Illumina Preset Protocol - Illumina DNA PCR-Free Library Prep Manual

The Illumina DNA PCR-Free Library Prep Manual workflow includes the following functionality.

• Preset Illumina DNA PCR-Free Library Prep Manual protocols that support the preparation of up to 96 dual-indexed paired-end single-stranded libraries from DNA using the Illumina DNA PCR-Free Library Prep workflow.
• 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: Sample Prep (Illumina DNA PCR-Free Library Prep Manual)

Protocol Type: Sample Prep

Master Step 1: Sort Sample (Illumina DNA PCR-Free Library Prep Manual)

Step Type: No Outputs

Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

Configured on master step
Record Details

Section

Subsection

Setting

Value

Specification
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Select a sample type and protocol type for each sample.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Sample Type for DNA PCR-Free Lib Prep

Text Dropdown
• Required
• Global field - derived sample
• Custom entries = no

Presets

• gDNA [default]
• Whole Blood
• Dried Blood Spot
• Saliva
   

Protocol Type

Text Dropdown
• Required
• Global field - derived sample
• Custom entries = no

Presets

• Thermal Cycler [default]
• Hybex
Automation

Automation Name

Setting

Value

Specification

Set Next Step - DNA PCR-Free Lib Prep

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 'if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::gDNA:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Qubit - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::gDNA:: && input.::Protocol Type:: == ::Hybex::) {nextStep = ::Qubit - Hybex (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Whole Blood:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Dried Blood Spot:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Saliva:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0)::}' \

-log {compoundOutputFileLuid0}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 2: Qubit - Hybex (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard QC

Queue/Ice Bucket

Setting

Value

Specification

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

Default

• 300
• Range = 300- 2000
   

Criteria 2 - Source Data Field

Text

Default

• Concentration
   

Criteria 2 - Operator

Text

Default

• <=
   

Criteria 2 - Threshold Value

Numeric

Default

• 2000
• Range = 300 -2000
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

Read Only

1. Upload or enter Concentration and Conc. Units.
2. Set Threshold Values then click on Assign QC Flags.

Step File Placeholders

 

File Name

Log

Auto
     

QC Log File

Auto
     

QC Result File

Manual
     

Upload File

Manual

Sample Table

 

Well sort order

Column

  
 

Table Columns

      
   

Concentration

Numeric

Global field - derived sample & measurement
   

Conc. Units

Text

Global field - derived sample & measurement
   

Sample Type

Text
• Global field - derived sample & measurement
• Calculated = copy "Sample Type for DNA PCR-Free Lib Prep" from previous step
Automation

Automation Name

Setting

Value

Specification

Copy Sample Type for DNA PCR-Free Lib Prep

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.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep:: ' \

-log {compoundOutputFileLuid0}"

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-REMOVE and Copy Concentration & Sample Type

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

Routing - gDNA Hybex

Trigger Location

Step

  
 

Trigger Style

Automatic upon exit

  
     

Command line

bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Sample Type' \

--FIELD_VALUE 'gDNA' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0' \

--STEP 'Tagment Genomic DNA (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'INPUTS'"

Next Step

Setting

Value

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 3: Qubit - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard QC

Queue/Ice Bucket

Setting

Value

Specification

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

Default

• 25
   

Criteria 2 - Source Data Field

Text

Default

• Concentration
   

Criteria 2 - Operator

Text

Default

• <=
   

Criteria 2 - Threshold Value

Numeric

Default

• 2000
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

1. Upload or enter Concentration and Conc. Units.
2. Set Threshold Values then click on Assign QC Flags.

Step File Placeholders

 

File Name

Log

Auto
     

QC Log File

Auto
     

QC Result File

Manual
     

Upload File

Manual

Sample Table

 

Well sort order

Column

  
 

Table Columns

Concentration

Numeric

Global field - derived sample & measurement
   

Conc. Units

Text

Global field - derived sample & measurement
   

Sample Type

Text
• Global field - derived sample & measurement
• Calculated = copy "Sample Type for DNA PCR-Free Lib Prep" from previous step
   

Prep Input Type

Text
• Global field - derived sample & measurement
• Calculated = if concentration is btw 25-99 then Prep Input Type = Low, if concentration is between 100-2000 then Prep Input Type = Standard
Automation

Automation Name

Setting

Value

Specification

Copy Sample Type for DNA PCR-Free Lib Prep

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.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep:: ' \

-log {compoundOutputFileLuid0}"

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 - REMOVE, Set Prep Input Type, Copy Concentration & Sample Type

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 -exp 'nextStep = ::REMOVE:: ; \

if (output.::Concentration:: >= 25 && output.::Concentration:: <= 99) {output.::Prep Input Type:: = ::Low::} ; \

if (output.::Concentration:: >= 100 && output.::Concentration:: <= 2000) {output.::Prep Input Type:: = ::Standard::} ; \

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

Routing - gDNA Thermal Cycler

Trigger Location

Step

  
 

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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Low' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0' \

--STEP 'Tagment Genomic DNA - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'INPUTS' \

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Standard' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0' \

--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'INPUTS'"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 4: Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Lysis Reagent Kit

Illumina

20042221
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

  
   

Total Samples

Numeric

Read Only

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

MLB (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 30 * 1.1
   

PK1 (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 2 * 1.1
   

Nuclease-free water (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 248 * 1.1
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

Read Only

1. Combine MLB, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube.
2. Add 280 µl of lysis master mix to a new 2 mL tube.
3. Add 20 µl to each 2 mL tube, mix by pipetting then vortex briefly and centrifuge.
4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly.
5. Add 135 µl of IPB, invert tube, vortex for 15 secs to mix.
6. Incubate at RT for 5 mins.
7. Place tube on magnetic stand for 5 mins then remove and discard supernatant.
8. Wash beads 2x with 500 µl of fresh 80% EtOH for each wash.
9. Air dry on magnetic stand for 5 mins.
10. Add 35 µl of RSB to each tube and vortex to mix.
11. Label a new 96 well PCR plate with LP1 and add 32 µl of supernatant of each sample to a new well.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

 

 

Table Columns

Sample Type

Text
• Global field - derived sample & measurement
• Calculated = copy "Sample Type for DNA PCR-Free Lib Prep" from previous step
Automation

Automation Name

Setting

Value

Specification

Calculate Whole Blood Lysis Master Mix & Copy Sample Type

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 'step.::Total Samples:: = step.::Total Samples:: + 1 ; \

step.::MLB (uL):: = step.::Total Samples:: * 30 *1.1 ; \

step.:: PK1 (uL):: = step.::Total Samples:: * 2 * 1.1 ; \

step.:: Nuclease-free water (uL):: = step.::Total Samples:: * 248 * 1.1 ; \

output.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep::’ \

-log {compoundOutputFileLuid0}"

Set Next Step - Remove

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 = ::REMOVE::' -log {compoundOutputFileLuid0}"

Routing - Whole Blood Lysis

Trigger Location

Step

  
 

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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Sample Type' \

--FIELD_VALUE 'Whole Blood' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 5: Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Lysis Reagent Kit

Illumina

20042221
Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

Configured on master step
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

80% EtOH Prep Date

Date

  
   

Total Samples

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

10x Lysis Buffer (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 30 * 1.1

 
   

PK1 (µl)

Numeric

Calculated = Total Samples * 2 * 1.1
   

Nuclease-free water (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 268 * 1.1
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

1. Combine 10x Lysis Buffer, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube.
2. Add 300 µl of lysis master mix to a new 2 mL tube.
3. Add 6 x 3 mm² punches to a each 2 ml tube, mix by pipetting then vortex briefly and centrifuge.
4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly.
5. Without removing the punches, transfer all supernatant from each tube to a new 2 ml tube.
6. Add 135 µl of IPB, invert tube, vortex for 15 secs to mix.
7. Incubate at RT for 5 mins.
8. Place tube on magnetic stand for 5 mins then remove and discard supernatant.
9. Wash beads 2x with 500 µl of fresh 80% EtOH for each wash.
10. Air dry on magnetic stand for 5 mins.
11. Add 35 µl of RSB to each tube and vortex to mix.
12. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear.
13. Label a new 96 well PCR plate with LP1 and add 32 µl of supernatant of each sample to a new well.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  

Table Columns

Sample Type

Text
• Global field - derived sample
• Calculated = copy "Sample Type for DNA PCR-Free Lib Prep" from previous step
Automation

Automation Name

Setting

Value

Specification

Calculate Dried Blood Lysis Master Mix & Copy Sample Type

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.::10x Lysis Buffer (uL):: = step.::Total Samples:: * 30 *1.1 ; \

step.:: PK1 (uL):: = step.::Total Samples:: * 2 * 1.1 ; \

step.:: Nuclease-free water (uL):: = step.::Total Samples:: * 268 * 1.1 ; \

output.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib::' \

-log {compoundOutputFileLuid0}"

Set Next Step - Remove

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 = ::REMOVE::' -log {compoundOutputFileLuid0}"

Routing - Dried Blood Spot Lysis

Trigger Location

Step

  
 

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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Sample Type' \

--FIELD_VALUE 'Dried Blood Spot' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 6: Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Lysis Reagent Kit

Illumina

20042221
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

  
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

1. Incubate saliva overnight at 50°C in a water bath or air incubator.
2. Add 250 µl of Nuclease-Free Water to a new 2 mL tube.
3. Invert each heat-treated saliva collection tube 5 times to mix, add 50 µl of Saliva to each 2 mL tube, pipette to mix, vortex briefly, and then centrifuge.
4. Add 135 µl of IPB, invert tube, and vortex for 15 secs to mix.
5. Incubate at RT for 5 mins.
6. Place tube on magnetic stand for 5 mins then remove and discard supernatant.
7. Wash beads 2x with 500 µl of fresh 80% EtOH for each wash.
8. Air dry on magnetic stand for 5 mins.
9. Add 35 µl of RSB to each tube and vortex to mix.
10. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear.
11. Label a new 96 well PCR plate with LP1 and add 32 µl of supernatant of each sample to a new well.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

 

Table Columns

Sample Type

Text
• Global field - derived sample
• Calculated = copy "Sample Type for DNA PCR-Free Lib Prep" from previous step
Automation

Automation Name

Setting

Value

Specification

Copy Sample Type for DNA PCR-Free Lib Prep

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.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep:: ' \

-log {compoundOutputFileLuid0}"

Set Next Step - Remove

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 = ::REMOVE::' -log {compoundOutputFileLuid0}"

Routing - Saliva Lysis

Trigger Location

Step

  
 

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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Sample Type' \

--FIELD_VALUE 'Saliva' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 2: Library Prep - Hybex (Illumina DNA PCR-Free Library Prep Manual)

Protocol Type: Library Prep

Master Step 1: Tagment Genomic DNA (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

User input
   

Desired DNA Input (ng)

Numeric
• Decimal places displayed = 0
• Range = 300 - 2000
• User input
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

1. Label a new 96 well MIDI plate with LP1 and add the calculated DNA and RSB volumes of each sample to a new well on the plate.
2. Add 10 µl of TB1 to each well.
3. Add 15 µl of BLT-PF to each well.
4. Seal and shake at 1800 rpm for 1 minute.
5. Incubate in pre-heated Hybex for 8 minutes.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  

 

Table Columns

Concentration

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Conc. Units

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

DNA (µl)

Numeric

Read Only

• Global field - derived sample
• Decimal places displayed = 0
• Calculated = Desired DNA Input (ng) / Concentration
   

RSB (µl) = 25 - DNA (µl)

    
   

Desired DNA Input (ng)

Numeric

Read Only

• Global field - derived sample & measurement
• Decimal places displayed = 0
• Calculated = copy from step field
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 DNA Volume & Copy Desired DNA Input

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 'output.::DNA (uL):: = output.::Desired DNA Input (ng):: / output.::Concentration:: ; \

output.::RSB (uL):: = 25 - step.::DNA (uL):: ; \

output.:: Desired DNA Input (ng):: = step.::Desired DNA Input (ng)::' \

-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 Cleanup (Illumina DNA PCR-Free Library Prep Manual)

Step Type: No Outputs

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

Configured on master step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Add 10 µl ST2 to each well.
• Seal and then shake at 1800 rpm for 1 minute.
• Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
• Without disturbing the bead pellet, remove and discard all supernatant from each well.
• Add 150 μl TWB to each well.
• Seal and shake at 1800 rpm for 1 minute.
• Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

Table Columns

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = copy from previous step
Automation

Automation Name

Setting

Value

Specification

Copy Desired DNA Input

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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \

-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: Ligate Indexes (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Add Labels

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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
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

Read Only

• Decimal places displayed = 0
• 0 (default)
• Calculated =Total Samples + 1
   

HP3 (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 6
   

Nuclease-free water (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 54
 

Multiline Text Fields

Instruction Notes for Hybex Protocol

Multiline Text

Read Only

Default

1. Remove and discard all supernatant.
2. Add 45 µl ELM to each well.
3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min.
4. Incubate in the preheated Hybex for 8 mins.
5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
6. Remove and discard all supernatant from each well.
7. Add 75 µl TWB onto the beads in each well then seal and shake at 1800 rpm for 1 minute.
8. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
9. Remove and discard all supernatant from each well.
10. Without disturbing the bead pellet, use a 20 µl pipette to remove and discard residual TWB from each well.
11. Add 45 µl diluted HP3 to each well then seal and shake at 1800 rpm for 1 minute.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  

 

Table Columns

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = Copy from previous step
Automation

Automation Name

Setting

Value

Specification

Calculate Dilution for HP3 & Copy Desired DNA Input

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 'output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng):: ; \

step.::Total Samples:: = step.::Total Samples:: + 1 ; \

step.::HP3 (uL):: = step.::Total Samples:: * 6 ; \

step.::Nuclease-free water (uL):: = step.::Total Samples:: * 54' \

-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: Clean Up Libraries (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

User select
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

1. Add 36 µl IPB to each well then seal and shake at 1800 rpm for 1 minute.
2. Incubate at room temperature for 2 minutes.
3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
4. Label a new 96-well MIDI plate LP2.
5. Add 42 µl IPB to each well of LP2.
6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of LP2 then seal and shake at 1800 rpm for 1 minute.
7. Discard LP1 and incubate LP2 at room temperature for 2 minutes.
8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
9. Without disturbing the bead pellet, remove and discard all supernatant from each well.
10. Wash beads 2x with 180 µl of fresh 80% EtOH for each wash.
11. Using a 20 µl pipette, remove residual EtOH from each well.
12. Air-dry on the magnetic stand (~4 minutes).
13. Add 22 µl of RSB onto the beads in each well then seal and shake at 1800 rpm for 1 minute.
14. Incubate at room temperature for 2 minutes.
15. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
16. Label a new PCR plate FLP.
17. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

 

Table Columns

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Protocol Type

Text
• Global field - derived sample
• Calculated = Hybex
Automation

Automation Name

Setting

Value

Specification

Copy Desired DNA Input

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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \

-log {compoundOutputFileLuid0}"

Set Next Step-Remove & Protocol Type

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 -exp 'nextStep = ::REMOVE:: ; \

output.::Prep Input Type:: = ::Standard::' -log {compoundOutputFileLuid0}"

Routing - Quantify & Pool Libraries

Trigger Location

Step

  
 

Trigger Style

Automatic upon exit

  
     

Command line

bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Standard' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Low' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'"

       
       
       
Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 3: Library Prep - Thermal Cycler Standard Input (Illumina DNA PCR-Free Library Prep Manual)

Protocol Type: Library Prep

Master Step 1: Tagment Genomic DNA - Standard Input

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

User input
   

Desired DNA Input (ng)

Numeric

Required

• Range = 100 - 2000
• User input
   

Thermal Cycler Program

Text

Default

• TAG
 

Multiline Text Fields

Thermal Cycler Program Notes

Multiline Text

Default

Read Only

1. Choose the preheat lid option and set to 100°C.
2. Set the reaction volume to 50 µl u 41°C for 5 minutes.
   

Instruction Notes

Multiline Text

Default

Read Only

1. Label a new 96 well PCR plate LP1.
2. Add the calculated DNA and RSB volumes to the LP1 plate and then pipette to mix.
3. Add the calculated BLT-PF to each well and pipette to mix.
4. Add 10 µl of TB1 to each well, pipette to mix, and then seal.
5. Place the LP1 plate on the thermo cycler and run the TAG program.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

 

Table Columns

Concentration

Numeric
• Global field - derived sample
• Calculated = if sample type = gDNA then copy from previous step otherwise NA
   

Conc. Units

Numeric
• Global field - derived sample
• Calculated = if sample type = gDNA then copy from previous step otherwise NA
   

Sample Type

Text
• Global field - derived sample
• Calculated = copy from previous step
   

DNA (µl)

Numeric
• Global field - derived sample
• Calculated = if sample type = gDNA then Desired DNA Input (ng) / Concentration otherwise = 30
   

RSB (µl)

Numeric
• Global field - derived sample
• Calculated = if sample type = gDNA then 25 - DNA (µl) otherwise = 0
   

BLT-PF (µl)

Numeric
• Global field - derived sample
• Calculated = if sample type = gDNA then = 15 otherwise = 10
   

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = copy from step field
Automation

Automation Name

Setting

Value

Specification

Copy Sample Type & 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.::Sample Type:: = input.::Sample Type:: ; \

if (output.::Sample Type:: == ::gDNA::) {output.::Concentration:: = input.::Concentration::} else {output.::Concentration:: = 0} ; \

if (output.::Sample Type:: == ::gDNA::) {output.::Conc. Units:: = input.::Conc. Units::} else {output.::Conc. Units:: = ::NA::}' \

-log {compoundOutputFileLuid0}"

Calculate Dilution & BLT-PF Volumes and Copy Desired DNA Input

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 'output.::Desired DNA Input (ng):: = step.::Desired DNA Input (ng):: ; \

if (output.::Sample Type:: == ::gDNA::) {output.::DNA (uL):: = output.::Desired DNA Input (ng):: / output.::Concentration::} ; \

if (output.::Sample Type:: == ::Whole Blood::) {output.::DNA (uL):: = 30} ; \

if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::DNA (uL):: = 30} ; \

if (output.::Sample Type:: == ::Saliva::) {output.::DNA (uL):: = 30} ; \

if (output.::Sample Type:: == ::gDNA::) {output.::RSB (uL):: = 25 - output.::DNA (uL)::} ; \

if (output.::Sample Type:: == ::Whole Blood::) {output.::RSB (uL):: = 0} ; \

if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::RSB (uL):: = 0} ; \

if (output.::Sample Type:: == ::Saliva::) {output.::RSB (uL):: = 0} ; \

if (output.::Sample Type:: == ::gDNA::) {output.::BLT-PF (uL):: = 15} ; \

if (output.::Sample Type:: == ::Whole Blood::) {output.::BLT-PF (uL):: = 10} ; \

if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::BLT-PF (uL):: = 10} ; \

if (output.::Sample Type:: == ::Saliva::) {output.::BLT-PF (uL):: = 10}' \

-log {compoundOutputFileLuid0}"

Set Next Step-Post TAG

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 true -h false \

-exp 'if (input.::Sample Type:: == ::Whole Blood::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (input.::Sample Type:: == ::Dried Blood Spot::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (input.::Sample Type:: == ::Saliva::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (input.::Sample Type:: == ::gDNA::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0)::}' \

-log {compoundOutputFileLuid0}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Tagment Genomic DNA - Low Input

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

User input
   

Desired DNA Input (ng)

Numeric

Required

• Default = 25 [user can change]
• Range = 25 - 99
   

Total Samples

Numeric

Read Only

• Decimal Places Displayed = 0
• Default = 0
• Calculated = Total Samples + 1
   

BLT-PF (µl)

Numeric

Read Only

• Decimal Places Displayed = 0
• Calculated = Total Samples * 11
   

TB1 (µl)

Numeric

Read Only

• Decimal Places Displayed = 0
• Calculated = Total Samples * 11
   

Thermal Cycler Program

Text

Default = TAG
 

Multiline Text Fields

Thermal Cycler Program Notes

Multiline Text

Read Only

Default

1. Choose the preheat lid option and set to 100°C.
2. Set the reaction volume to 50 µl u 41°C for 5 minutes.
   

Instruction Notes

Multiline Text

Read Only

Default

1. Label a new 96 well MIDI plate LP1.
2. Combine the calculated BLT-PF and TB1 volumes into a 1.5 mL tube to prepare the Tagmentation Master Mix.
3. Add the calculated DNA and RSB volumes to the LP1 plate and then pipette to mix.
4. Add 20 µl Tagmentation Master Mix to each well, pipette to mix, and then seal.
5. Place the LP1 plate on the thermo cycler and run the TAG program.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

Table Columns

Concentration

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Conc. Units

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Sample Type

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

DNA (µl)

Numeric
• Global field - derived sample
• Calculated = Desired DNA Input (ng) / Concentration
   

RSB (µl)

Numeric
• Global field - derived sample
• Calculated = 30 - DNA (µl)
   

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = copy from step field
Automation

Automation Name

Setting

Value

Specification

Calculate Tag Master Mix and Copy Concentration & Sample Type

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:: ; \

output.::Sample Type:: = input.::Sample Type:: ; \

step.::Total Samples:: = step.::Total Samples:: + 1 ; \

step.::BLT-PF (uL):: = step.::Total Samples:: * 11 ; \

step.::TB1 (uL):: = step.::Total Samples:: * 11' \

-log {compoundOutputFileLuid0}"

Calculate Dilution and Copy Desired DNA Input

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 'output.::Desired DNA Input (ng):: = step.::Desired DNA Input (ng):: ; \

output.::DNA (uL):: = output.::Desired DNA Input (ng):: / output.::Concentration:: ; \

output.::RSB (uL):: = 30 - output.::DNA (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: Post Tagmentation Cleanup

Step Type: No Outputs

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

 
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

1. Add 10 µl ST2 to each well, seal, and then shake at 1800 rpm for 1 minute.
2. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
3. Without disturbing the bead pellet, remove and discard all supernatant from each well.
4. Add 150 μl TWB to each well, seal, and shake at 1800 rpm for 1 minute.
5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

 

Table Columns

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Sample Type

Text
• Global field - derived sample
• Calculated = copy from previous step
Automation

Automation Name

Setting

Value

Specification

Copy Desired DNA Input & Sample Type

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.::Sample Type:: = input.::Sample Type:: ; \

output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \

-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: Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Add Labels

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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
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

Read Only

• Decimal places displayed = 0
• Default = 0
• Calculated =Total Samples + 1
   

HP3 (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 6
   

Nuclease-free water (µl)

Numeric

Read Only

• Decimal places displayed = 0
• Calculated = Total Samples * 54
   

Thermal Cycler Program

Text

Default = ELM
 

Multiline Text Fields

Thermal Cycler Program Notes

Multiline Text

Default

ELM Program

1. Choose the preheat lid option and set to 100°C.
2. Set the reaction volume to 50 μl.
3. Run for 37°C for 5 minutes.
4. Run for 50°C for 5 minutes.
   

Instruction Notes

Multiline Text

Default

Read Only

1. Remove and discard all supernatant.
2. Add 45 µl ELM to each well and pipette to mix.
3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min.
4. Place on the preprogrammed thermal cycler and run the ELM program.
5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
6. Remove and discard all supernatant from each well.
7. Add 75 µl TWB onto the beads in each well then pipette to mix.
8. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
9. Remove and discard all supernatant from each well.
10. Seal and centrifuge at 280 x g for 10 seconds and then place on magnetic stand.
11. Without disturbing the bead pellet, remove and discard residual TWB from each well.
12. Remove the plate from the magnetic stand.
13. Add 45 µl diluted HP3 to each well, pipette to mix, and then incubate at RT for 2 mins.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  

Table Columns

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = Copy from previous step
Automation

Automation Name

Setting

Value

Specification

Calculate Dilution for HP3 & Copy Desired DNA Input

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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng):: ; \

step.::Total Samples:: = step.::Total Samples:: + 1 ; \

step.::HP3 (uL):: = step.::Total Samples:: * 6 ; \

step.::Nuclease-free water (uL):: = step.::Total Samples:: * 54' \

-log {compoundOutputFileLuid0}"

Set Next Step - Clean Up Libraries

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 true -h false \

-exp 'if (output.::Desired DNA Input (ng):: >= 100) {nextStep = ::Clean Up Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)::} ; \

if (output.::Desired DNA Input (ng):: <= 99) {nextStep = ::Clean Up Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0)::}' \

-log {compoundOutputFileLuid0}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 5: Clean Up Libraries - Thermal Cycler

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

User select
 

Multiline Text Fields

Instruction Notes for Standard Input

Multiline Text

Read Only

Default

1. Add 36 µl IPB to each well containing BLT-PF beads and pipette to mix.
2. Incubate at room temperature for 2 minutes.
3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
4. Label a new 96-well PCR plate LP2.
5. Add 42 µl IPB to each well of LP2.
6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of the LP2, pipette to mix and remove LP1 from the magnetic stand, then discard.
7. Incubate LP2 at room temperature for 2 minutes.
8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
9. Without disturbing the bead pellet, remove and discard all supernatant from each well.
10. Wash beads 2x with 180 µl fresh 80% ethanol per well for each wash.
11. Seal and then centrifuge 280 x g for 10 seconds.
12. Place on the magnetic stand, and then wait 10 seconds.
13. Remove residual EtOH from each well, air-dry on the magnetic stand (~2 minutes) then remove from the magnetic stand.
14. Add 22 µl RSB onto the beads in each well and pipette to mix.
15. Incubate at room temperature for 2 minutes.
16. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
17. Label a new PCR plate FLP.
18. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

  

Well Sort Order

Column

  

Table Columns

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Prep Input Type

Text
• Global field - derived sample
• Calculated = If Desired DNA Input (ng) is >= 300 then Prep Input Type = Standard else Prep Input Type = Low
Automation

Automation Name

Setting

Value

Specification

Copy Desired DNA Input

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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \

-log {compoundOutputFileLuid0}"

Set Next Step-Remove & Prep Input Type

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 -exp 'nextStep = ::REMOVE:: ; \

if (output.::Desired DNA Input (ng):: >= 300) {output.::Prep Input Type:: = ::Standard::} else {output.::Prep Input Type:: = ::Low::}' \

-log {compoundOutputFileLuid0}"

Routing - Quantify & Pool Libraries

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Standard' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS' \

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Low' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 6: Clean Up Libraries - Thermal Cycler

Step Type: Standard

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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

80% EtOH Prep Date

Date

User select
 

Multiline Text Fields

Instruction Notes for Low Input

Multiline Text

Read Only

Default

1. Add 81 µl IPB to each well and pipette to mix.
2. Incubate at room temperature for 5 minutes.
3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
4. Remove and discard all supernatant from each well.
5. Wash beads 2x with 180 µl of fresh 80% EtOH for each wash.
6. Seal plate then centrifuge 280 x g for 10 seconds.
7. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
8. Remove residual EtOH from each well and air-dry on the magnetic stand (~2 minutes).
9. Remove the plate from the magnetic stand.
10. Add 15 µl RSB onto the beads in each well and pipette to resuspend.
11. Incubate at room temperature for 2 minutes.
12. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
13. Transfer 14 µl supernatant from each well of the plate to the corresponding well of a new PCR plate.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

Table Columns

Desired DNA Input (ng)

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Prep Input Type

Text
• Global field - derived sample
• Calculated = If Desired DNA Input (ng) is >= 300 then Prep Input Type = Standard else Prep Input Type = Low
Automation

Automation Name

Setting

Value

Specification

Copy Desired DNA Input

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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \

-log {compoundOutputFileLuid0}"

Set Next Step-Remove & Prep Input Type

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 -exp 'nextStep = ::REMOVE:: ; \

if (output.::Desired DNA Input (ng):: >= 300) {output.::Prep Input Type:: = ::Standard::} else {output.::Prep Input Type:: = ::Low::}' \

-log {compoundOutputFileLuid0}"

Routing - Quantify & Pool Libraries

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Standard' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS' \

\

--FIELD_NAME 'Prep Input Type' \

--FIELD_VALUE 'Low' \

--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v0.1' \

--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 4: Quantify and Pool Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual)

Protocol Type: Library Prep

Master Step 1: Pool Samples (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Pooling

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina DNA PCR-Free Library Prep

Illumina

24 - 20041794; 96 - 20041795
Queue/Ice Bucket

Setting

Value

Specification

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

Read Only

1. Combine 9 µl of each library in a 1.5 or 1.7 ml microcentrifuge tube.
2. Vortex to mix and then centrifuge at 280 x g for 1 minute.

Step File Placeholders

 

File Name

Log

Auto
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: Qubit (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard QC

Queue/Ice Bucket

Setting

Value

Specification

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

  

Well Sort Order

Column

  

 

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

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 5: Quantify and Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual)

Protocol Type: Library Prep

Master Step 1: KAPA Sample Prep - Illumina/Universal

Step Type: Standard

Reagent Kits

Kit Name

Supplier

10 mM Tris-HCl, pH 8.0 – 8.5 (25°C)

  

0.05% Tween® 20

  
Control Types

Control Name

Supplier

Cat. #

Specification

KAPA Library Quantification Dilution Control

Roche

KK4906-07960417001

Concentration = 200 pM

NTC

    

Concentration = 0

KAPA DNA Standard 1

Roche

KK4828-07960166001

Concentration = 20 pM

KAPA DNA Standard 2

Roche

KK4828-07960166001

Concentration = 2 pM

KAPA DNA Standard 3

Roche

KK4828-07960166001

Concentration = 0.2pM

KAPA DNA Standard 4

Roche

KK4828-07960166001

Concentration = 0.02 pM

KAPA DNA Standard 5

Roche

KK4828-07960166001

concentration = 0.002 pM

KAPA DNA Standard 6

Roche

KK4828-07960166001

concentration = 0.0002 pM
Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

 
Placement

Setting

Value

Specification

Destination Containers
• 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

DNA Dilution Buffer Prep Date

Date

  
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Prepare the appropriate library dilutions (using DNA dilution buffer). Depending on the expected concentration of the library, 1:1,000 – 1:100,000 dilutions may be appropriate.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  
 

 

Sample Display Default

Expand

  

 

Table Columns

Desired DNA Input (ng)

Numeric

Calculated = copy from previous step
   

Expected Concentration (pM)

Numeric
• Range = 0 - 333000
• Calculated = Desired DNA Input (ng) * 3.36 * 1000
   

Dilution Factor

Numeric
• Range = 1000 - 100000
• Calculated = if Expected Concentration (pM) <= 188000 and >= 84000 then dilution factor = 10,000; if Desired DNA Input (ng) is >=189000 and <=333000 dilution factor = 20,000; if Expected Concentration (pM) <= 83000 then dilution factor = 0
Automation

Automation Name

Setting

Value

Specification

Copy Desired DNA Input for KAPA

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 'if (output.name.contains(::KAPA DNA Standard 1::)) {output.::Desired DNA Input (ng):: = 0.005952} ; \

if (output.name.contains(::KAPA DNA Standard 2::)) {output.::Desired DNA Input (ng):: = 0.000595} ; \

if (output.name.contains(::KAPA DNA Standard 3::)) {output.::Desired DNA Input (ng):: = 0.00006} ; \

if (output.name.contains(::KAPA DNA Standard 4::)) {output.::Desired DNA Input (ng):: = 0.000006} ; \

if (output.name.contains(::KAPA DNA Standard 5::)) {output.::Desired DNA Input (ng):: = 0.0000006} ; \

if (output.name.contains(::KAPA DNA Standard 6::)) {output.::Desired DNA Input (ng):: = 0.00000006} ; \

if (output.name.contains(::KAPA Library Quantification Dilution Control::)) {output.::Desired DNA Input (ng):: = 0.059524} ; \

if (output.name.contains(::KAPA NTC::)) {output.::Desired DNA Input (ng):: = 0} ; \

if (!output.name.contains(::KAPA::)) {output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::}' \

-log {compoundOutputFileLuid0}"

Calculate Expected Concentration & Dilution Factor

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 true -h false \

-exp 'output.::Expected Concentration (pM):: = output.::Desired DNA Input (ng):: * 3.36 * 1000 ; \

if (output.::Expected Concentration (pM):: >= 84000 && output.::Expected Concentration (pM):: <= 188000) {output.::Dilution Factor:: = 10000} ; \

if (output.::Expected Concentration (pM):: >= 189000 && output.::Expected Concentration (pM):: <= 333000) {output.::Dilution Factor:: = 20000} ; \

if (output.::Expected Concentration (pM):: <= 83000) {output.::Dilution Factor:: = 0}' \

-log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false \

-exp 'if (output.::Expected Concentration (pM):: > 333000) {fail(::Expected Concentration is too high, please edit Dilution Factor and recalculate::)}' \

-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: KAPA qPCR Prep & Analysis - Illumina/Universal

Step Type: Standard QC

Measurement Generation = Variable

Reagent Kits

Kit Name

Supplier

Cat. #

KAPA qPCR library quantification kit - Illumina/Universal

Roche

KK4828-07960166001
Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

 
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

qPCR Master Mix with Primer Prep Date

Numeric

  
   

Reaction Volume (µl)

Numeric Dropdown
• 20 (default)
• 10
   

Adding Rox?

Text Dropdown
• Yes (default)
• No
   

Total Samples

Numeric

Read Only

• Decimal Places Displayed = 0
• 0 (default)
• Calculated = Total Samples + 1
   

KAPA SYBR FAST qPCR Master Mix + Primer Premix (µl)

Numeric

Read Only

• Decimal Places Displayed = 0
• Calculated = if Reaction Volume (µl) is 20 then total samples * 12 else total samples * 6
   

ROX (µl)

Numeric

Read Only

• Decimal Places Displayed = 0
• Calculated = if Reaction Volume (µl) is 20 and Adding Rox? is Yes then total samples * 0.4, if Reaction Volume (µl) is 20 and Adding Rox? is No then 0, if Reaction Volume (µl) is 10 and Adding Rox? is Yes then total samples * 0.2, if Reaction Volume (µl) is 10 and Adding Rox? is No then 0
   

PCR-grade water (µl)

Numeric

Read Only

• Decimal Places Displayed = 0
• Calculated = if Reaction Volume (µl) is 20 and Adding Rox? is Yes then total samples * 3.6, if Reaction Volume (µl) is 20 and Adding Rox? is No then total samples * 4, if Reaction Volume (µl) is 10 then 0
   

Criteria 1 - Source Data Field

Text

Default

• Cq
   

Criteria 1 - Operator

Text

Default

• >=
   

Criteria 1 - Threshold Value

Numeric

Default

• 25
   

Criteria 2 - Source Data Field

Text

Default

• Cq
   

Criteria 2 - Operator

Text

Default

• <=
   

Criteria 2 - Threshold Value

Numeric

Default

• 25
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

1. Set reaction volume and adding Rox? then click on calculate qPCR Master Mix and Sample Volume button.
2. Combine KAPA SYBR FAST qPCR Master Mix + Primer Premix, ROX and PCR-grade water to produce the qPCR Master Mix.
3. Add the qPCR Master Mix and Sample volume to a new 96 well PCR plate, seal the plate, vortex briefly to mix, and then centrifuge.
4. Place the plate on the qPCR instrument.
5. Select Absolute Quantification and run the following cycling protocol:
– Initial denaturation Temp. at 95C fro 5 mins for 1 cycle
– Denaturation Temp. at 95C for 30 secs for 35 cycles
– Annealing/Extension/Data acquisition Temp. at 95C for 45 sec for 35 cycles
– Melt curve analysis (C) between 65C - 95C
6. Upload Cq values.
7. Set Cq Thresholds then click on Assign QC Flags button.
8. Review Cq values and remove outliers.
9. Click on Calculate Average Cq.

Step File Placeholders

 

File Name

Log

Auto
     

QC Log File

Auto
     

QC Result File

Manual
     

Upload File

Manual

Sample Table

 

Well Sort Order

Column

  
   

Sample Display Default

Expand

  

 

Table Columns

qPCR Master Mix (µl)

Numeric
• Global field - derived sample
• Calculated = if Reaction Volume (µl) = 20 then qPCR Master Mix (µl) = 16; if Reaction Volume (µl) = 10 and Adding Rox? = Yes then qPCR Master Mix (µl) = 6.2; if Reaction Volume (µl) = 10 and Adding Rox? = No then qPCR Master Mix (µl) = 6
   

Sample (µl)

Numeric
• Global field - derived sample
• Calculated = 4
   

Cq

Numeric
• Global field - derived sample & measurement
• User input or parse from Cq Result file
   

Average Cq

Numeric
• Global field - derived sample & measurement
• Calculated = Average Cq of replicate
   

Dilution Factor

Numeric
• Global field - derived sample
• Calculated = copied from previous step
Automation

Automation Name

Setting

Value

Specification

Calculate qPCR Master Mix & Sample Volume

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} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false \

-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; \

if (step.::Reaction Volume (uL):: == 20) {step.::KAPA SYBR FAST qPCR Master Mix + Primer Premix (uL):: = step.::Total Samples:: * 12} else {step.::KAPA SYBR FAST qPCR Master Mix + Primer Premix (uL):: = step.::Total Samples:: * 6} ; \

if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::Yes::) {step.::ROX (uL):: = step.::Total Samples:: * 0.4} ; \

if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::No::) {step.::ROX (uL):: = 0} ; \

if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::Yes::) {step.::ROX (uL):: = step.::Total Samples:: * 0.2} ; \

if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::No::) {step.::ROX (uL):: = 0} ; \

if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::Yes::) {step.::PCR-grade water (uL):: = step.::Total Samples:: * 3.6} ; \

if (step.::Reaction Volume (uL):: == 20 && step.::Adding Rox?:: == ::No::) {step.::PCR-grade water (uL):: = step.::Total Samples:: * 4} ; \

if (step.::Reaction Volume (uL):: == 10) {step.::PCR-grade water (uL):: = 0} ; \

if (step.::Reaction Volume (uL):: == 20) {output.::qPCR master mix (uL):: = 16} ; \

if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::Yes::) {output.::qPCR master mix (uL):: = 6.2} ; \

if (step.::Reaction Volume (uL):: == 10 && step.::Adding Rox?:: == ::No::) {output.::qPCR master mix (uL):: = 6} ; \

output.::Sample (uL):: = 4' \

-log {compoundOutputFileLuid0}"

Calculate Average Cq

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} \

-u {username} \

-p {password} \

script:computeReplicateAverage \

-src 'Cq' \

-dest 'Average Cq' \

-log {compoundOutputFileLuid0}"

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 - Advance & Copy Dilution Factor and Average Cq

      
 

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

 

Master Step 3: KAPA Library Quantification - Illumina/Universal

Step Type: Standard QC

Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

 
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Library Length (bp)

Numeric

Default

• 450
   

Standard Curve Slope

Numeric

User input
   

Standard Curve Y-Intercept

  

User input
   

Library Length Ratio

Numeric

Read Only

• Decimal Places Displayed = 0
• Calculated = 452 / Library Length (bp)
   

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

 

Well Sort Order

Column

  

 

Table Columns

Average Cq

Numeric
• Global field - derived sample & measurement
• Calculated = copy from previous step
   

Diluted Avg. Conc. (pM)

Numeric
• Global field - derived sample & measurement
• Calculated = POWER(10,(Average Cq - Standard Curve Y-Intercept) / standard curve slope
   

Concentration (nM)

Numeric
• Global field - derived sample & measurement
• Calculated = Diluted Avg. Conc. (pM) * Library Length Ratio * Dilution Factor * 0.001
   

Dilution Factor

Numeric
• Global field - derived sample & measurement
• Calculated = copy from previous step
Automation

Automation Name

Setting

Value

Specification

Calculate Library Length Ratio & Copy Dilution Factor and Average Cq

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.::Average Cq:: = input.::Average Cq:: ; \

output.::Dilution Factor:: = input.::Dilution Factor:: ; \

step.::Library Length Ratio:: = 452 / step.::Library Length (bp)::' \

-log {compoundOutputFileLuid0}"

Calculate Concentration

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} -u {username} -p {password} \

script:evaluateDynamicExpression \

-t true \

-h false \

-exp 'output.::Diluted Avg. Conc. (pM):: = 10 ** ((output.::Average Cq:: - step.::Standard Curve Y-Intercept::) / step.::Standard Curve Slope::) ; \

if (output.::Dilution Factor::.toFloat() >= 1) {output.::Concentration (pM):: = output.::Diluted Avg. Conc. (pM):: * step.::Library Length Ratio:: * output.::Dilution Factor::.toFloat()} else {output.::Concentration (pM):: = output.::Diluted Avg. Conc. (pM):: * step.::Library Length Ratio::} ; \

output.::Concentration (nM):: = 0.001 * output.::Concentration (pM)::' \

-log {compoundOutputFileLuid0}"

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:: ; input.::Sample Type:: = output.::Sample Type::' -log {compoundOutputFileLuid0}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 4: Dilute Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Standard

Reagent Kits

Kit Name

Supplier

RSB

Illumina
Queue/Ice Bucket

Setting

Value

Specification

Well Sort Order

Column

 
Placement

Setting

Value

Specification

Destination Containers
• 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

Final Concentration (nM)

Numeric

Default = 2
   

Final Volume (µl)

Numeric

  

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

 

Table Columns

Concentration (nM)

Numeric
• Global field - derived sample
• Calculated = copy from previous step
   

Sample (µl)

Numeric
• Global field - derived sample
• Calculated = Final Concentration (nM) * Final Volume (µl) / Concentration (nM)
   

RSB (µl)

Numeric
• Global field - derived sample
• Calculated = Final Volume (µl) - Sample (µl)
   

Final Concentration (nM)

Numeric
• Global field - derived sample
• Calculated = copy from step field
Automation

Automation Name

Setting

Value

Specification

Copy Concentration (nM)

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 (nM):: = input.::Concentration (nM)::' \

-log {compoundOutputFileLuid0}"

Calculate Dilution 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 'output.::Sample (uL):: = (step.::Final Concentration (nM):: * step.::Final Volume (uL)::) / output.::Concentration (nM):: ; \

output.::RSB (uL):: = step.::Final Volume (uL):: - output.::Sample (uL):: ; \

output.::Final Concentration (nM):: = step.::Final Concentration (nM)::' \

-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 5: Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual)

Step Type: Pooling

Queue/Ice Bucket

Setting

Value

Specification

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

Sample Volume (µl)

Numeric

  
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

1. Combine 9 µl of each library in a 1.5 or 1.7 ml microcentrifuge tube.
2. Vortex to mix and then centrifuge at 280 x g for 1 minute.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

 

Well Sort Order

Column

  

 

Table Columns

Concentration (nM)

Numeric
• Global field - derived sample
• Calculated = copy "final concentration (nM)" from previous step
Automation

Automation Name

Setting

Value

Specification

Copy Final Concentration (nM)

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 (nM):: = input.::Final Concentration (nM)::' \

-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

End of Protocol