MiniSeq v1.0

Overview

The MiniSeq workflow includes the following functionality.

• Preconfigured protocols that support the preparation of small genome, amplicon, targeted enrichment, and RNA sequencing using low library volumes for sequencing.

• 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: Select Normalization Variation (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Select Normalization Variation (MiniSeq v1.0)

• Master Step Name = Select Normalization Variation (MiniSeq v1.0)

• Step Type = No Outputs

Automations

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 Script for Normalization Variation

• 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 'Normalization Variation' \
--FIELD_VALUE 'Standard Normalization' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Create Normalized Library Pool (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Normalization Variation' \
--FIELD_VALUE 'Bead-Based Normalization' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Normalization Variation' \
--FIELD_VALUE 'AmpliSeq for Illumina Normalization' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Dilute Libraries - AmpliSeq (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Normalization Variation' \
--FIELD_VALUE 'AmpliSeq Library Equalizer for Illumina Normalization' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Pool Libraries - AmpliSeq Equalizer (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'INPUTS' "

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

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
  Comment	Multiline Text
  Instruction Notes	Multiline Text	Read Only	Default = Select a Normalization Variation protocol 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	Sample Name	Built-in
    Derived Sample	Normalization Variation	Text Dropdown	Required Field	Presets Standard Normalization Bead-Based Normalization AmpliSeq for Illumina Normalization AmpliSeq Library Equalizer for Illumina Normalization Default = Standard Normalization
    Project	Project Name	Built-in

Protocol 2: Standard Normalization (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Create Normalized Library Pool (MiniSeq v1.0)

• Master Step Name = Create Normalized Library Pool (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Resuspension Buffer (RSB)

Automations

Calculate Library & RSB Volumes - MiniSeq

• 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.::Library (uL):: = (step.::Final Molarity (nM):: * step.::Final Volume (uL)::) / output.::Library Molarity (nM):: ; \
output.::RSB (uL):: = step.::Final Volume (uL):: - output.::Library (uL):: ; 
output.::Final Molarity (nM):: = step.::Final Molarity (nM)::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Molarity (nM)	Numeric		Default = 10 Range = 1 To 10 Decimal Places Displayed = 0
  Final Volume (uL)	Numeric		Default = 10 Range = 10 To 400 Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = - Enter in Final Molarity (nM), Final Volume (uL) and Library Molarity (nM) before clicking on calculation button. - Add 10 uL of each 10 nM library to a new microcentrifuge tube.

• 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	Final Molarity (nM)	Numeric
    Derived Sample	Library Molarity (nM)	Numeric	Required Field
    Derived Sample	Library (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: Dilute Library Pool (MiniSeq v1.0)

• Master Step Name = Dilute Library Pool (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Resuspension Buffer (RSB)

Automations

Copy Library Pool Molarity

• 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 true -h false \
-exp 'output.::Library Pool (nM):: = input.::Final Molarity (nM)::' \
-log {compoundOutputFileLuid0}"

Calculate Library Pool & RSB Volumes - MiniSeq

• 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.::Library Pool (uL):: = (step.::Final Molarity (nM):: * step.::Final Volume (uL)::) / output.::Library Pool (nM):: ; \
output.::RSB (uL):: = step.::Final Volume (uL):: - output.::Library Pool (uL)::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Molarity (nM)	Numeric		Default = 1
  Final Volume (uL)	Numeric		Default = 100
  Instruction Notes	Multiline Text	Read Only	Default = Enter in Final Molarity (nM), and Final Volume (uL) before clicking on calculation button.

• 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	Library Pool (nM)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Library Pool (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 3: Denature Library Pool (MiniSeq v1.0)

• Master Step Name = Denature Library Pool (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • 0.1 N NaOH

  • 200 mM Tris-HCl, pH 7.0

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

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Instruction Notes	Multiline Text	Read Only	Default = - Combine 5 uL of 1 nM Library Pool and 5 uL of 0.1 N NaOH in a microcentridge tube. - Vortex briefly and then centrifuge at 280 × g for 1 minute. - Incubate at room temperature for 5 minutes. - Add 5 µl 200 mM Tris-HCl, pH 7.0. Vortex briefly and then centrifuge at 280 × 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 4: Dilute to Loading Concentration (MiniSeq v1.0)

• Master Step Name = Dilute to Loading Concentration (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Hybridization Buffer

    • Supplier = Illumina; part of MiniSeq High Output Reagent Kit

    • Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Routing Script for MiniSeq

• 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 {compoundOutputLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'No' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Run Setup (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'Yes' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

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

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = 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
  Add PhiX Control	Text Dropdown	Required Field	Presets No Yes Default = No
  Instruction Notes	Multiline Text	Read Only	Default = - Add 985 µl of prechilled Hybridization Buffer to the tube of denatured library. - The total volume is 1 ml at 5 pM. - Vortex briefly and then centrifuge at 280 × g for 1 minute. - Transfer 180 µl diluted library to a new microcentrifuge tube. - Add 320 µl prechilled Hybridization Buffer. - The total volume is 500 µl at 1.8 pM.
  Loading Concentration (pM)	Numeric		Default = 1.8
  Total Volume (uL)	Numeric		Default = 500

• 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

Protocol 3: Bead-Based Normalization (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0)

• Master Step Name = Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Hybridization Buffer

    • Supplier = Illumina; part of MiniSeq High Output Reagent Kit

    • Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003

    • Website = www.illumina.com

Automations

Calculate Prechilled Hybridization Buffer Volumes - MiniSeq

• 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.::Prechilled Hybridization Buffer (uL):: = output.::Final Volume (uL):: - output.::Library Pool (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}"

Routing Script for MiniSeq

• 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 {compoundOutputLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'No' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Run Setup (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'Yes' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

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

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = 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
  Add PhiX Control	Text Dropdown	Required Field	Presets No Yes Default = No
  Instruction Notes	Multiline Text	Read Only	Default = - Enter in Library Pool and Final Volume before clicking on calculation button. - Vortex briefly and then centrifuge at 280 × g for 1 minute. - Transfer 250 µl diluted library to a new microcentrifuge tube. - Add 250 µl prechilled Hybridization Buffer. - Vortex briefly and then centrifuge at 280 × g for 1 minute. - To denture the diluted library pool, place the tube in the incubator for 2 minutes. Immediately cool on ice and leave it on ice 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	Final Volume (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Library Pool (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Prechilled Hybridization Buffer (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 4: AmpliSeq for Illumina Normalization (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Dilute Libraries - AmpliSeq (MiniSeq v1.0)

• Master Step Name = Dilute Libraries - AmpliSeq (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Low TE

    • Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit

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

    • Website = www.illumina.com

Automations

Copy Final Molarity

• 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.::Final Molarity (nM):: = step.::Final Molarity (nM)::' \
-log {compoundOutputFileLuid0}"

Calculate Starting Molarity and Library & Low TE Volumes - MiniSeq

• 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.::Starting Molarity (nM):: = (output.::Concentration:: * 1000000) / (660 * output.::Average Library Size (bp)::) ; \
output.::Library (uL):: = (output.::Final Molarity (nM):: * step.::Final Volume (uL)::) / output.::Starting Molarity (nM):: ; \
output.::Low TE (uL):: = step.::Final Volume (uL):: - output.::Library (uL)::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Molarity (nM)	Numeric		Default = 2
  Final Volume (uL)	Numeric		Default = 50
  Instruction Notes	Multiline Text	Read Only	Default = Enter in the concentration and average library size before clicking on the calculation button.

• 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	Average Library Size (bp)	Numeric		Default = 350
    Derived Sample	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Final Molarity (nM)	Numeric
    Derived Sample	Library (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Low TE (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Starting Molarity (nM)	Numeric		Decimal Places Displayed = 0
    Project	Project Name	Built-in

Step 2: Pool Libraries - AmpliSeq (MiniSeq v1.0)

• Master Step Name = Pool Libraries - AmpliSeq (MiniSeq v1.0)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

• Reagent Kits

  • Low TE

    • Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit

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

    • Website = www.illumina.com

Automations

Copy Molarity - MiniSeq

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

Calculate DNA to RNA - MiniSeq

• 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 'if (step.::AmpliSeq for Illumina Panel:: == ::Myeloid Panel::) {step.::DNA to RNA Ratio:: = ::8:1::} ; \
if (step.::AmpliSeq for Illumina Panel:: == ::Childhood Cancer Panel::) {step.::DNA to RNA Ratio:: = ::5:1::} ; \
if (step.::AmpliSeq for Illumina Panel:: == ::Focus Panel::) {step.::DNA to RNA Ratio:: = ::7:3::} ; \
if (step.::AmpliSeq for Illumina Panel:: == ::Comprehensive Panel v3::) {step.::DNA to RNA Ratio:: = ::25:1::}' \
-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
  AmpliSeq for Illumina Panel	Text Dropdown	Required Field	Presets Childhood Cancer Panel Comprehensive Panel Focus Panel Myeloid Panel Default = Childhood Cancer Panel
  DNA to RNA Ratio	Text
  Instruction Notes	Multiline Text	Read Only	Default = - Select the AmpliSeq for Illumina Panel before clicking on Calculate DNA to RNA ratio button. - Combine equal library volumes to a 1.5 mL LoBind tube. - User different tubes for DNA and RNA Libraries. - Combine DNA to RNA ratio in a new tube.
  Transfer Library 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	Molarity (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 3: Denature Libraries - AmpliSeq (MiniSeq v1.0)

• Master Step Name = Denature Libraries - AmpliSeq (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Low TE

    • Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit

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

    • Website = www.illumina.com

Automations

Copy Molarity - MiniSeq

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

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  0.2 N NaOH	Numeric		Default = 10
  0.2 N NaOH Prep Date	Date
  200 mM Tris-HCl, pH 7.0	Numeric		Default = 10
  Instruction Notes	Multiline Text	Read Only	Default = - Combine Pool Libraries and 0.2 N NaOH volumes. - Vortex briefly. Incubate at room temp for 5 mins. - Add 10 µl 200 mM Tris-HCl, pH 7.0 to the tube. Vortex briefly.
  Pool Libraries (uL)	Numeric		Default = 10

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

Step 4: Dilute Denatured Libraries to Loading Concentration - AmpliSeq (MiniSeq v1.0)

• Master Step Name = Dilute Denatured Libraries to Loading Concentration - AmpliSeq (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • HT1

    • Supplier = Illumina; part of AmpliSeq Library Plus Kit

    • Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 Website - www.illumina.com

    • Website = www.illumina.com

Automations

Calculate Diluted Denatured Libraries & Prechilled HT1 Volumes - MiniSeq

• 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.::Diluted Denatured Libraries (uL):: = (step.::Loading Concentration (pM):: * step.::Final Volume (uL)::) / output.::Diluted Denatured Libraries (pM):: ; \
output.::Prechilled HT1(uL):: = step.::Final Volume (uL):: - output.::Diluted Denatured Libraries (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}"

Routing Script for MiniSeq

• 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 {compoundOutputLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'No' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Run Setup (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'Yes' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

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

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = 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
  200 mM Tris-HCl, pH 7.0	Numeric		Default = 10
  Add PhiX Control	Text Dropdown	Custom Entries	Presets No Yes Default = No
  Final Volume (uL)	Numeric		Default = 1000
  Instruction Notes	Multiline Text	Read Only	Default = - Enter Loading Concentration before clicking on the calculation button. - Add 970 uL of prechilled HT1 to denatured library pool. - Dilute to loading concentration by combining Diluted Denatured Libraries and Prechilled HT1 volumes.
  Loading Concentration (pM)	Numeric		Default = 1.8 Range = 1.1 To 1.9

• 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	Diluted Denatured Libraries (pM)	Numeric		Default = 20
    Derived Sample	Diluted Denatured Libraries (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Prechilled HT1(uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 5: AmpliSeq Library Equalizer for Illumina Normalization (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Pool Libraries - AmpliSeq Equalizer (MiniSeq v1.0)

• Master Step Name = Pool Libraries - AmpliSeq (MiniSeq v1.0)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

• Reagent Kits

  • Low TE

    • Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit

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

    • Website = www.illumina.com

Automations

Copy Molarity - MiniSeq

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

Calculate DNA to RNA - MiniSeq

• 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 'if (step.::AmpliSeq for Illumina Panel:: == ::Myeloid Panel::) {step.::DNA to RNA Ratio:: = ::8:1::} ; \
if (step.::AmpliSeq for Illumina Panel:: == ::Childhood Cancer Panel::) {step.::DNA to RNA Ratio:: = ::5:1::} ; \
if (step.::AmpliSeq for Illumina Panel:: == ::Focus Panel::) {step.::DNA to RNA Ratio:: = ::7:3::} ; \
if (step.::AmpliSeq for Illumina Panel:: == ::Comprehensive Panel v3::) {step.::DNA to RNA Ratio:: = ::25:1::}' \
-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
  AmpliSeq for Illumina Panel	Text Dropdown	Required Field	Presets Childhood Cancer Panel Comprehensive Panel Focus Panel Myeloid Panel Default = Childhood Cancer Panel
  DNA to RNA Ratio	Text
  Instruction Notes	Multiline Text	Read Only	Default = - Select the AmpliSeq for Illumina Panel before clicking on Calculate DNA to RNA ratio button. - Combine equal library volumes to a 1.5 mL LoBind tube. - User different tubes for DNA and RNA Libraries. - Combine DNA to RNA ratio in a new tube.
  Transfer Library 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	Molarity (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 2: Denature Libraries - AmpliSeq Equalizer (MiniSeq v1.0)

• Master Step Name = Denature Libraries - AmpliSeq (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Low TE

    • Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit

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

    • Website = www.illumina.com

Automations

Copy Molarity - MiniSeq

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

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  0.2 N NaOH	Numeric		Default = 10
  0.2 N NaOH Prep Date	Date
  200 mM Tris-HCl, pH 7.0	Numeric		Default = 10
  Instruction Notes	Multiline Text	Read Only	Default = - Combine Pool Libraries and 0.2 N NaOH volumes. - Vortex briefly. Incubate at room temp for 5 mins. - Add 10 µl 200 mM Tris-HCl, pH 7.0 to the tube. Vortex briefly.
  Pool Libraries (uL)	Numeric		Default = 10

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

Step 3: Dilute Denatured Libraries to Loading Concentration - AmpliSeq Equalizer (MiniSeq v1.0)

• Master Step Name = Dilute Denatured Libraries to Loading Concentration - AmpliSeq (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • HT1

    • Supplier = Illumina; part of AmpliSeq Library Plus Kit

    • Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 Website - www.illumina.com

    • Website = www.illumina.com

Automations

Calculate Diluted Denatured Libraries & Prechilled HT1 Volumes - MiniSeq

• 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.::Diluted Denatured Libraries (uL):: = (step.::Loading Concentration (pM):: * step.::Final Volume (uL)::) / output.::Diluted Denatured Libraries (pM):: ; \
output.::Prechilled HT1(uL):: = step.::Final Volume (uL):: - output.::Diluted Denatured Libraries (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}"

Routing Script for MiniSeq

• 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 {compoundOutputLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'No' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Run Setup (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Add PhiX Control' \
--FIELD_VALUE 'Yes' \
--WORKFLOW 'MiniSeq v1.0' \
--STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

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

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = 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
  200 mM Tris-HCl, pH 7.0	Numeric		Default = 10
  Add PhiX Control	Text Dropdown	Custom Entries	Presets No Yes Default = No
  Final Volume (uL)	Numeric		Default = 1000
  Instruction Notes	Multiline Text	Read Only	Default = - Enter Loading Concentration before clicking on the calculation button. - Add 970 uL of prechilled HT1 to denatured library pool. - Dilute to loading concentration by combining Diluted Denatured Libraries and Prechilled HT1 volumes.
  Loading Concentration (pM)	Numeric		Default = 1.8 Range = 1.1 To 1.9

• 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	Diluted Denatured Libraries (pM)	Numeric		Default = 20
    Derived Sample	Diluted Denatured Libraries (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Prechilled HT1(uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 6: Add PhiX Control (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Dilute Stock PhiX (MiniSeq v1.0)

• Master Step Name = Dilute Stock PhiX (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Resuspension Buffer (RSB)

• Control Types

  • PhiX v3

    • Supplier = Illumina

    • Catalog Number = FC-110-3001

    • Website = http://www.illumina.com/products/phix_control_v3.ilmn

Automations

Calculate Stock PhiX and RSB Volumes

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Stock PhiX Volume (uL):: = (step.::Final PhiX Molarity (nM):: * step.::Final PhiX Volume (uL)::) / step.::Stock PhiX Molarity (nM):: ; \
step.::RSB (uL):: = step.::Final PhiX Volume (uL):: - step.::Stock PhiX Volume (uL)::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final PhiX Molarity (nM)	Numeric		Default = 4
  Final PhiX Volume (uL)	Numeric		Default = 25
  Instruction Notes	Multiline Text	Read Only	Default = Enter Stock PhiX (nM), Final PhiX Molarity (nM), and Final Volume (uL) before clicking on calculation button.
  RSB (uL)	Numeric		Decimal Places Displayed = 0
  Stock PhiX Molarity (nM)	Numeric		Default = 10
  Stock PhiX Volume (uL)	Numeric		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
    Project	Project Name	Built-in

Step 2: Denature PhiX (MiniSeq v1.0)

• Master Step Name = Denature PhiX (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • 0.1 N NaOH

  • 200 mM Tris-HCl, pH 7.0

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

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Instruction Notes	Multiline Text	Read Only	Default = - Combine 5uL of 4 nM PhiX and 5 uL of 0.1 N NaOH. - Vortex briefly and then pulse centrifuge. - Incubate at room temperature for 5 minutes. - Add 5 µl 200 mM Tris-HCl, pH 7.0. - Vortex briefly and then centrifuge at 280 × 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 3: Dilute PhiX to Loading Concentration and Combine Library & PhiX (MiniSeq v1.0)

• Master Step Name = Dilute PhiX to Loading Concentration and Combine Library & PhiX (MiniSeq v1.0)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

• Reagent Kits

  • Hybridization Buffer * Supplier = Illumina; part of MiniSeq High Output Reagent Kit * Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003 * Website = www.illumina.com

Automations

Calculate Denatured PhiX and Prechilled Hybridization Buffer Volumes

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Denatured PhiX Volume (uL):: = (step.::Final Denatured PhiX Molarity (pM):: * step.::Final Volume (uL)::) / step.::Denatured PhiX Molarity (pM):: ; \
step.::Prechilled Hybridization Buffer (uL):: = step.::Final Volume (uL):: - step.::Denatured PhiX Volume (uL)::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

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
  Denatured PhiX Molarity (pM)	Numeric		Default = 20
  Denatured PhiX Volume (uL)	Numeric		Decimal Places Displayed = 0
  Final Denatured PhiX Molarity (pM)	Numeric		Default = 1.8
  Final Volume (uL)	Numeric		Default = 500
  Instruction Notes	Multiline Text	Read Only	Default = - Enter Denatured PhiX Molarity (pM), Final Denatured PhiX Molarity (pM) and Final Volume (uL) before clicking on calculation button. - Add Denatured PhiX and Prechilled Hybridization Buffer volumes too dilute the denatured PhiX. - Invert to mix and then centrifuge at 280 × g for 1 minute. - Combine 5 uL of denatured and diluted PhiX control with 500 uL of denatured and diluted library pool.
  Prechilled Hybridization Buffer (uL)	Numeric		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
    Project	Project Name	Built-in

Protocol 7: Run Setup (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Run Setup (MiniSeq v1.0)

• Master Step Name = Run Setup (MiniSeq v1.0)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • MiniSeq High Output Reagent Kit

    • Supplier = Illumina

    • Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003

    • Website = www.illumina.com

Automations

1. miniseq sample sheet

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {processURI:v2} -u {username} -p {password} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/miniSeq_Samplesheet.csv \
-o {compoundOutputFileLuid1}.csv \
-q true \
-destLIMSID {compoundOutputFileLuid1} \
-l {compoundOutputFileLuid2}"

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 = Row

  • Placement Pattern = Row

• Destination Containers

  • MiniSeq Reagent Cartridge

  

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Adapter	Text		Default = CTGTCTCTTATACACATCT
  BaseSpace Sequence Hub	Text Dropdown	Custom Entries	Presets Run Monitoring Only Run Monitoring and Storage
  Chemistry	Text
  Custom Primer for Read 1	Text
  Customer Primer for Index 1	Text
  Customer Primer for Read 2	Text
  Customer Primer for Index 2	Text
  Experiment Name	Text	Required Field
  Index 1 (i7 Primer) Cycles	Text	Required Field	Default = 8
  Index 2 (i5 Primer) Cycles	Text	Required Field	Default = 8
  Instruction Notes	Multiline Text	Read Only	Default = Add 500 uL of prepared libraries to reagent cartridge.
  Local Run Manager Analysis Id	Text
  Manifest File Name	Text
  Module	Text
  Output Folder Location	Text
  Read 1 Cycles	Numeric	Required Field	Range = 0 To 151
  Read 2 Cycles	Numeric	Required Field	Range = 0 To 151
  Read Type	Text Dropdown	Required Field	Presets Paired End Read Single Read Default = Paired End Read
  Workflow	Text		Default = GenerateFASTQ

• Step File Placeholders

  • Log - Automatically attached

  • LRM Sample Sheet - Automatically attached

  • Manual Sample Sheet - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = 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