gDNA:: && input.::Protocol Type:: == ::Hybex::) {nextStep = ::Qubit - Hybex (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Whole Blood:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Dried Blood Spot:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Saliva:: && input.::Protocol Type::  == ::Thermal Cycler::) {nextStep = ::Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::}' \
-log {compoundOutputFileLuid0}"

ℹ️ The version of the nextStep step names may be different depending on the version of IPP installed.

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
  Instruction Notes	Multiline Text	Read Only	Default = Select a sample type and protocol type for each sample.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • 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	Protocol Type	Text Dropdown	Required Field	Presets Thermal Cycler Hybex Default = Thermal Cycler
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Type for DNA PCR-Free Lib Prep	Text Dropdown	Required Field	Presets gDNA Whole Blood Dried Blood Spot Saliva Default = gDNA
    Project	Project Name	Built-in

Step 2: Qubit - Hybex (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Qubit - Hybex (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

ℹ️ The version of Qubit - Hybex master step name may be different depending on the version of IPP installed.

Automations

Copy Sample Type for DNA PCR-Free Lib Prep

• 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.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep:: ' \
-log {compoundOutputFileLuid0}"

Assign QC flags

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

Set Next Step-REMOVE and Copy Concentration & Sample Type

• 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 = ::REMOVE:: ; input.::Concentration:: = output.::Concentration:: ; input.::Conc. Units:: = output.::Conc. Units:: ; input.::Sample Type:: = output.::Sample Type::' -log {compoundOutputFileLuid0}"

Routing - gDNA Hybex

• Trigger Location = Step

• Trigger Style = Automatic upon exit

bash -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 'Sample Type' \
--FIELD_VALUE 'gDNA' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'INPUTS'"

ℹ️ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.

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
  Criteria 1 - Operator	Text		Default = >=
  Criteria 1 - Source Data Field	Text		Default = Concentration
  Criteria 1 - Threshold Value	Numeric		Default = 300 Range = 300 To 2000
  Criteria 2 - Operator	Text		Default = <=
  Criteria 2 - Source Data Field	Text		Default = Concentration
  Criteria 2 - Threshold Value	Numeric		Default = 2000 Range = 300 To 2000
  Instruction Notes	Multiline Text	Read Only	Default = 1. Upload or enter Concentration and Conc. Units. 2. Set Threshold Values then click on Assign QC Flags.

• 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 = Column

  • File Column Options

    • File Column Display = Show

    • File Attachment Method = Manual

  • 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
    Derived Sample	Sample Type	Text Dropdown	Required Field Custom Entries	Presets DNA RNA
    Measurement	Concentration	Numeric		Decimal Places Displayed = 2
    Measurement	Conc. Units	Text
    Measurement	Sample Type	Text
    Project	Project Name	Built-in

Step 3: Qubit - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Qubit - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

ℹ️ The version of Qubit - Thermal Cycler master step name may be different depending on the version of IPP installed.

Automations

Copy Sample Type for DNA PCR-Free Lib Prep

• 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.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep:: ' \
-log {compoundOutputFileLuid0}"

Assign QC flags

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

Set Next Step-REMOVE, Set Prep Input Type, Copy Concentration & Sample Type

• 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 -exp 'nextStep = ::REMOVE:: ; \
if (output.::Concentration:: >= 25 && output.::Concentration:: <= 99) {output.::Prep Input Type:: = ::Low::} ; \
if (output.::Concentration:: >= 100 && output.::Concentration:: <= 2000) {output.::Prep Input Type:: = ::Standard::} ; \
input.::Concentration:: = output.::Concentration:: ; input.::Conc. Units:: = output.::Conc. Units:: ; input.::Sample Type:: = output.::Sample Type::' -log {compoundOutputFileLuid0}"

Routing - gDNA Thermal Cycler

• 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 'Prep Input Type' \
--FIELD_VALUE 'Low' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Prep Input Type' \
--FIELD_VALUE 'Standard' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'INPUTS'"

ℹ️ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.

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
  Criteria 1 - Operator	Text		Default = >=
  Criteria 1 - Source Data Field	Text		Default = Concentration
  Criteria 1 - Threshold Value	Numeric		Default = 25
  Criteria 2 - Operator	Text		Default = <=
  Criteria 2 - Source Data Field	Text		Default = Concentration
  Criteria 2 - Threshold Value	Numeric		Default = 2000
  Instruction Notes	Multiline Text	Read Only	Default = 1. Upload or enter Concentration and Conc. Units. 2. Set Threshold Values then click on Assign QC Flags.

• 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 = Column

  • File Column Options

    • File Column Display = Show

    • File Attachment Method = Manual

  • 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	Prep Input Type	Text
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Type	Text Dropdown	Required Field Custom Entries	Presets DNA RNA
    Measurement	Concentration	Numeric		Decimal Places Displayed = 2
    Measurement	Conc. Units	Text
    Measurement	Prep Input Type	Text
    Measurement	Sample Type	Text
    Project	Project Name	Built-in

Step 4: Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina Lysis Reagent Kit

    • Supplier = Illumina

    • Catalog Number = 20042221

    • Website = www.illumina.com

ℹ️ The version of Whole Blood Lysis master step name may be different depending on the version of IPP installed.

Automations

Calculate Whole Blood Lysis Master Mix & Copy Sample Type

• 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 'step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::MLB (uL):: = step.::Total Samples:: * 30 *1.1 ; \
step.:: PK1 (uL):: = step.::Total Samples:: * 2 * 1.1 ; \
step.:: Nuclease-free water (uL):: = step.::Total Samples:: * 248 * 1.1 ; \
output.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Remove

• 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 = ::REMOVE::' -log {compoundOutputFileLuid0}"

Routing - Whole Blood Lysis

• 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 'Sample Type' \
--FIELD_VALUE 'Whole Blood' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.

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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • 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
  80% EtOH Prep Date	Date
  Instruction Notes	Multiline Text	Read Only	Default = 1. Combine MLB, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube. 2. Add 280 µl of lysis master mix to a new 2 mL tube. 3. Add 20 uL to each 2 mL tube, mix by pipetting then vortex briefly and centrifuge. 4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly. 5. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix. 6. Incubate at RT for 5 mins. 7. Place tube on magnetic stand for 5 mins then remove and discard supernatant. 8. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash. 9. Air dry on magnetic stand for 5 mins. 10. Add 35 uL of RSB to each tube and vortex to mix. 11. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
  MLB (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Nuclease-free water (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  PK1 (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Total Samples	Numeric	Read Only	Default = 0 Decimal Places Displayed = 0

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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
    Derived Sample	Sample Type	Text Dropdown	Required Field Custom Entries	Presets DNA RNA
    Project	Project Name	Built-in

Step 5: Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina Lysis Reagent Kit

    • Supplier = Illumina

    • Catalog Number = 20042221

    • Website = www.illumina.com

ℹ️ The version of Dried Blood Spot Lysis master step name may be different depending on the version of IPP installed.

Automations

Calculate Dried Blood Lysis Master Mix

• 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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::10x Lysis Buffer (uL):: = step.::Total Samples:: * 30 *1.1 ; \
step.:: PK1 (uL):: = step.::Total Samples:: * 2 * 1.1 ; \
step.:: Nuclease-free water (uL):: = step.::Total Samples:: * 268 * 1.1 ; \
output.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Remove

• 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 = ::REMOVE::' -log {compoundOutputFileLuid0}"

Routing - Dried Blood Spot Lysis

• 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 'Sample Type' \
--FIELD_VALUE 'Dried Blood Spot' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.

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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • 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
  10x Lysis Buffer (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  80% EtOH Prep Date	Date
  Instruction Notes	Multiline Text	Read Only	Default = 1. Combine 10x Lysis Buffer, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube. 2. Add 300 uL of lysis master mix to a new 2 mL tube. 3. Add 6 x 3 mm² punches to a each 2 ml tube, mix by pipetting then vortex briefly and centrifuge. 4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly. 5. Without removing the punches, transfer all supernatant from each tube to a new 2 ml tube. 6. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix. 7. Incubate at RT for 5 mins. 8. Place tube on magnetic stand for 5 mins then remove and discard supernatant. 9. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash. 10. Air dry on magnetic stand for 5 mins. 11. Add 35 uL of RSB to each tube and vortex to mix. 12. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear. 13. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
  Nuclease-free water (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  PK1 (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Total Samples	Numeric	Read Only	Default = 0 Decimal Places Displayed = 0

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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
    Derived Sample	Sample Type	Text Dropdown	Required Field Custom Entries	Presets DNA RNA
    Project	Project Name	Built-in

Step 6: Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina Lysis Reagent Kit

    • Supplier = Illumina

    • Catalog Number = 20042221

    • Website = www.illumina.com

ℹ️ The version of Saliva Lysis master step name may be different depending on the version of IPP installed.

Automations

Copy Sample Type for DNA PCR-Free Lib Prep

• 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.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep:: ' \
-log {compoundOutputFileLuid0}"

Set Next Step - Remove

• 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 = ::REMOVE::' -log {compoundOutputFileLuid0}"

Routing - Saliva Lysis

• 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 'Sample Type' \
--FIELD_VALUE 'Saliva' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.

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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • 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
  80% EtOH Prep Date	Date
  Instruction Notes	Multiline Text	Read Only	Default = 1. Incubate saliva overnight at 50°C in a water bath or air incubator. 2. Add 250 uL of Nuclease-Free Water to a new 2 mL tube. 3. Invert each heat-treated saliva collection tube 5 times to mix, add 50 uL of Saliva to each 2 mL tube, pipette to mix, vortex briefly and then centrifuge. 4. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix. 5. Incubate at RT for 5 mins. 6. Place tube on magnetic stand for 5 mins then remove and discard supernatant. 7. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash. 8. Air dry on magnetic stand for 5 mins. 9. Add 35 uL of RSB to each tube and vortex to mix. 10. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear. 11. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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
    Derived Sample	Sample Type	Text Dropdown	Required Field Custom Entries	Presets DNA RNA
    Project	Project Name	Built-in

Protocol 2: Library Prep - Hybex (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Tagment Genomic DNA (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Tagment Genomic DNA (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Tagment Genomic DNA master step name may be different depending on the version of IPP installed.

Automations

Copy Concentration

• 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::' \
-log {compoundOutputFileLuid0}"

Calculate DNA Volume & Copy Desired DNA 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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'output.::DNA (uL):: = (step.::Desired DNA Input (ng):: / output.::Concentration::) ; \
output.::RSB (uL):: = (25 - output.::DNA (uL)::) ; \
output.::Desired DNA Input (ng):: = step.::Desired DNA Input (ng)::' \
-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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • 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
  80% EtOH Prep Date	Date
  Desired DNA Input (ng)	Numeric		Range = 300 To 2000 Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = 1. Label a new 96 well MIDI plate with LP1 and add the calculated DNA and RSB volumes of each sample to a new well on the plate. 2. Add 10 uL of TB1 to each well. 3. Add 15 uL of BLT-PF to each well. 4. Seal and shake at 1800 rpm for 1 minute. 5. Incubate in pre-heated Hybex for 8 minutes.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	DNA (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 2: Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = No Outputs

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Post Tagmentation Cleanup master step name may be different depending on the version of IPP installed.

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}"

Copy Desired DNA Input

• 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 'output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-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
  Instruction Notes	Multiline Text	Read Only	Default = 1. Add 10 µl ST2 to each well then seal and shake at 1800 rpm for 1 minute. 2. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 3. Without disturbing the bead pellet, remove and discard all supernatant from each well. 5. Add 150 μl TWB to each well then seal and shake at 1800 rpm for 1 minute. 6. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 3: Ligate Indexes (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Ligate Indexes (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Add Labels

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}-{AppliedReagentLabels}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Ligate Indexes master step name may be different depending on the version of IPP installed.

Automations

Calculate Dilution for HP3 & Copy Desired DNA Input

• 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng):: ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::HP3 (uL):: = step.::Total Samples:: * 6 ; \
step.::Nuclease-free water (uL):: = step.::Total Samples:: * 54' \
-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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • Placement Pattern = Column

• Destination Containers

  • 96 well plate

Add Labels

• Label Groups

  • IDT for Illumina Nextera DNA UD Indexes Set A for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set A-D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • Illumina DNA-RNA UD Indexes Set A B C D Tagmentation

  • Illumina DNA-RNA UD Indexes Set A Tagmentation

  • Illumina DNA-RNA UD Indexes Set B Tagmentation

  • Illumina DNA-RNA UD Indexes Set C Tagmentation

  • Illumina DNA-RNA UD Indexes Set D Tagmentation

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  HP3 (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Instruction Notes for Hybex Protocol	Multiline Text	Read Only	Default = 1. Remove and discard all supernatant. 2. Add 45 µl ELM to each well. 3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min. 4. Incubate in the preheated Hybex for 8 mins. 5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 6. Remove and discard all supernatant from each well. 7. Add 75 µl TWB onto the beads in each well then seal and shake at 1800 rpm for 1 minute. 8. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 9. Remove and discard all supernatant from each well. 10. Without disturbing the bead pellet, use a 20 µl pipette to remove and discard residual TWB from each well. 11. Add 45 µl diluted HP3 to each well then seal and shake at 1800 rpm for 1 minute.
  Nuclease-free water (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Total Samples	Numeric	Read Only	Default = 0 Decimal Places Displayed = 0

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 4: Clean Up Libraries (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Clean Up Libraries (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Clean Up Libraries master step name may be different depending on the version of IPP installed.

Automations

Copy Desired DNA Input

• 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-log {compoundOutputFileLuid0}"

Set Next Step-Remove & Prep Input Type

• 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 -exp 'nextStep = ::REMOVE:: ; \
output.::Prep Input Type:: = ::Standard::' -log {compoundOutputFileLuid0}"

Routing - Quantify & Pool Libraries

• Trigger Location = Step

• Trigger Style = Automatic upon exit

bash -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 'Prep Input Type' \
--FIELD_VALUE 'Standard' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Prep Input Type' \
--FIELD_VALUE 'Low' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The actual version of the workflows and steps in the routing automation script may be different depending on the version of IPP installed.

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
  80% EtOH Prep Date	Date
  Instruction Notes	Multiline Text	Read Only	Default = 1. Add 36 µl IPB to each well then seal and shake at 1800 rpm for 1 minute. 2. Incubate at room temperature for 2 minutes. 3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes). 4. Label a new 96-well MIDI plate LP2. 5. Add 42 µl IPB to each well of LP2. 6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of LP2 then seal and shake at 1800 rpm for 1 minute. 7. Discard LP1 and incubate LP2 at room temperature for 2 minutes. 8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes). 9. Without disturbing the bead pellet, remove and discard all supernatant from each well. 10. Wash beads 2x with 180 uL of fresh 80% EtOH for each wash. 11. Using a 20 µl pipette, remove residual EtOH from each well. 12. Air-dry on the magnetic stand (~4 minutes). 13. Add 22 µl of RSB onto the beads in each well then seal and shake at 1800 rpm for 1 minute. 14. Incubate at room temperature for 2 minutes. 15. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 16. Label a new PCR plate FLP. 17. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.

• Step File Placeholders

  • Log - 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Prep Input Type	Text
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 3: Library Prep - Thermal Cycler Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Tagment Genomic DNA - Standard Input v1.0.2

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Tagment Genomic DNA - Standard Input master step name may be different depending on the version of IPP installed.

Automations

Copy Sample Type & Concentration

• 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.::Sample Type:: = input.::Sample Type:: ; \
if (output.::Sample Type:: == ::gDNA::) {output.::Concentration:: = input.::Concentration::} else {output.::Concentration:: = 0} ; \
if (output.::Sample Type:: == ::gDNA::) {output.::Conc. Units:: = input.::Conc. Units::} else {output.::Conc. Units:: = ::NA::}' \
-log {compoundOutputFileLuid0}"

Calculate Dilution & BLT-PF Volumes and Copy Desired DNA 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.::Desired DNA Input (ng):: = step.::Desired DNA Input (ng):: ; \
if (output.::Sample Type:: == ::gDNA::) {output.::DNA (uL):: = output.::Desired DNA Input (ng):: / output.::Concentration::} ; \
if (output.::Sample Type:: == ::Whole Blood::) {output.::DNA (uL):: = 30} ; \
if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::DNA (uL):: = 30} ; \
if (output.::Sample Type:: == ::Saliva::) {output.::DNA (uL):: = 30} ; \
if (output.::Sample Type:: == ::gDNA::) {output.::RSB (uL):: = 25 - output.::DNA (uL)::} ; \
if (output.::Sample Type:: == ::Whole Blood::) {output.::RSB (uL):: = 0} ; \
if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::RSB (uL):: = 0} ; \
if (output.::Sample Type:: == ::Saliva::) {output.::RSB (uL):: = 0} ; \
if (output.::Sample Type:: == ::gDNA::) {output.::BLT-PF (uL):: = 15} ; \
if (output.::Sample Type:: == ::Whole Blood::) {output.::BLT-PF (uL):: = 10} ; \
if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::BLT-PF (uL):: = 10} ; \
if (output.::Sample Type:: == ::Saliva::) {output.::BLT-PF (uL):: = 10}' \
-log {compoundOutputFileLuid0}"

Set Next Step-Post TAG

• 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 true -h false \
-exp 'if (input.::Sample Type:: == ::Whole Blood::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type:: == ::Dried Blood Spot::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type:: == ::Saliva::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type:: == ::gDNA::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::}' \
-log {compoundOutputFileLuid0}"

ℹ️ The actual version of the nextStep step names may be different depending on the version of IPP installed.

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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • 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
  80% EtOH Prep Date	Date
  Desired DNA Input (ng)	Numeric	Required Field	Range = 100 To 2000
  Instruction Notes	Multiline Text	Read Only	Default = 1. Label a new 96 well PCR plate LP1. 2. Add the calculated DNA, and RSB volumes to the LP1 plate and then pipette to mix. 3. Add the calculated BLT-PF to each well and pipette to mix. 4. Add 10 uL of TB1 to each well, pipette to mix and then seal. 5. Place the LP1 plate on the thermo cycler and run the TAG program.
  Thermal Cycler Program	Text		Default = TAG
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = 1. Choose the preheat lid option and set to 100°C 2. Set the reaction volume to 50 µl u 41°C for 5 minutes.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • 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	BLT-PF (uL)	Numeric
    Derived Sample	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	DNA (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Type	Text Dropdown	Requird Field Custom Entries	Presets DNA RNA
    Project	Project Name	Built-in

Step 2: Tagment Genomic DNA - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Tagment Genomic DNA - Low Input v1.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

Automations

Calculate Tag Master Mix and Copy Concentration & Sample Type

• 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 Type:: = input.::Sample Type:: ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::BLT-PF (uL):: = step.::Total Samples:: * 11 ; \
step.::TB1 (uL):: = step.::Total Samples:: * 11' \
-log {compoundOutputFileLuid0}"

Calculate Dilution and Copy Desired DNA 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.::Desired DNA Input (ng):: = step.::Desired DNA Input (ng):: ; \
output.::DNA (uL):: = output.::Desired DNA Input (ng):: / output.::Concentration:: ; \
output.::RSB (uL):: = 30 - output.::DNA (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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • 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
  80% EtOH Prep Date	Date
  BLT-PF (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Desired DNA Input (ng)	Numeric	Required Field	Default = 25 Range = 25 To 99</li
  Instruction Notes	Multiline Text	Read Only	Default = 1. Label a new 96 well MIDI plate LP1. 2. Combine the calculated BLT-PF and TB1 volumes into a 1.5 mL tube to prepare the Tagmentation Master Mix. 3. Add the calculated DNA, and RSB volumes to the LP1 plate and then pipette to mix. 4. Add 20 µl Tagmentation Master Mix to each well, pipette to mix and then seal. 5. Place the LP1 plate on the thermo cycler and run the TAG program.
  TB1 (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Thermal Cycler Program	Text		Default = TAG
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = 1. Choose the preheat lid option and set to 100°C 2. Set the reaction volume to 50 µl u 41°C for 5 minutes.
  Total Samples	Numeric	Read Only	Default = 0 Decimal Places Displayed = 0

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	DNA (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Type	Text Dropdown	Requird Field Custom Entries	Presets DNA RNA
    Project	Project Name	Built-in

Step 3: Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Post Tagmentation Cleanup v1.0.1

• Step Type = No Outputs

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Post Tagmentation Cleanup master step name may be different depending on the version of IPP installed.

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}"

Copy Desired DNA Input & Sample Type

• 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 'output.::Sample Type:: = input.::Sample Type:: ; \
output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-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
  Instruction Notes	Multiline Text	Read Only	Default = 1. Add 10 µl ST2 to each well, seal and then shake at 1800 rpm for 1 minute. 2. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 3. Without disturbing the bead pellet, remove and discard all supernatant from each well. 4. Add 150 μl TWB to each well, seal and shake at 1800 rpm for 1 minute. 5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Type	Text Dropdown	Requird Field Custom Entries	Presets DNA RNA
    Project	Project Name	Built-in

Step 4: Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Add Labels

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}-{AppliedReagentLabels}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.

Automations

Calculate Dilution for HP3 & Copy Desired DNA Input

• 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng):: ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::HP3 (uL):: = step.::Total Samples:: * 6 ; \
step.::Nuclease-free water (uL):: = step.::Total Samples:: * 54' \
-log {compoundOutputFileLuid0}"

Set Next Step - Clean Up Libraries

• 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 true -h false \
-exp 'if (output.::Desired DNA Input (ng):: >= 100) {nextStep = ::Clean Up Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (output.::Desired DNA Input (ng):: <= 99) {nextStep = ::Clean Up Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::}' \
-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

Add Labels

• Label Groups

  • IDT for Illumina Nextera DNA UD Indexes Set A for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set A-D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000

  • Illumina DNA-RNA UD Indexes Set A B C D Tagmentation

  • Illumina DNA-RNA UD Indexes Set A Tagmentation

  • Illumina DNA-RNA UD Indexes Set B Tagmentation

  • Illumina DNA-RNA UD Indexes Set C Tagmentation

  • Illumina DNA-RNA UD Indexes Set D Tagmentation

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  HP3 (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = 1. Remove and discard all supernatant. 2. Add 45 µl ELM to each well and pipette to mix. 3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min. 4. Place on the preprogrammed thermal cycler and run the ELM program. 5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 6. Remove and discard all supernatant from each well. 7. Add 75 µl TWB onto the beads in each well then pipette to mix. 8. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 9. Remove and discard all supernatant from each well. 10. Seal and centrifuge at 280 x g for 10 seconds and then place on magnetic stand. 11. Without disturbing the bead pellet, remove and discard residual TWB from each well. 12. Remove the plate from the magnetic stand. 13. Add 45 µl diluted HP3 to each well, pipette to mix and then incubate at RT for 2 mins.
  Nuclease-free water (uL)	Numeric	Read Only	Decimal Places Displayed = 0
  Thermal Cycler Program	Text		Default = ELM
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = ELM Program 1. Choose the preheat lid option and set to 100°C 2. Set the reaction volume to 50 μl 3. Run for 37°C for 5 minutes 4. Run for 50°C for 5 minutes
  Total Samples	Numeric	Read Only	Default = 0 Decimal Places Displayed = 0

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 5: Clean Up Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Clean Up Libraries - Thermal Cycler v1.0.2

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Clean Up Libraries - Thermal Cycler master step name may be different depending on the version of IPP installed.

Automations

Copy Desired DNA Input

• 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-log {compoundOutputFileLuid0}"

Set Next Step-Remove & Prep Input Type

• 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 -exp 'nextStep = ::REMOVE:: ; \
if (output.::Desired DNA Input (ng):: >= 300) {output.::Prep Input Type:: = ::Standard::} else {output.::Prep Input Type:: = ::Low::}' \
-log {compoundOutputFileLuid0}"

Routing - Quantify & Pool Libraries

• Trigger Location = Step

• Trigger Style = Automatic upon exit

bash -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 'Prep Input Type' \
--FIELD_VALUE 'Standard' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Prep Input Type' \
--FIELD_VALUE 'Low' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.

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
  80% EtOH Prep Date	Date
  Instruction Notes for Standard Input	Multiline Text	Read Only	Default = 1. Add 36 µl IPB to each well containing BLT-PF beads and pipette to mix. 2. Incubate at room temperature for 2 minutes. 3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes). 4. Label a new 96-well PCR plate LP2. 5. Add 42 µl IPB to each well of LP2. 6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of the LP2, pipette to mix and remove LP1 from the magnetic stand, and then discard. 7. Incubate LP2 at room temperature for 2 minutes. 8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes). 9. Without disturbing the bead pellet, remove and discard all supernatant from each well. 10. Wash beads 2x with 180 µl fresh 80% ethanol per well for each wash. 11. Seal and then centrifuge 280 x g for 10 seconds. 12. Place on the magnetic stand, and then wait 10 seconds. 13. Remove residual EtOH from each well, air-dry on the magnetic stand (~2 minutes) then remove from the magnetic stand. 14. Add 22 µl RSB onto the beads in each well and pipette to mix. 15. Incubate at room temperature for 2 minutes. 16. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 17. Label a new PCR plate FLP. 18. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Prep Input Type	Text
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 6: Clean Up Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Clean Up Libraries - Thermal Cycler v1.0.2

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Clean Up Libraries - Thermal Cycler master step name may be different depending on the version of IPP installed.

Automations

Copy Desired DNA Input

• 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-log {compoundOutputFileLuid0}"

Set Next Step-Remove & Prep Input Type

• 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 -exp 'nextStep = ::REMOVE:: ; \
if (output.::Desired DNA Input (ng):: >= 300) {output.::Prep Input Type:: = ::Standard::} else {output.::Prep Input Type:: = ::Low::}' \
-log {compoundOutputFileLuid0}"

Routing - Quantify & Pool Libraries

• Trigger Location = Step

• Trigger Style = Automatic upon exit

bash -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 'Prep Input Type' \
--FIELD_VALUE 'Standard' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Prep Input Type' \
--FIELD_VALUE 'Low' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.

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
  80% EtOH Prep Date	Date
  Instruction Notes for Low Input	Multiline Text	Read Only	Default = 1. Add 81 µl IPB to each well and pipette to mix. 2. Incubate at room temperature for 5 minutes. 3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes). 4. Remove and discard all supernatant from each well. 5. Wash beads 2x with 180 uL of fresh 80% EtOH for each wash. 6. Seal plate then centrifuge 280 x g for 10 seconds. 7. Place on the magnetic stand and wait until the liquid is clear (~5 minutes). 8. Remove residual EtOH from each well and air-dry on the magnetic stand (~2 minutes). 9. Remove the plate from the magnetic stand. 10. Add 15 µl RSB onto the beads in each well and pipette to resuspend. 11. Incubate at room temperature for 2 minutes. 12. Place on the magnetic stand and wait until the liquid is clear (~2 minutes). 13. Transfer 14 µl supernatant from each well of the plate to the corresponding well of a new PCR plate.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Prep Input Type	Text
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 4: Quantify and Pool Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Pool Samples (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

• Reagent Kits

  • Illumina DNA PCR-Free Library Prep

    • Supplier = Illumina

    • Catalog Number = 24 - 20041794; 96 - 20041795

    • Website = www.illumina.com

ℹ️ The version of Pool Samples (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.

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}"

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

Pooling

• Label Uniqueness = On

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Instruction Notes	Multiline Text	Read Only	Default = 1. Combine 9 µl of each library in a 1.5 or 1.7 ml microcentrifuge tube. 2. Vortex to mix, and then centrifuge at 280 x g for 1 minute.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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 2: Qubit (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Qubit (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

ℹ️ The version of Qubit (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.

Automations

Assign QC flags

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

Set Next Step-Remove 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 = ::REMOVE:: ; input.::Concentration:: = output.::Concentration:: ; input.::Conc. Units:: = output.::Conc. Units::' -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
  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 = Column

  • 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

Protocol 5: Quantify and Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = KAPA Sample Prep - Illumina/Universal v1.0.1

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • 0.05% Tween® 20

  • 10 mM Tris-HCl, pH 8.0 – 8.5 (25°C)

• Control Types

  • KAPA DNA Standard 1

    • Supplier = Roche

    • Catalog Number = KK4828-07960166001

    • Website = www.roche.com

    • Conc. = 20 pM

    • Control Use

      • Single step only = No

  • KAPA DNA Standard 2

    • Supplier = Roche

    • Catalog Number = KK4828-07960166001

    • Website = www.roche.com

    • Conc. = 2 pM

    • Control Use

      • Single step only = No

  • KAPA DNA Standard 3

    • Supplier = Roche

    • Catalog Number = KK4828-07960166001

    • Website = www.roche.com

    • Conc. = 0.2 pM

    • Control Use

      • Single step only = No

  • KAPA DNA Standard 4

    • Supplier = Roche

    • Catalog Number = KK4828-07960166001

    • Website = www.roche.com

    • Conc. = 0.02 pM

    • Control Use

      • Single step only = No

  • KAPA DNA Standard 5

    • Supplier = Roche

    • Catalog Number = KK4828-07960166001

    • Website = www.roche.com

    • Conc. = 0.002 pM

    • Control Use

      • Single step only = No

  • KAPA DNA Standard 6

    • Supplier = Roche

    • Catalog Number = KK4828-07960166001

    • Website = www.roche.com

    • Conc. = 0.0002

    • Control Use

      • Single step only = No

  • KAPA Library Quantification Dilution Control

    • Supplier = Roche

    • Catalog Number = KK4906

    • Website = www.roche.com

    • Conc. = 200 pM

    • Control Use

      • Single step only = No

  • KAPA NTC

    • Control Use

      • Single step only = No

ℹ️ The version of KAPA Sample Prep - Illumina/Universal master step name may be different depending on the version of IPP installed.

Automations

Copy Desired DNA Input for KAPA

• 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 'if (output.name.contains(::KAPA DNA Standard 1::)) {output.::Desired DNA Input (ng):: = 0.005952} ; \
if (output.name.contains(::KAPA DNA Standard 2::)) {output.::Desired DNA Input (ng):: = 0.000595}  ; \
if (output.name.contains(::KAPA DNA Standard 3::)) {output.::Desired DNA Input (ng):: = 0.00006}  ; \
if (output.name.contains(::KAPA DNA Standard 4::)) {output.::Desired DNA Input (ng):: = 0.000006}  ; \
if (output.name.contains(::KAPA DNA Standard 5::)) {output.::Desired DNA Input (ng):: = 0.0000006}  ; \
if (output.name.contains(::KAPA DNA Standard 6::)) {output.::Desired DNA Input (ng):: = 0.00000006}  ; \
if (output.name.contains(::KAPA Library Quantification Dilution Control::)) {output.::Desired DNA Input (ng):: = 0.059524} ; \
if (output.name.contains(::KAPA NTC::)) {output.::Desired DNA Input (ng):: = 0} ; \
if (!output.name.contains(::KAPA::)) {output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::}' \
-log {compoundOutputFileLuid0}"

Calculate Expected Concentration & Dilution Factor

• 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 'output.::Expected Concentration (pM):: = output.::Desired DNA Input (ng):: * 3.36 * 1000 ; \
if (output.::Expected Concentration (pM):: >= 84000 && output.::Expected Concentration (pM):: <= 188000) {output.::Dilution Factor:: = 10000} ; \
if (output.::Expected Concentration (pM):: >= 189000 && output.::Expected Concentration (pM):: <= 333000) {output.::Dilution Factor:: = 20000} ; \
if (output.::Expected Concentration (pM):: <= 83000) {output.::Dilution Factor:: = 0}' \
-log {compoundOutputFileLuid0} && /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 (output.::Expected Concentration (pM):: > 333000) {fail(::Expected Concentration is too high, please edit Dilution Factor and recalculate::)}' \
-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
  DNA Dilution Buffer Prep Date	Date
  Instruction Notes	Multiline Text	Read Only	Default = - Prepare the appropriate library dilutions (using DNA dilution buffer). - Depending on the expected concentration of the library, 1:1,000 – 1:100,000 dilutions may be appropriate.

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • 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	Desired DNA Input (ng)	Numeric Dropdown	Custom Entries	Presets 99 50 25 0.005952 0.000595 0.00006 0.000006 6e-7 6e-8 0.059524
    Derived Sample	Dilution Factor	Text Dropdown	Required Field Custom Entries	Presets 1:10000 1:20000
    Derived Sample	Expected Concentration (pM)	Numeric		Decimal Places Displayed = 4
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 2: KAPA qPCR Prep & Analysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = KAPA qPCR Prep & Analysis - Illumina/Universal v1.0

• Step Type = Standard QC

• Measurement Generation = Variable

• Naming Convention = {InputItemName}

• Reagent Kits

  • KAPA qPCR library quantification kit - Illumina/Universal

    • Supplier = Roche

    • Catalog Number = 07960166001

    • Website = www.roche.com

Automations

Assign QC flags

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

Calculate Average Cq

• 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:computeReplicateAverage \
 -src 'Cq' \
 -dest 'Average Cq' \
 -log {compoundOutputFileLuid0}"

Calculate qPCR Master Mix & Sample Volume

• 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 'step.::Total Samples:: = step.::Total Samples:: + 1 ; \
if (step.::Reaction Volume (uL):: == 20) {step.::KAPA SYBR FAST qPCR Master Mix + Primer Premix (uL):: = step.::Total Samples:: * 12} else {step.::KAPA SYBR FAST qPCR Master Mix + Primer Premix (uL):: = step.::Total Samples:: * 6} ; \
if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::Yes::) {step.::ROX (uL):: = step.::Total Samples:: * 0.4} ; \
if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::No::) {step.::ROX (uL):: = 0} ; \
if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::Yes::) {step.::ROX (uL):: = step.::Total Samples:: * 0.2} ; \
if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::No::) {step.::ROX (uL):: = 0} ; \
if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::Yes::) {step.::PCR-grade water (uL):: = step.::Total Samples:: * 3.6} ; \
if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::No::) {step.::PCR-grade water (uL):: = step.::Total Samples:: * 4} ; \
if (step.::Reaction Volume (uL):: == 10) {step.::PCR-grade water (uL):: = 0} ; \
if (step.::Reaction Volume (uL):: == 20) {output.::qPCR master mix (uL):: = 16} ; \
if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::Yes::) {output.::qPCR master mix (uL):: = 6.2} ; \
if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::No::) {output.::qPCR master mix (uL):: = 6} ; \
output.::Sample (uL):: = 4' \
-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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • 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
  Adding Rox?	Text Dropdown		Presets Yes No Default = Yes
  Criteria 1 - Operator	Text		Default = >=
  Criteria 1 - Source Data Field	Text		Default = Cq
  Criteria 1 - Threshold Value	Numeric		Default = 7
  Criteria 2 - Operator	Text		Default = <=
  Criteria 2 - Source Data Field	Text		Default = Cq
  Criteria 2 - Threshold Value	Numeric		Default = 25
  Instruction Notes	Multiline Text	Read Only	Default = 1. Set reaction volume and adding Rox? then click on calculate qPCR Master Mix and Sample Volume button. 2. Combine KAPA SYBR FAST qPCR Master Mix + Primer Premix, ROX and PCR-grade water to produce the qPCR Master Mix. 3. Add the qPCR Master Mix and Sample volume to a new 96 well PCR plate, seal the plate, vortex briefly to mix and then centrifuge. 4. Place the plate on the qPCR instrument. 5. Select Absolute Quantification and run the following cycling protocol: - Initial denaturation Temp. at 95C fro 5 mins for 1 cycle - Denaturation Temp. at 95C for 30 secs for 35 cycles - Annealing/Extension/Data acquisition Temp. at 95C for 45 sec for 35 cycles - Melt curve analysis (C) between 65C - 95C 6. Upload Cq values 7. Set Cq Thresholds then click on Assign QC Flags button. 8. Review Cq values and remove outliers. 9. Click on Calculate Average Cq.
  KAPA SYBR FAST qPCR Master Mix + Primer Premix (uL)	Numeric	Read Only	Decimal Places Displayed = 2
  PCR-grade water (uL)	Numeric	Read Only	Decimal Places Displayed = 2
  qPCR Master Mix with Primer Prep Date	Date
  Reaction Volume (uL)	Numeric Dropdown		Presets 20 10 Default = 20 Decimal Places Displayed = 0
  ROX (uL)	Numeric	Read Only	Decimal Places Displayed = 2
  Total Samples	Numeric	Read Only	Default = 0

• 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 = Column

  • 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	Average Cq	Numeric		Decimal Places Displayed = 0
    Derived Sample	Cq	Numeric		Decimal Places Displayed = 2
    Derived Sample	Dilution Factor	Text Dropdown	Required Field Custom Entries	Presets 1:10000 1:20000
    Derived Sample	qPCR master mix (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample (uL)	Numeric		Decimal Places Displayed = 0
    Measurement	Cq	Numeric		Decimal Places Displayed = 2
    Measurement	qPCR master mix (uL)	Numeric
    Measurement	Sample (uL)	Numeric
    Project	Project Name	Built-in

Step 3: KAPA Library Quantification (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = KAPA Library Quantification - Illumina/Universal v1.0

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Calculate Library Length Ratio & Copy Dilution Factor and Average Cq

• 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 'step.::Library Length Ratio:: = 452 / step.::Library Length (bp):: ; \
output.::Average Cq:: = input.::Average Cq:: ; \
output.::Dilution Factor:: = input.::Dilution Factor::' \
-log {compoundOutputFileLuid0}"

Assign QC flags

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

Calculate Concentration

• 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.::Diluted Avg. Conc. (pM)::  = 10 ** ((output.::Average Cq:: - step.::Standard Curve Y-Intercept::) / step.::Standard Curve Slope::) ; \
if (output.::Dilution Factor::.toFloat() >= 1) {output.::Concentration (pM):: = output.::Diluted Avg. Conc. (pM):: * step.::Library Length Ratio:: * output.::Dilution Factor::.toFloat()} else {output.::Concentration (pM):: = output.::Diluted Avg. Conc. (pM):: * step.::Library Length Ratio::} ; \
output.::Concentration (nM):: = 0.001 * output.::Concentration (pM)::' \
-log {compoundOutputFileLuid0}"

Set Next Step-KAPA Lib Quant

• 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 'if (output.name.contains(::KAPA::)) {nextStep = ::REMOVE::} else {nextStep = ::ADVANCE::} ; \
input.::Concentration (nM):: = output.::Concentration (nM)::' \
-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
  Criteria 1 - Operator	Text		Default = >=
  Criteria 1 - Source Data Field	Text		Default = Concentration (nM)
  Criteria 1 - Threshold Value	Numeric		Default = 25
  Criteria 2 - Operator	Text		Default = <=
  Criteria 2 - Source Data Field	Text		Default = Concentration (nM)
  Criteria 2 - Threshold Value	Numeric		Default = 99
  Instruction Notes	Multiline Text	Read Only	Default = 1. Enter the Slope and the Y-Intercept for your standard curve then click on the Calculate Concentration button. 2. Set your Concentration Threshold then click on the Assign QC Flogs button.
  Library Length (bp)	Numeric		Default = 450
  Library Length Ratio	Numeric	Read Only	Decimal Places Displayed = 0
  Standard Curve Slope	Numeric
  Standard Curve Y-Intercept	Numeric

• Step File Placeholders

  • Log - Automatically attached

  • QC Log File - Automatically attached

  • QC Result File - Manually uploaded

  • Upload File - Manually uploaded

• Sample Table

  • Enable QC Flags = Yes

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • File Column Options

    • File Column Display = Show

    • File Attachment Method = Manual

  • 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	Average Cq	Numeric		Decimal Places Displayed = 2
    Derived Sample	Concentration (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Concentration (pM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Diluted Avg. Conc. (pM)	Numeric
    Derived Sample	Dilution Factor	Text Dropdown	Required Field Custom Entries	Presets 1:10000 1:20000
    Derived Sample	Sample Name	Built-in
    Measurement	Average Cq	Numeric		Decimal Places Displayed = 2
    Measurement	Concentration (nM)	Numeric		Decimal Places Displayed = 2
    Measurement	Concentration (pM)	Numeric		Decimal Places Displayed = 2
    Measurement	Diluted Avg. Conc. (pM)	Numeric		Decimal Places Displayed = 4
    Measurement	Dilution Factor	Text	Read Only
    Project	Project Name	Built-in

Step 4: Dilute Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Dilute Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Resuspension Buffer (RSB)

Automations

Copy Concentration (nM)

• 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 (nM):: = input.::Concentration (nM)::' \
-log {compoundOutputFileLuid0}"

Calculate Dilution Volumess

• 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.::Sample (uL):: = (step.::Final Concentration (nM):: * step.::Final Volume (uL)::) / output.::Concentration (nM):: ; \
output.::RSB (uL):: = step.::Final Volume (uL):: - output.::Sample (uL):: ; \
output.::Final Concentration (nM):: = step.::Final Concentration (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 Concentration (nM)	Numeric		Default = 2
  Final Volume (uL)	Numeric

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • 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 (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Final Concentration (nM)	Numeric
    Derived Sample	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 5: Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Master Step Name = Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

Automations

Copy Final Concentration (nM)

• 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 (nM):: = input.::Final Concentration (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

Pooling

• Label Uniqueness = On

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Instruction Notes	Multiline Text	Read Only	Default = 1. Combine libraries equimolarly to a 2 nM final concentration. 2. Vortex to mix, and then centrifuge at 280 x g for 1 minute.
  Sample Volume (uL)	Numeric

• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • 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 (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in