Illumina Preset Protocol - Illumina COVIDSeq Workflow

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)

Master Step 1: Prep Sample - COVIDSeq

Step Type: Standard

Input = Purified Total RNA

Output = Denatured purified total RNA

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina COVIDSeq Positive Control HT

Illumina

  

Illumina COVIDSeqTest Box 4

Illumina

20043436
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

1.7 mL Tube

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step

Placement Screen

Auto

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

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

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Spin Purification of RNA - COVIDSeq

Step Type: Standard

Input = Swab sample

Output = Purified viral RNA

Reagent Kits

Kit Name

Supplier

Cat. #

QIAmp Viral RNA Mini kit

Qiagen

(50) - 52904; (250) - 52906
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step

Placement Screen

Auto

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

% EtOH

Numeric
• 96 (default)
• Range = 96-100
   

% EtOH Prep Date

Date

  
   

No. of Preps

Numeric Dropdown
• 50 (default)
• 250
   

AW1 Concentrate (mL)

Numeric

Logic = If No. of Preps = 50 then 19; if No. of Preps = 250 then 98
   

% EtOH for AW1 (mL)

Numeric

Logic = If No. of Preps = 50 then 25; if No. of Preps = 250 then 130
   

AW2 Concentrate (mL)

Numeric

Logic = If No. of Preps = 50 then 13; if No. of Preps = 250 then 66
   

% EtOH for AW2 (mL)

Numeric

Logic = If No. of Preps = 50 then 30; if No. of Preps = 250 then 160
   

Total Samples

Numeric
• 0 (default)
• Calculated = Total Samples + 1
   

Buffer AVL (mL)

Numeric

Calculated = 0.56 * Total Samples
   

Carrier RNA-Buffer AVE (uL)

Numeric

Calculated = Buffer AVL (mL) * 10
   

Elution Incubation Start Time

Text

  
   

Elution Incubation End Time

Text

  
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Elute in 30 µl Buffer AVE instead of 60 µl. Incubate elution for at least 1 minute.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Calculate Total Samples

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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 and AVL Reagent Volumes

Trigger Location

Record Details

  
 

Trigger Style

Manual button

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 3: Anneal Sample - COVIDSeq

Step Type: Standard

Input = Purified viral RNA

Output = Annealed samples

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina COVIDSeq Test Box 5 – 3072 Samples

Illumina

20043648
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• COVIDSeq Anneal
 

Multiline Text Fields

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.
   

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.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 2: cDNA Synthesis (Illumina COVIDSeq Workflow)

Master Step 1: Synthesize First Strand cDNA - COVIDSeq

Step Type: Standard

Input = Annealed RNA

Output = cDNA

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina COVIDSeq Test Box 6 – 3072 Samples

Illumina

20043647
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step and step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric

Default = 0

Calculated = Total Samples + 1

 

 

FSA (uL)

Numeric

Calculated = 9 * Total Samples

 

  

RVT (uL)

Numeric

Calculated = 1 * Total Samples
   

Thermal Cycler Program

Text

Default = COVIDSeq FSS
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Add 8 µl master mix to each well of the CDNA1 plate.
   

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.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Calculate First Strand cDNA Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Protocol 3: Target Amplification (Illumina COVIDSeq Workflow)

Master Step 1: Amplify cDNA - COVIDSeq

Step Type: Standard

Input = cDNA

Output = Amplified Targeted cDNA

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina COVIDSeq Test Box 5 – 3072 Samples

Illumina

20043648

Illumina COVIDSEQ Test Box 7 – 3072 Samples

Illumina

20043439
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step and step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric

Default = 0

Calculated = Total Samples + 1

 

 

IPM HT for COVIDSeq PCR 1 Master Mix (uL)

Numeric

Calculated = 15 * Total Samples

 

  

CPP1 HT for COVIDSeq PCR 1 Master Mix (uL)

Numeric

Calculated = 4.3 * Total Samples
   

Nuclease-free water for COVIDSeq PCR 1 Master Mix (uL)

Numeric

Calculated = 4.7 * Total Samples
   

IPM HT for COVIDSeq PCR 2 Master Mix (uL)

Numeric

Calculated = 15 * Total Samples
   

CPP2 HT for COVIDSeq PCR 2 Master Mix (uL)

Numeric

Calculated = 4.3 * Total Samples
   

Nuclease-free water for COVIDSeq PCR 2 Master Mix (uL)

Numeric

Calculated = 4.7 * Total
   

Thermal Cycler Program

Text

Default = COVIDSeq PCR
 

Multiline Text Fields

COVIDSeq PCR Program 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.
   

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.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Calculate COVIDSeq PCR 1 and PCR2 Master Mixes

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 4: Library Prep (Illumina COVIDSeq Workflow)

Master Step 1: Tagment PCR Amplicons - COVIDSeq

Step Type: Standard

Input = Amplified Targeted cDNA

Output = Tagmented amplicons

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina COVIDSeqTest Box 3 – 3072 Samples

Illumina

20043646

Illumina COVIDSEQ Test Box 7 – 3072 Samples

Illumina

20043439
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step and step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric

Default = 0

Calculated = Total Samples + 1

 

 

TB1 (uL)

Numeric

Calculated = 12 * Total Samples

 

  

eBLT (uL)

Numeric

Calculated = 4 * Total Samples
   

Nuclease-free water (uL)

Numeric

Calculated = 20 * Total Samples
   

Thermal Cycler Program

Text

Default = COVIDSeq TAG
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Add 30 µl Tagmentation Master Mix to each well in TAG1 plate.
   

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.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Calculate Tagmentation Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Post Tagmentation Clean Up - COVIDSeq

Step Type: No Input

Input = Tagmented amplicons

Output = Cleaned up tagmented amplicons

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina COVIDSeq Test Box 2 – 3072 Samples

Illumina

20043434

Illumina COVIDSeqTest Box 4 – 3072 Samples

Illumina

20043436
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  
         
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 3: Amplify Tagmented Amplicons - COVIDSeq

Step Type: Add Labels

Input = Cleaned up tagmented amplicons

Output = Amplified tagmented amplicons

Reagent Kit

Kit Name

Supplier

Cat. #

llumina COVIDSeq Test Box 5 – 3072 Samples

Illumina

20043648
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step and step
Add Labels

Setting

Value

Label Groups

IDT for Illumina Nextera DNA Unique Dual Indexes Set A for NovaSeq, MiSeq, HiSeq 2500, HiSeq 2000
 

IDT for Illumina Nextera DNA Unique Dual Indexes Set B for NovaSeq, MiSeq, HiSeq 2500, HiSeq 2000
 

IDT for Illumina Nextera DNA Unique Dual Indexes Set C for NovaSeq, MiSeq, HiSeq 2500, HiSeq 2000
 

IDT for Illumina Nextera DNA Unique Dual Indexes Set D for NovaSeq, MiSeq, HiSeq 2500, HiSeq 2000
 

IDT for Illumina Nextera DNA Unique Dual Indexes Set A-D for NovaSeq, MiSeq, HiSeq 2500, HiSeq 2000
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric
• 0 (default)
• Calculated = Total Samples + 1

 

 

EPM HT (uL)

Numeric

Calculated = 24 * Total Samples

 

  

Nuclease-free water (uL)

Numeric

Calculated = 24 * Total Samples
   

Thermal Cycler Program

Text

Default

• COVIDSeq TAG PCR
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Add 40 µl PCR Master Mix to each well.
   

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.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Calculate PCR Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 4: Pool and Clean Up Libraries - COVIDSeq

Step Type: Pooling

Input = Amplified tagmented amplicons

Output = Pooled and cleaned up libraries

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina COVIDSeqTest Box 3 – 3072 Samples

Illumina

20043646
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Pooling

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers
• 1x8 strip tube
• 96 well plate

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Date

Date

  

 

 

Total Pooled ITB Tubes

Numeric

Calculated = logic (total pooled ITB tubes)

 

  

ITB (uL)

Numeric

Calculated = 0.9 * (55 * Total Pooled ITB Tubes)
 

Multiline Text Fields

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.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Calculate ITB Volume

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Protocol 5: Quantification (Illumina COVIDSeq Workflow)

Master Step 1: Quantify Libraries - COVIDSeq Qubit

Step Type: Standard QC

Input = Pooled and cleaned up libraries

Output = Measurement

Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

Text

Default

• Concentration
   

Criteria 1 - Operator

Text

Default

• >=
   

Criteria 1 - Threshold Value

Numeric

  
   

Criteria 2 - Source Data Field

Text

Default

• Concentration
   

Criteria 2 - Operator

Text

Default

• <=
   

Criteria 2 - Threshold Value

Numeric

  

Step File Placeholders

 

File Name

Log

Auto
     

QC Log File

Auto
     

QC Result File

Manual
     

Upload File

Manual

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample & measurement
   

Conc. Units

Text

Global field - derived sample & measurement
Automation

Automation Name

Setting

Value

Specification

Assign QC Flags

Trigger Location

Record Details

  
 

Trigger Style

Manual

  
     

Command line

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

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Manual

Must move forward

Optional

• Dilute Libraries (Illumina COVIDSeq Workflow)
• Normalize Libraries (Illumina COVIDSeq Workflow)

Master Step 2: Dilute Libraries - COVIDSeq

Step Type: Standard

Input = DNA with concentration

Output = Diluted DNA

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina COVIDSeqTest Box 3 – 3072 Samples

Illumina

20043646
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Final Volume (uL)

Numeric

Default

• 10

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Concentration

Numeric
• Global field - derived sample
• Copied from previous step

 
   

Conc. Units

Text
• Global field - derived sample
• Copied from previous step
   

SampleVolume (uL)

Numeric
• Global field - derived sample
• Calculated = Final Volume (uL) * 0.1
   

RSB (uL)

Numeric
• Global field - derived sample
• Calculated = Final Volume (uL) - SampleVolume (uL)
Automation

Automation Name

Setting

Value

Specification

Copy Concentration

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

  
     

Command line

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 'input.::SampleVolume (uL):: = 0.1 * step.::Final Volume (uL):: ; input.::RSB (uL):: = step.::Final Volume (uL):: - input.::SampleVolume (uL):: ' -log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 3: Quantify Libraries - COVIDSeq Qubit

Step Type: Standard QC

Input = Diluted libraries

Output = Measurement

Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

Text

Default

• Concentration
   

Criteria 1 - Operator

Text

Default

• >=
   

Criteria 1 - Threshold Value

Numeric

  
   

Criteria 2 - Source Data Field

Text

Default

• Concentration
   

Criteria 2 - Operator

Text

Default

• <=
   

Criteria 2 - Threshold Value

Numeric

  

Step File Placeholders

 

File Name

Log

Auto
     

QC Log File

Auto
     

QC Result File

Manual
     

Upload File

Manual

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample

   

Conc. Units

Text

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Assign QC Flags

Trigger Location

Record Details

  
 

Trigger Style

Manual

  
     

Command line

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

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Manual

Must move forward

 

Master Step 3: Normalize Libraries - COVIDSeq

Step Type: Standard

Input = Quantified diluted library

Output = Normalized library

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina COVIDSeqTest Box 3 – 3072 Samples

Illumina

20043646
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Final Molarity (nM)

Numeric

Default

• 4
   

Final Volume (uL)

Numeric

Default

• 30

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Concentration

Numeric

Copied from previous step
   

Conc. Units

Text

Copied from previous step
   

Starting Molarity (nM)

Numeric

Calculated = Concentration / 660 * 400
   

Library Pool (uL)

Numeric

Calculated = (Final Molarity (nM) * Final Volume (uL))/Starting Molarity (nM)
   

RSB (uL)

Numeric

Calculated = Final Volume (uL) - Library Pool (uL)
Automation

Automation Name

Setting

Value

Specification

Calculate Molarity, Library Pool & RSB

Trigger Location

Record Details

  
 

Trigger Style

Manual

  
     

Command line

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 'input.::Starting Molarity (nM):: = input.::Concentration:: / (660 * 400) ; input.::Library Pool (uL):: = (step.::Final Molarity (nM):: * step.::Final Volume (uL)::) / step.::Starting Molarity (nM):: ; input.::RSB (uL):: = step.::Final Volume (uL):: - input.::Library Pool (uL):: ' -log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 4: Pool and Dilute Libraries - COVIDSeq

Step Type: Pooling

Input = Normalized library

Output = Pooled and diluted libraries

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina COVIDSeqTest Box 3 – 3072 Samples

Illumina

20043646
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step

Well Sort Order

Column

Configured on master step

Placement Pattern

Column

Configured on master step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes

Multiline Text

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, then centrifuge briefly. The resulting final loading concentration is 100 pM.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

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

Master Step 1: NovaSeq Denature & ExAmp - COVIDSeq

Step Type: Standard

Reagent Kit

Kit Name

Supplier

Cat. #

DPX1

Illumina

20019916

DPX2

Illumina

20019917

DPX3

Illumina

20019918
Placement

Setting

Value

Specification

Destination Containers

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

No. of Lanes

Numeric

Default

• 4
   

DPX1 Volume (ul)

Numeric

Calculated = 315 * No. of Lanes
   

DPX2 Volume (ul)

Numeric

Calculated = 45 * No. of Lanes
   

DPX3 Volume (ul)

Numeric

Calculated = 165 * No. of Lanes
   

0.2 N NaOH Prep Date

Date

  
   

0.2 N NaOH per pool (uL)

Numeric

Default

• 7
   

400 mM Tris-HCI Prep Date

Date

  
   

400 mM Tris-HCI per pool (uL)

Numeric

Default

• 8
   

Flowcell Type

Numeric

Default

Read Only

• S4

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Flowcell Type

Numeric

Calculated = step Flowcell Type
Automation

Automation Name

Setting

Value

Specification

Calculate DPX1, DPX2, DPX3

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Load to Flowcell (Illumina COVIDSeq Workflow)

For configuration details, see the Clarity LIMS NovaSeq Integration documentation.

Protocol 7: Automated - NovaSeq Run (Illumina COVIDSeq Workflow)

Master Step 1: Automated - NovaSeq Run (Illumina COVIDSeq Workflow)

For configuration details, see the Clarity LIMS NovaSeq Integration documentation.

Protocol 8: Analysis Results (Illumina COVIDSeq Workflow)

Master Step 1: COVIDSeq analysis results

Step Type: Demultiplexing

Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step File Placeholders

 

File Name

Log

Auto
     

COVIDSeq TSV_Run Report File

Manual
     

COVIDSeq_RunReport PDF

Manual
     

Parsing Log

Auto

Sample Table

 

Well Sort Order

Column

  

Sample Table

Table Columns

Quality Control

Text
• Parsed via custom script
• Measurement
   

Internal Control

Text
• Parsed via custom script
• Measurement
   

Result

Text
• Parsed via custom script
• Measurement
   

Consensus sequence

Text
• Parsed via custom script
• Measurement
   

Lane

Numeric
• Parsed via custom script
• Measurement
   

Index Set

Text
• Parsed via custom script
• Measurement
   

Index ID

Text
• Parsed via custom script
• Measurement
   

Control Type

Text
• Parsed via custom script
• Measurement
   

Human Control

Text
• Parsed via custom script
• Measurement
   

SARS-CoV-2 for Control Sample

Text
• Parsed via custom script
• Measurement
   

Lane for Control Sample

Numeric
• Parsed via custom script
• Measurement
   

Index Set for Control Sample

Text
• Parsed via custom script
• Measurement
   

Index ID for Control Sample

Text
• Parsed via custom script
• Measurement
Automation

Automation Name

Setting

Value

Specification

CovidSEQ: Parse TSV Run Report

Trigger Location

Record Details

 

 

Trigger Style

Manual Button

 

 

 

 

Command line

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

End of Workflow