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

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

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}"

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}"

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

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

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}"

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

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}"

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