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

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}"

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' "

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

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}"

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

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}"

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}"

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

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

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}"

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'"

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