Real-Time RT-PCR Prep v1.1

Overview

The Real-Time RT-PCR Prep CDC COVID-19 RT-PCR is part of the CDC COVID-19 RT-PCR protocols that include the following functionality.

Preconfigured CDC COVID-19 RT-PCR protocols that support the preparation of upper and lower respiratory specimens for real-time RT-PCR qualitative detection of nucleic acid from the 2019-nCoV.
Automated calculation of sample and buffer volumes.
Automated calculation or display of reagents at every step in the protocol.
Automatic step transition when required.
Automatic placement of samples when necessary.
Automated assignment of QC Pass/Fail, based on user-selected threshold values.

Protocol 1: Real-Time RT-PCR Prep (CDC COVID-19 RT-PCR v1.1)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Assay Set Up Sorting (CDC COVID-19 RT-PCR v1.1)

Master Step Name = Assay Set Up Sorting (CDC COVID-19 RT-PCR v1.1)
Step Type = No Outputs

Automations

Set Next Step - RT-qPCR

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 'if (input.::RT-qPCR Assay:: == ::Thermofisher TaqPath 1-Step RT-qPCR::) {nextStep = ::Thermofisher TaqPath 1-Step RT-qPCR (CDC COVID-19 RT-PCR v1.1)::}  ; if (input.::RT-qPCR Assay:: == ::Promega GoTaq Probe 1-Step RT qPCR::) {nextStep = ::Promega GoTaq Probe 1-Step RT qPCR (CDC COVID-19 RT-PCR v1.1)::} ; if (input.::RT-qPCR Assay:: == ::Quantabio qScript XLT One-Step RT-qPCR Tough Mix::) {nextStep = ::Quantabio qScript XLT One-Step RT-qPCR Tough Mix (CDC COVID-19 RT-PCR v1.1)::} ; if (input.::RT-qPCR Assay:: == ::Quantabio UltraPlex 1-Step ToughMix (4X)::) {nextStep = ::Quantabio UltraPlex 1-Step ToughMix (4X) (CDC COVID-19 RT-PCR v1.1)::}' \
-log {compoundOutputFileLuid0}"

Queue/Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
Sample Table (Column Headers)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 2: Thermofisher TaqPath 1-Step RT-qPCR (CDC COVID-19 RT-PCR v1.1)

Master Step Name = Thermofisher TaqPath 1-Step RT-qPCR (CDC COVID-19 RT-PCR v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 3
Naming Convention = {InputItemName}-{LIST:N1,N2,P}
Reagent Kits
- Thermofisher TaqPath 1-Step RT-qPCR Master Mix, CG
  - Supplier = Thermofisher
  - Catalog Number = A15299; A15300
Control Types
- HSC
- nCoVPC
- NTC

Automations

auto placement for 7500 Covid-19 assay

Trigger Location = Placement
Trigger Style = Automatic upon entry

/usr/bin/python3 /opt/gls/clarity/extensions/conf/covid/autoPlaceCovid7500.py -u {username} -p {password} -s {stepURI}

Calculate Total Samples and 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} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Nuclease-free Water (uL):: = step.::Total Samples:: * 8.5 ; \
step.::Combined Primer/Probe Mix (uL):: = step.::Total Samples:: * 1.5 ; \
step.::TaqPath 1-Step RT-qPCR Master Mix (4x) (uL):: = step.::Total Samples:: * 5 ; \
step.::Total Volume (uL):: = step.::Total Samples:: * 15' \
-log {compoundOutputFileLuid0}"

Set Next Step - RT-qPCR Assay

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 = ::ABI 7500 Fast DX (CDC COVID-19 RT-PCR v1.1)::' \
-log {compoundOutputFileLuid0}"

copy PCR Product LIMSID to Sample

Trigger Location = Step
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'submittedSample.::PCR Product LIMSID:: = input.node.@limsid' \
-log {compoundOutputFileLuid0}"

Queue/Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
Sample Table (Column Headers)

Placement = Enabled

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
- Placement Pattern = Row
Destination Containers
- 96 well plate

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 3: Promega GoTaq Probe 1-Step RT qPCR (CDC COVID-19 RT-PCR v1.1)

Master Step Name = Promega GoTaq Probe 1-Step RT qPCR (CDC COVID-19 RT-PCR v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 3
Naming Convention = {InputItemName}-{LIST:N1,N2,P}
Reagent Kits
- Promega GoTaq Probe 1- Step RT-qPCR System
  - Catalog Number = A6120; A6121
Control Types
- HSC
- nCoVPC
- NTC

Automations

auto placement for 7500 Covid-19 assay

Trigger Location = Placement
Trigger Style = Automatic upon entry

/usr/bin/python3 /opt/gls/clarity/extensions/conf/covid/autoPlaceCovid7500.py -u {username} -p {password} -s {stepURI}

Calculate Total Samples and 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} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Nuclease-free Water (uL):: = step.::Total Samples:: * 3.1 ; \
step.::Combined Primer/Probe Mix (uL):: = step.::Total Samples:: * 1.5 ; \
step.::GoTaq Probe qPCR Master Mix with dUTP (uL):: = step.::Total Samples:: * 10 ; \
step.::Go Script RT Mix for 1-Step RT-qPCR (uL):: = step.::Total Samples:: * 0.4 ; \
step.::Total Volume (uL):: = step.::Total Samples:: * 15' \
-log {compoundOutputFileLuid0}"

Set Next Step - RT-qPCR Assay

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 = ::ABI 7500 Fast DX (CDC COVID-19 RT-PCR v1.1)::' \
-log {compoundOutputFileLuid0}"

copy PCR Product LIMSID to Sample

Trigger Location = Step
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'submittedSample.::PCR Product LIMSID:: = input.node.@limsid' \
-log {compoundOutputFileLuid0}"

Queue/Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
Sample Table (Column Headers)

Placement = Enabled

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
- Placement Pattern = Row
Destination Containers
- 96 well plate

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 4: Quantabio qScript XLT One-Step RT-qPCR Tough Mix (CDC COVID-19 RT-PCR v1.1)

Master Step Name = Quantabio qScript XLT One-Step RT-qPCR Tough Mix (CDC COVID-19 RT-PCR v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 3
Naming Convention = {InputItemName}-{LIST:N1,N2,P}
Reagent Kits
- Quantabio qScript XLT One-Step RT-qPCR ToughMix
  - Catalog Number = 95132-100; 95132-02K; 95132-500
Control Types
- HSC
- nCoVPC
- NTC

Automations

auto placement for 7500 Covid-19 assay

Trigger Location = Placement
Trigger Style = Automatic upon entry

/usr/bin/python3 /opt/gls/clarity/extensions/conf/covid/autoPlaceCovid7500.py -u {username} -p {password} -s {stepURI}

Calculate Total Samples and 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} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Nuclease-free Water (uL):: = step.::Total Samples:: * 3.5 ; \
step.::Combined Primer/Probe Mix (uL):: = step.::Total Samples:: * 1.5 ; \
step.::qScript XLT One-Step RT-qPCR Tough Mix (uL):: = step.::Total Samples:: * 10 ; \
step.::Total Volume (uL):: = step.::Total Samples:: * 15' \
-log {compoundOutputFileLuid0}"

Set Next Step - RT-qPCR Assay

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 = ::ABI 7500 Fast DX (CDC COVID-19 RT-PCR v1.1)::' \
-log {compoundOutputFileLuid0}"

copy PCR Product LIMSID to Sample

Trigger Location = Step
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'submittedSample.::PCR Product LIMSID:: = input.node.@limsid' \
-log {compoundOutputFileLuid0}"

Queue/Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
Sample Table (Column Headers)

Placement = Enabled

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
- Placement Pattern = Row
Destination Containers
- 96 well plate

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 5: Quantabio UltraPlex 1-Step ToughMix (4X) (CDC COVID-19 RT-PCR v1.1)

Master Step Name = Quantabio UltraPlex 1-Step ToughMix (4X) (CDC COVID-19 RT-PCR v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 3
Naming Convention = {InputItemName}-{LIST:N1,N2,P}
Reagent Kits
- Quantabio UltraPlex 1-Step ToughMix (4X)
  - Catalog Number = 95166-100; 95166-500; 95166-01K
Control Types
- HSC
- nCoVPC
- NTC

Automations

auto placement for 7500 Covid-19 assay

Trigger Location = Placement
Trigger Style = Automatic upon entry

/usr/bin/python3 /opt/gls/clarity/extensions/conf/covid/autoPlaceCovid7500.py -u {username} -p {password} -s {stepURI}

Calculate Total Samples and 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} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Nuclease-free Water (uL):: = step.::Total Samples:: * 8.5 ; \
step.::Combined Primer/Probe Mix (uL):: = step.::Total Samples:: * 1.5 ; \
step.::UltraPlex 1-Step ToughMix (4x) (uL):: = step.::Total Samples:: * 5 ; \
step.::Total Volume (uL):: = step.::Total Samples:: * 15' \
-log {compoundOutputFileLuid0}"

Set Next Step - RT-qPCR Assay

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 = ::ABI 7500 Fast DX (CDC COVID-19 RT-PCR v1.1)::' \
-log {compoundOutputFileLuid0}"

copy PCR Product LIMSID to Sample

Trigger Location = Step
Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'submittedSample.::PCR Product LIMSID:: = input.node.@limsid' \
-log {compoundOutputFileLuid0}"

Queue/Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
Sample Table (Column Headers)

Placement = Enabled

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
- Placement Pattern = Row
Destination Containers
- 96 well plate

Record Details

Step Data (Master Step Fields)
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 6: ABI 7500 Fast DX (CDC COVID-19 RT-PCR v1.1)

Master Step Name = ABI 7500 Fast DX (CDC COVID-19 RT-PCR v1.1)
Step Type = No Outputs

Automations

Generate ABI Instrument File

Trigger Location = Record Details
Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -cp /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar driver_file_generator -i {processURI} -u {username} -p {password} -t /opt/gls/clarity/extensions/conf/covid/7900sds_container_setup_template.txt -o {compoundOutputFileLuid1}.txt -l {compoundOutputFileLuid0}.html && /opt/gls/clarity/bin/java -cp /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar addBlankLines -i {stepURI} -u {username} -p {password} -f {compoundOutputFileLuid1}.txt -l {compoundOutputFileLuid0}.html -sep TAB -b '0' -h 7 -c PLACEMENT"

PLACEHOLDER - Parse ABI File

Trigger Location = Record Details
Trigger Style = Manual button

need parser

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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
- Instrument Driver File - Automatically attached
- Upload Result File - Manually upload
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 7: Report Results (CDC COVID-19 RT-PCR v1.1)

Master Step Name = Report Results (CDC COVID-19 RT-PCR v1.1)
Step Type = No Outputs

Automations

Assign Pos/Neg for Ct Value

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 true -h false -exp 'if (step.::Criteria - Ct Threshold:: >= input.::Ct Value::.toFloat()) {input.::Result:: = ::Positive::} ; if (step.::Criteria - Ct Threshold:: < input.::Ct Value::.toFloat()) {input.::Result:: = ::Negative::} ; ' -log {compoundOutputFileLuid0}"

Generate Results File

Trigger Location = Record Details
Trigger Style = Manual button

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

Set Next Step - Advance

Trigger Location = Record Details
Trigger Style = Automatic upon exit

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

Queue/Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
Sample Table (Column Headers)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
- Results File (.csv) - Automatically attached
- File Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Column
- Table Columns - Global Fields

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