Illumina COVIDSeq v1.6

Overview

The Illumina COVIDSeq Workflow workflow includes the following functionality:

• Preset Illumina COVIDSeq Workflow protocols that track samples through the quantitative detection of SARS-CoV-2 RNA from nasopharyngeal (NP), oropharyngeal (OP), and nasal swab as well as mid-turbinate specimens collected from individuals who meet COVID-19 clinical or epidemiological criteria.

• 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.

• Sample preparation for use on the NovaSeq 6000 Sequencing System.

• Parsing of analysis results into Clarity LIMS.

Protocol 1: RNA Extraction (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Prep Sample (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Prep Sample (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina COVIDSeq Positive Control HT

    • Supplier = Illumina

    • Website = www.illumina.com

  • Illumina COVIDSeqTest Box 4

    • Supplier = Illumina

    • Catalog Number = 20043436

    • 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}"

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 = For each sample, add 140 µl patient sample, dilution 3 CPC HT, or ELB HT (no template control) to a new 1.7 ml microcentrifuge tube.

• Step File Placeholders

  • Log File - 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: Purification of RNA - Spin (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Spin Purification of RNA (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • QIAmp Viral RNA Mini kit

    • Supplier = Qiagen

    • Catalog Number = (50) - 52904; (250) - 52906

    • Website = https://www.qiagen.com

Automations

Calculate Total Samples

• 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 \
-t false \
-h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0}"

Calculate AW1, AW2, AVL Reagent 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 'if (step.::No. of Preps:: == 50) {step.::AW1 Concentrate (mL):: = 19 ; step.::EtOH for AW1 (mL):: = 25 ; step.::AW2 Concentrate (mL):: = 13 ; step.::EtOH for AW2 (mL):: = 30} else {step.::AW1 Concentrate (mL):: = 98 ; step.::EtOH for AW1 (mL):: = 130 ; step.::AW2 Concentrate (mL):: = 66 ; step.::EtOH for AW2 (mL):: = 160} ; \
step.::Buffer AVL (mL):: = 0.56 * step.::Total Samples:: ; step.::Carrier RNA-Buffer AVE (uL):: = 10 * step.::Buffer AVL (mL):: ' \
-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
  % EtOH	Numeric		Default = 96 Range = 96 To 100
  % EtOH Prep Date	Date
  AW1 Concentrate (mL)	Numeric
  AW2 Concentrate (mL)	Numeric
  Buffer AVL (mL)	Numeric		Decimal Places Displayed = 0
  Carrier RNA-Buffer AVE (uL)	Numeric		Decimal Places Displayed = 0
  Elution Incubation End Time	Numeric
  Elution Incubation Start Time	Numeric
  EtOH for AW1 (mL)	Numeric
  EtOH for AW2 (mL)	Numeric
  Instruction Notes	Multiline Text	Read Only	Default = Enter in No. of Preps before clicking on "Calculate AW1, AW2, AVL Reagent Volumes". Elute in 30 µl Buffer AVE instead of 60 µl. Incubate elution for at least 1 minute.
  No. of Preps	Numeric Dropdown	Required Field Custom Entries	Presets 250 50 Default = 250
  Total Samples	Numeric		Default = 0

• Step File Placeholders

  • Log File - 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: Anneal Sample (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Anneal Sample (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina COVIDSeq Test Box 5 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043648

    • 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}"

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
  COVIDSeq Anneal Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option Set the reaction volume to 17 µl 65°C for 3 minutes Hold at 4°C
  Instruction Notes	Multiline Text	Read Only	Default = Add 8.5 µl EPH3 HT to each well. Add 8.5 µl eluted sample to each well. Seal and shake at 1600 rpm for 1 minute. Centrifuge at 1000 × g for 1 minute.
  Thermal Cycler Program Notes	Text		Default = COVIDSeq Anneal

• Step File Placeholders

  • Log File - 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 2: cDNA Synthesis (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Synthesize First Strand cDNA (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Synthesize First Strand cDNA (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina COVIDSeq Test Box 6 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043647

    • Website = www.illumina.com

Automations

Calculate First Strand Synthesis Master Mix

• 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 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::FSA (uL):: = 9 * step.::Total Samples:: ; step.::RVT (uL):: = 1 * step.::Total Samples:: ' -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
  COVIDSeq FSS Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option Set the reaction volume to 25 µl 25°C for 5 minutes 50°C for 10 minutes 80°C for 5 minutes Hold at 4°C
  FSA (uL)	Numeric
  Instruction Notes	Multiline Text	Read Only	Default = Add 8 µl First Strand cDNA Master Mix to each well of the CDNA1 plate.
  RVT (uL)	Numeric
  Total Samples	Numeric	Required Field	Default = 0
  Thermal Cycler Program	Text		Default = COVIDSeq FSS

• Step File Placeholders

  • Log File - 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: Target Amplification (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Amplify cDNA (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Amplify cDNA (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina COVIDSeq Test Box 5 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043648

    • Website = www.illumina.com

  • Illumina COVIDSEQ Test Box 7 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043439

    • Website = www.illumina.com

Automations

Calculate COVIDSeq PCR 1 and PCR2 Master Mixes

• 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.::IPM HT for COVIDSeq PCR 1 Master Mix (uL):: = 15 * step.::Total Samples:: ; step.::CPP1 HT for COVIDSeq PCR 1 Master Mix (uL):: = 4.3 * step.::Total Samples:: ; step.::Nuclease-free water for COVIDSeq PCR 1 Master Mix (uL):: = 4.7 * step.::Total Samples:: ; step.::IPM HT for COVIDSeq PCR 2 Master Mix (uL):: = 15 * step.::Total Samples:: ; step.::CPP2 HT for COVIDSeq PCR 2 Master Mix (uL):: = 4.3 * step.::Total Samples:: ; step.::Nuclease-free water for COVIDSeq PCR 2 Master Mix (uL):: = 4.7 * step.::Total Samples::' -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
  COVIDSeq PCR Notes	Multiline Text	Read Only	Default = Choose the preheat lid option Set the reaction volume to 25 µl 98°C for 3 minutes 35 cycles of: 98°C for 15 seconds 65°C for 5 minutes Hold at 4°C
  CPP1 HT for COVIDSeq PCR 1 Master Mix (uL)	Numeric		Decimal Places Displayed = 0
  CPP2 HT for COVIDSeq PCR 2 Master Mix (uL)	Numeric		Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = Add 20 µl COVIDSeq PCR 1 Master Mix to each well of the COV1 plate corresponding to each well of the CDNA1 plate. Add 5 µl first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of the COV1 plate. Add 20 µl COVIDSeq PCR 2 Master Mix to each well of the COV2 plate corresponding to each well of the CDNA1 plate. Add 5 µl first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of the COV2 plate.
  IPM HT for COVIDSeq PCR 1 Master Mix (uL)	Numeric		Decimal Places Displayed = 0
  IPM HT for COVIDSeq PCR 2 Master Mix (uL)	Numeric		Decimal Places Displayed = 0
  Nuclease-free water for COVIDSeq PCR 1 Master Mix (uL)	Numeric		Decimal Places Displayed = 0
  Nuclease-free water for COVIDSeq PCR 2 Master Mix (uL)	Numeric		Decimal Places Displayed = 0
  Thermal Cycler Program	Text		Default = COVIDSeq PCR
  Total Samples	Numeric		Default = 0

• Step File Placeholders

  • Log File - 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 4: Library Prep (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Tagment PCR Amplicons (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Tagment PCR Amplicons (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina COVIDSEQ Test Box 7 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043439

    • Website = www.illumina.com

  • Illumina COVIDSeqTest Box 3 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043646

    • Website = www.illumina.com

Automations

Calculate Tagmentation 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.::TB1 (uL):: = 12 * step.::Total Samples:: ; step.::eBLT (uL):: = 4 * step.::Total Samples:: ; step.::Nuclease-free water (uL):: = 20 * step.::Total Samples::' -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
  COVIDSeq TAG Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option Set the reaction volume to 50 µl 55°C for 5 minutes Hold at 10°C
  eBLT (uL)	Numeric		Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = Add 30 µl Tagmentation Master Mix to each well in TAG1 plate.
  Nuclease-free water (uL)	Numeric		Decimal Places Displayed = 0
  TB1 (uL)	Numeric		Decimal Places Displayed = 0
  Thermal Cycler Program	Text		Default = COVIDSeq TAG
  Total Samples	Numeric		Default = 0

• Step File Placeholders

  • Log File - 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: Post Tagmentation Clean Up (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Post Tagmentation Clean Up (Illumina COVIDSeq Workflow v1.6)

• Step Type = No Outputs

• Reagent Kits

  • Illumina COVIDSeq Test Box 2 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043434

    • Website = www.illumina.com

  • Illumina COVIDSeqTest Box 4

    • Supplier = Illumina

    • Catalog Number = 20043436

    • 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}"

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 File Placeholders

  • Log File - 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: Amplify Tagmented Amplicons (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Amplify Tagmented Amplicons (Illumina COVIDSeq Workflow v1.6)

• Step Type = Add Labels

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}-{AppliedReagentLabels}

• Reagent Kits

  • Illumina COVIDSeq Test Box 5 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043648

    • Website = www.illumina.com

Automations

Calculate PCR 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.::EPM HT (uL):: = 24 * step.::Total Samples:: ; step.::Nuclease-free water (uL):: = 24 * step.::Total Samples::' -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

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
  COVIDSeq TAG PCR Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 100°C Set the reaction volume to 50 µl 72°C for 3 minutes 98°C for 3 minutes 7 cycles of: 98°C for 20 seconds 60°C for 30 seconds 72°C for 1 minute 72°C for 3 minutes Hold at 10°C
  EPM HT (uL)	Numeric
  Instruction Notes	Multiline Text	Read Only	Default = Add 40 µl PCR Master Mix to each well.
  Nuclease-free water (uL)	Numeric
  Thermal Cycler Program	Text		Default = COVIDSeq TAG PCR
  Total Samples	Numeric		Default = 0

• Step File Placeholders

  • Log File - 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: Pool and Clean Up Libraries (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Pool and Clean Up Libraries (Illumina COVIDSeq Workflow v1.6)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

• Reagent Kits

  • Illumina COVIDSeqTest Box 3 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043646

    • Website = www.illumina.com

Automations

Calculate ITB 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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'step.::ITB (uL):: = 0.9 * (55 * step.::Total Pooled ITB Tubes::) ' -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

Placement = Enabled

• Defaults

  • Well Sort Order = Column

  • Placement Pattern = Column

• Destination Containers

  • 1x8 Strip Tube

  

  • 96 well plate

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  80% EtOH Date	Date
  Instruction Notes	Multiline Text	Read Only	Default = Enter in Total Pooled ITB Tubes to Calculate ITB (uL) volume. Transfer 50 µl supernatant from each Pooled ITB tube to a new microcentrifuge tube.
  ITB (uL)	Numeric
  Total Pooled ITB Tubes	Numeric

• Step File Placeholders

  • Log File - 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 5: Quantification (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Quantify Libraries (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Quantify Libraries - Qubit (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Assign QC flags (Qubit QC)

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:assignQC -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}"

Copy Concentration From QC Step

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

Set Next Step and Copy Concentration

• 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:: ; 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		Default = 2
  Criteria 2 - Operator	Text		Default = <>=
  Criteria 2 - Source Data Field	Text		Default = Concentration
  Criteria 2 - Threshold Value	Numeric		Default = 30

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

Step 2: Dilute Libraries (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Dilute Libraries (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina COVIDSeqTest Box 3 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043646

    • Website = www.illumina.com

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 Sample & 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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'output.::SampleVolume (uL):: = 0.1 * step.::Final Volume (uL):: ; output.::RSB (uL):: = step.::Final Volume (uL):: - output.::SampleVolume (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
  Dilution Factor	Text	Read Only	Default = 1:10
  Final Volume (uL)	Numeric
  Instruction Notes	Multiline Text		Default = Enter Final Volume (uL) then click on "Calculate Sample & RSB Volumes" for dilution volumes.

• 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	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	SampleVolume (uL)	Numeric		Decimal Places Displayed = 0
    Project	Project Name	Built-in

Step 3: Quantify Libraries (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Quantify Libraries - Qubit (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Assign QC flags (Qubit QC)

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:assignQC -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}"

Set Next Step and Copy Concentration

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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

Copy Concentration From QC Step

• 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 '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		Default = 2
  Criteria 2 - Operator	Text		Default = <>=
  Criteria 2 - Source Data Field	Text		Default = Concentration
  Criteria 2 - Threshold Value	Numeric		Default = 30

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

Step 4: Normalize Libraries (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Normalize Libraries (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina COVIDSeqTest Box 3 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043646

    • Website = www.illumina.com

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 Molarity, Library Pool & RSB

• 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:: / (660 * 400)) * 1000000 ; output.::Library Pool (uL):: = step.::Final Molarity (nM):: * step.::Final Volume (uL):: / output.::Starting Molarity (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 = 4
  Final Volume (uL)	Numeric		Default = 30

• 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	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	Starting Molarity (nM)	Numeric		Default = 0
    Derived Sample	Library Pool (uL)	Numeric		Default = 0
    Project	Project Name	Built-in

Step 5: Pool and Dilute Libraries (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Pool and Dilute Libraries (Illumina COVIDSeq Workflow v1.6)

• Step Type = Pooling

• Aliquot Generation = Fixed, 1

• Naming Convention = {PoolName}

• Reagent Kits

  • Illumina COVIDSeqTest Box 3 – 3072 Samples

    • Supplier = Illumina

    • Catalog Number = 20043646

    • 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}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table (Column Headers)

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

Pooling

• Label Uniqueness = On

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Instruction Notes	Multiline Text	Read Only	Default = For each set of 384 samples, combine 25 µl of each normalized pool containing index adapter set A, B, C, D in a new microcentrifuge tube. Do not combine pools with the same index adapter set. This step produces a final pool of 384 samples for one flow cell lane. For each flow cell lane, dilute each 384 sample pool with RSB HT as follows. These steps produce 30 µl 0.5 nM pooled library. Combine 3.75 µl library from the 384-sample pool with 26.25 µl RSB. Vortex, and then centrifuge briefly. The resulting final loading concentration is 100 pM.

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

Protocol 6: NovaSeq 6000 Xp Setup (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: NovaSeq Denature & ExAmp (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = NovaSeq Denature & ExAmp (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}_WP{OutputItemNumber}

• Reagent Kits

  • DPX1

    • Supplier = Illumina

    • Catalog Number = 20019916

  • DPX2

    • Supplier = Illumina

    • Catalog Number = 20019917

  • DPX3

    • Supplier = Illumina

    • Catalog Number = 20019918

  • NaOH

  • Tris-HCl

Automations

Calculate DPX1, DPX2, DPX3

• 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 'step.::DPX1 Volume (ul):: = 315 * step.::No. of Lanes:: ; step.::DPX2 Volume (ul):: = 45 * step.::No. of Lanes:: ; step.::DPX3 Volume (ul):: = 165 * step.::No. of Lanes:: ; output.::Flowcell Type:: = step.::Flowcell Type::; output.::Loading Workflow Type:: = step.::Loading Workflow Type::' -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	Well	Built-in
    Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Loading Workflow Type	Text Dropdown	Required Field	Presets NovaSeq Standard NovaSeq Xp [Remove from workflow]
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	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 per pool (uL)	Numeric	Read Only	Default = 7
  0.2 N NaOH Prep Date	Date
  400 mM Tris-HCl per pool (uL)	Numeric	Read Only	Default = 8
  400 mM Tris-HCl Prep Date	Date
  DPX1 Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 0
  DPX2 Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 0
  DPX3 Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 0
  Flowcell Type	Text	Read Only	Default = S4
  Instruction Notes	Multiline Text	Read Only	Default = Enter the No. of Lanes for your S4 flow cell to calculate DPX 1, DPX2 and DPX 3 volumes.
  Loading Workflow Type	Text	Read Only	Default = NovaSeq Xp
  No. of Lanes	Numeric	Required Field	Default = 4

• Step File Placeholders

  • Log File - 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	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Loading Workflow Type	Text Dropdown	Required Field	Presets NovaSeq Standard NovaSeq Xp [Remove from workflow]
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 2: Load to Flowcell (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = Load to Flowcell (Illumina COVIDSeq Workflow v1.6)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Validate Inputs and Selected Container

• Trigger Location = Step

• 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 script:validate_flowcell_for_input_pools -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -validateSelectedContainer true"

Validate Flowcell Barcode

• Trigger Location = Placement

• 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 'if (input.::Flowcell Type:: == ::SP:: && !output.container.name.matches( ::[A-Z0-9]{5}DR[A-Z0-9]{2}:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; if (input.::Flowcell Type:: == ::S1:: && !output.container.name.matches( ::[A-Z0-9]{5}DR[A-Z0-9]{2}:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; if (input.::Flowcell Type:: == ::S2:: && !output.container.name.matches( ::[A-Z0-9]{5}DM[A-Z0-9]{2}:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; if (input.::Flowcell Type:: == ::S4:: && !output.container.name.matches( ::[A-Z0-9]{5}DS[A-Z0-9]{2}:: ) ) {fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) } ; output.::Flowcell Type:: = input.::Flowcell Type:: ; output.::Loading Workflow Type:: = input.::Loading Workflow Type::' \
        -log {compoundOutputFileLuid1}"

Validate Run Setup and Generate 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/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
    script:evaluateDynamicExpression \
        -exp '{udf:Validation Script}' \
        -log {compoundOutputFileLuid1} \
        -t true \
&& if [ '{udf:Reverse Complement Workflow}' == 'true' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Reverse_Complement_Samplesheet.csv'; \
elif [ '{udf:Reverse Complement Workflow}' == 'false' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Samplesheet.csv'; \
else echo 'UDF Reverse Complement Workflow is empty or undefined.'; exit -1; fi \
&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
    script:driver_file_generator \
        -t /opt/gls/clarity/extensions/conf/driverfiletemplates/$templateFilename \
        -o {compoundOutputFileLuid0}.csv \
        -q true \
        -destLIMSID {compoundOutputFileLuid0} \
        -l {compoundOutputFileLuid1}"

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	Well	Built-in
    Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

  • Placement Pattern = Column

• Destination Containers

  • S4

  

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  BaseSpace Sequence Hub Configuration	Text Dropdown	Required Field	Presets Not Used Run Monitoring Only Run Monitoring And Storage
  Custom Recipe Path	Text
  Experiment Name	Text	Required Field
  Index Read 1	Numeric Dropdown	Required Field Custom Entries	Presets 0 6 8 Range = 0 To 20 Decimal Places Displayed = 0
  Index Read 2	Numeric Dropdown	Required Field Custom Entries	Presets 0 6 8 Range = 0 To 20 Decimal Places Displayed = 0
  Library Tube Barcode	Text	Required Field
  Output Folder	Text	Required Field
  Paired End	Text Dropdown	Required Field	Presets True False
  Read 1 Cycles	Numeric Dropdown	Required Field Custom Entries	Presets 251 151 101 51 Range = 1 To 251 Decimal Places Displayed = 0
  Read 2 Cycles	Numeric Dropdown	Required Field Custom Entries	Presets 251 151 101 51 Range = 0 To 251 Decimal Places Displayed = 0
  Reverse Complement Workflow	Toggle Switch	Required Field	Default = Yes
  UMI - Read 1 Length	Numeric		Range From 1
  UMI - Read 1 Start From Cycle	Numeric		Range From 1
  UMI - Read 2 Length	Numeric		Range From 1
  UMI - Read 2 Start From Cycle	Numeric		Range From 1
  Use Custom Index Read 1 Primer	Toggle Switch		Default = None Set
  Use Custom Read 1 Primer	Toggle Switch		Default = None Set
  Use Custom Read 2 Primer	Toggle Switch		Default = None Set
  Use Custom Recipe	Toggle Switch	Required Field	Default = No
  Workflow	Text	Required Field	Default = GenerateFASTQ
  Workflow Type	Text Dropdown	Required Field	Presets No Index Single Index Dual Index Custom

• Step File Placeholders

  • Sample Sheet - Automatically attached

  • Log File - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Loading Workflow Type	Text Dropdown	Required Field	Presets NovaSeq Standard NovaSeq Xp [Remove from workflow]
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 7: AUTOMATED - NovaSeq Run (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: AUTOMATED - NovaSeq Run (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = AUTOMATED - NovaSeq Run (Illumina COVIDSeq Workflow v1.6)

• Step Type = Analysis

• Measurement Generation = Variable

• Naming Convention = Lane {InputWellLocation}

• Reagent Kits

  • Buffer Cartridge

    • Supplier = Illumina

  • Cluster Cartridge

    • Supplier = Illumina

  • SBS Cartridge

    • Supplier = Illumina

Automations

Update Lane Number

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "sleep 10 && /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 'if (input.::Loading Workflow Type:: == ::NovaSeq Standard::) { output.::name:: = ::Lane :: + step.::Lane Counter::; output.::name:: = output.::name::.split(::\\.::)[0] + :::1::; step.::Lane Counter::++; }' \
        -log {compoundOutputFileLuid3} \
        -t true \
&& /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 'step.::Lane Counter:: = 1' \
        -log {compoundOutputFileLuid3} \
        -t true"

Set Next Steps

• 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 -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	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Current Cycle	Numeric	Read Only	Decimal Places Displayed = 0
  Current Read	Numeric	Read Only	Decimal Places Displayed = 0
  Firmware Version	Text	Read Only
  Flow Cell Expiration Date	Date	Read Only
  Flow Cell ID	Text	Read Only
  Flow Cell Lot Number	Text	Read Only
  Flow Cell Mode	Text	Read Only
  Flow Cell Part Number	Text	Read Only
  Flow Cell Side	Text	Read Only
  Instrument Control Software Version	Text	Read Only
  Instrument ID	Text	Read Only
  Instrument Type	Text	Read Only
  Loading Workflow Type	Text	Read Only
  Output Folder	Text	Read Only
  RTA Version	Text	Read Only
  Run ID	Text	Read Only
  Run Status	Text	Read Only
  Sequencing Log	Multiline Text	Read Only

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Row

  • File Column Options

    • File Column Display = Hide

    • File Attachment Method = Auto

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	% Aligned R1	Numeric	Read Only	Decimal Places Displayed = 2
    Container	% Aligned R2	Numeric	Read Only	Decimal Places Displayed = 2
    Container	% Bases >=Q30 R1	Numeric	Read Only	Decimal Places Displayed = 2
    Container	% Bases >=Q30 R2	Numeric	Read Only	Decimal Places Displayed = 2
    Container	% Error Rate R1	Numeric	Read Only	Decimal Places Displayed = 2
    Container	% Error Rate R2	Numeric	Read Only	Decimal Places Displayed = 2
    Container	% Phasing R1	Numeric	Read Only	Decimal Places Displayed = 3
    Container	% Phasing R2	Numeric	Read Only	Decimal Places Displayed = 3
    Container	% Prephasing R1	Numeric	Read Only	Decimal Places Displayed = 3
    Container	% Prephasing R2	Numeric	Read Only	Decimal Places Displayed = 3
    Container	%PF R1	Numeric	Read Only	Decimal Places Displayed = 2
    Container	%PF R2	Numeric	Read Only	Decimal Places Displayed = 2
    Container	Container Name	Built-in
    Container	Cluster Density (K/mm^2) R1	Numeric	Read Only
    Container	Cluster Density (K/mm^2) R2	Numeric	Read Only
    Container	Intensity Cycle 1 R1	Numeric	Read Only
    Container	Intensity Cycle 1 R2	Numeric	Read Only
    Container	LIMS ID (Container)	Built-in
    Container	Reads PF (M) R1	Numeric	Read Only	Decimal Places Displayed = 2
    Container	Reads PF (M) R2	Numeric	Read Only	Decimal Places Displayed = 2
    Container	Well	Built-in
    Container	Yield PF (Gb) R1	Numeric	Read Only	Decimal Places Displayed = 2
    Container	Yield PF (Gb) R2	Numeric	Read Only	Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 8: Analysis Results (Illumina COVIDSeq Workflow v1.6)

Protocol Type = Data Analysis

Next Steps Configuration

Step 1: Analysis Results (Illumina COVIDSeq Workflow v1.6)

• Master Step Name = COVIDSeq Analysis Results (Illumina COVIDSeq Workflow v1.6)

• Step Type = Demultiplexing

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName} (FASTQ reads) {AppliedReagentLabels}

Automations

CovidSEQ: Parse TSV Run Report

• Trigger Location = Record Details

• Trigger Style = Manual button

/usr/bin/python3 /opt/gls/clarity/extensions/conf/covid/parseCovidSeqRunReport.py -u {username} -p {password} -s {stepURI} -f {compoundOutputFileLuid1} -g {compoundOutputFileLuid3}

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table (Column Headers)

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

Record Details

• Step Data

  • Step Data Heading = WARNING: It might take several minutes to parse the TSV Run Report

• Step File Placeholders

  • Log - Automatically attached

  • COVIDSeq TSV_Run Report File - Manually uploaded

  • COVIDSeq_RunReport PDF - Manually uploaded

  • Parsing Log - Automatically attached

• Sample Table

  • 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	Sample Name	Built-in
    Measurement	Consensus Sequence	Text	Read Only
    Measurement	Control Type	Text	Read Only
    Measurement	Human Control	Text	Read Only
    Measurement	Index ID	Text	Read Only
    Measurement	Index ID for Control Sample	Text	Read Only
    Measurement	Index Set	Text	Read Only
    Measurement	Index Set for Control Sample	Text	Read Only
    Measurement	Internal Control	Text	Read Only
    Measurement	Lane	Numeric	Read Only
    Measurement	Lane for Control Sample	Numeric	Read Only
    Measurement	Quality Control	Text	Read Only
    Measurement	Result	Text	Read Only
    Measurement	SARS-CoV-2	Text	Read Only
    Measurement	SARS-CoV-2 for Control Sample	Text	Read Only
    Project	Project Name	Built-in