Illumina Preset Protocol - Illumina RNA Prep with Enrichment (L) Tagmentation

The Illumina RNA Prep with Enrichment (L) Tagmentation workflow includes the following functionality.

• Preset Illumina RNA Prep with Enrichment (L) Tagmentation protocols that support the generation of enriched libraries for dual-indexed, paired-end sequencing.
• 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: Prep Sample (Illumina RNA Prep with Enrichment (L) Tagmentation)

Master Step 1: Denature RNA

Step Type: Standard

Input = Purified Total RNA

Output = Denatured purified total RNA

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Final Diluted Concentration (ng/µl)

Numeric

Default

• 10
• Range = 10 - 100
• Requires user input

 

 

Final Volume (µl)

Numeric

Default

• 8.5
• Requires user input

 

  

Thermal Cycler Program

Text

Default

• DEN_RNA
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

Read Only

• Enter Final Diluted Concentration and Starting Concentration before clicking on the Calculate Sample and Water Volume button.
   

Thermal Cycler Program Notes

Multiline Text

Default

Read Only

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 17 µl.
• 65°C for 5 minutes.
• Hold at 4°C.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Well Sort Order

Column

  
   

Starting Concentration (ng/µl)

Numeric
• Global field - derived sample
• Requires user input
   

SampleVolume (µl)

  
• Global field - derived sample
• Calculated = (Final Diluted Concentration (ng/µl) * Final Volume (µl))/Starting Concentration (ng/µl)

 

  

Nuclease-free Ultrapure Water (µl)

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

Automation Name

Setting

Value

Specification

Calculate Sample and Water Volume

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.::SampleVolume (µl):: = (step.::Final Diluted Concentration (ng/uL):: * step.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; output.::Nuclease-free Ultrapure Water (uL):: = step.::Final Volume (uL):: - output.::SampleVolume (uL)::' \

-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Synthesize First Strand cDNA

Step Type: No Output

Input = Denatured purified total RNA

Output = First strand complementary DNA (cDNA)

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric

Default

• 0
• Calculated = Total Samples + 1

 

 

FSA (µl)

Numeric
• Decimal Places Displayed
• Calculated = 9 * Total Samples

 

  

RVT (µl)

Numeric
• Decimal Places Displayed
• Calculated = 1 * Total Samples
   

Thermal Cycler Program

Text

Default

• FSS
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Add 8 µl of First Strand Synthesis Master Mix to each sample.
   

Thermal Cycler Program Notes

Multiline Text

Default

Read Only

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 25 µl.
• 25°C for 10 minutes.
• 42°C for 15 minutes.
• 70°C for 15 minutes.
• Hold at 4°C.

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate First Strand Synthesis Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::FSA (uL):: = 9 * step.::Total Samples:: ; step.::RVT (uL):: = 1 * step.::Total Samples:: ' -log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 3: Synthesize Second Strand cDNA

Step Type: Standard

Input = First strand complementary DNA (cDNA)

Output = Blunt-ended, double-stranded cDNA fragments

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537

Agencourt AMPure XP, 5 ml

Beckman Coulter

A63880
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Prep Date

Date

  

 

 

Thermal Cycler Program

Text

Default

• SSS
 

Multiline Text Fields

Thermal Cycler Program Notes

Multiline Text

Read Only

Default

• Choose the preheat lid option and set to 40°C.
• Reaction volume is 50 µl u 16°C for 1 hour.
• Hold at 4°C.

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

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 2: Library Prep (Illumina RNA Prep with Enrichment (L) Tagmentation)

Master Step 1: Tagment cDNA

Step Type: No Output

Input = Blunt-ended, double-stranded cDNA fragments

Output = Tagmented double-stranded cDNA

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric
• Calculated = Total Samples + 1

Default

• 0

 

 

TB1 (µl)

Numeric
• Decimal Places Displayed = 0
• Calculated = 11.5 * Total Samples

 

  

EBLTL (µl)

Numeric
• Decimal Places Displayed = 0
• Calculated = 11.5 * Total Samples
   

Nuclease-free ultrapure water (µl)

Numeric
• Decimal Places Displayed = 0
• Calculated = 14.5 * Total Samples
   

Thermal Cycler Program

Text

Default

• TAG
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Add 32.5 µl Tagmentation Master Mix to each well.
   

Thermal Cycler Program Notes

Multiline Text

Default

Read Only

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 50 µl u 55°C for 5 minutes.
• Hold at 10°C.

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate Tagmentation Master Mix - RNA 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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \

-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::TB1 (uL):: = 11.5 * step.::Total Samples:: ; step.::EBLTL (uL):: = 11.5 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 14.5 * step.::Total Samples::' \

-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Amplify Tagmented cDNA

Step Type: Add Labels

Input = Tagmented double-stranded cDNA

Output = Amplified tagmented cDNA

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Add Labels

Setting

Value

Label Groups

IDT-Ilmn DNA-RNA UD Indexes Set A Tagmentation
 

IDT-Ilmn DNA-RNA UD Indexes Set B Tagmentation
 

IDT-Ilmn DNA-RNA UD Indexes Set C Tagmentation
 

IDT-Ilmn DNA-RNA UD Indexes Set D Tagmentation
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric
• Calculated = Total Samples + 1

Default

• 0

 

 

EPM (µl)

Numeric
• Decimal Places Displayed = 0
• Calculated = 23 * Total Samples

 

  

Nuclease-free ultrapure water (µl)

Numeric
• Decimal Places Displayed = 0
• Calculated = 23 µl * Total Samples
   

Thermal Cycler Program

Text

Default

• TAG_PCR
   

PCR Cycles

Text Dropdown
• 14 cycles for High-quality RNA with DV200 > 80%
• 17 cycles for FFPE and RNA with DV200 < 80%
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Add 40 µl PCR Master Mix to each well.
   

Thermal Cycler Program Notes

Multiline Text

Default

Read Only

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 50 µl u 72°C for 3 minutes u 98°C for 3 minutes.
• X cycles of: u 98°C for 20 seconds u 60°C for 30 seconds.
• 72°C for 1 minute u 72°C for 3 minutes.
• Hold at 10°C.

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate PCR Master Mix - RNA 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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \

-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::EPM (uL):: = 23 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 23 * step.::Total Samples::' \

-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 3: Clean Library

Step Type: Standard

Input = Amplified Tagmented cDNA

Output = Purified Tagmented library

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537

Agencourt AMPure XP, 5 ml

Beckman Coulter

A63880
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Prep Date

Date

  

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

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 3: Normalize Libraries (Illumina RNA Prep with Enrichment (L) Tagmentation)

Master Step 1: Qubit Library QC

Step Type: Standard QC

Input = Purified tagmented libraries

Output = Quatified tagmented libraries

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

• 200
   

Criteria 2 - Source Data Field

Text

Default

• Concentration
   

Criteria 2 - Operator

Text

Default

• <=
   

Criteria 2 - Threshold Value

Numeric

Default

• 1500

Step File Placeholders

 

File Name

Log

Auto
     

QC Result File

Manual

Sample Table

Table Columns

Concentration

Numeric

Global fields - derived sample & measurement
   

Concentration

Numeric

Global fields - derived sample & measurement
Automation

Automation Name

Setting

Value

Specification

Assign QC flags

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 -u {username} -p {password} \

script:assignQC \

-i {processURI:v2} \

-log {compoundOutputFileLuid1} \

-qcResult {compoundOutputFileLuid2}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Manual

Safe Stopping Point

Optional:

• Step 2: Bioanalyzer Library QC (Illumina RNA Prep with Enrichment (L) Tagmentation)
• Step 3: Dilute Libraries (Illumina RNA Prep with Enrichment (L) Tagmentation)
• Step 4: Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation)

Master Step 2: Bioanalyzer Library QC

Step Type: Standard QC

Input = Enriched DNA Fragments

Output = Result File

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

 

Default

• Peak 2 Size - bp
   

Criteria 1 - Operator

  

Default

• >=
   

Criteria 1 - Threshold

  

Default

• 300
   

Criteria 2 - Source Data Field

  

Default

• Peak 2 Size - bp
   

Criteria 2 - Operator

  

Default

• <=
   

Criteria 2 - Threshold

  

Default

• 400
   

Use strict matching for Bioanalyzer results

  

Toggle switch

Step File Placeholders

 

File Name

Bioanalyzer Input File

Auto
     

Bioanalyzer Input File Generation Log File

Auto
     

Bioanalyzer XML Result File
• Required
• Manual
     

Result File
• Optional
• Manual
     

PDF Summary File
• Optional
• Manual
     

Bioanalyzer XML Parsing Log File

Auto
     

QC Assignment Log File

Auto
     

QC Assignment Report

Auto

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample & measurement
   

Concentration

Numeric

Global field - derived sample & measurement
Automation

Automation Name

Setting

Value

Specification

Generate Bioanalyzer Input File

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/DriverFileGenerator.jar script:driver_file_generator -i {processURI:v2} -u {username} -p {password} -t /opt/gls/clarity/extensions/ngs-common/v5/EPP/conf/readonly/bioA_driver_file_template.csv -o {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:addBlankLines -i {stepURI:v2} -u {username} -p {password} -f {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} -sep COMMA -b ',False,' -h 1 -c LIMSID -pre 'Sample '"

Parse Bioanalyzer XML and assign QC flags

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 {processURI:v2} -u {username} -p {password} script:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' script:assignQC -log {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

Optional

• Step 3: Dilute Libraries (Illumina RNA Prep with Enrichment (L) Tagmentation)
• Step 4: Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation)

Master Step 3: Dilute Libraries

Step Type: Standard

Input = Quantified Libraries

Output = Diluted Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Enrichment Plexity

Numeric Dropdown

Requires user input

Presets

• 1
• 3
   

Final Diluted Concentration (ng/µl)

Numeric

Requires user input

Default

• 200
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• In one well of a new PCR plate, combine the applicable number of 200 ng libraries.
• If Enrichment Plexity is 1 then add 7.5 µl of the sample.
• If Enrichment Plexity is 3 then add 2.5 µl of each sample to one well with a final volume of 7.5 µl.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Starting Concentration (ng/µl)

Numeric
• Global field - derived sample
• Decimal Places Displayed = 0
• Pulled in from previous step
   

SampleVolume (µl)

Numeric
• Global field - derived sample
• Decimal Places Displayed = 0
• Calculated = (Final Diluted Concentration (ng/µl) * Final Volume (µl))/Starting Concentration (ng/µl)
   

RSB (µl)

Numeric
• Global field - derived sample
• Decimal Places Displayed = 0
• Calculated = Final Volume (µl) - SampleVolume (µl)
   

Final Volume (µl)

Numeric Dropdown
• Global field - derived sample
• Decimal Places Displayed = 0
• Logic: if Enrichment Plexity = 1 then Final Volume = 7.5; if Enrichment Plexity = 3 then Final Volume = 2.5
Automation

Automation Name

Setting

Value

Specification

Calculate Sample, RSB, & Final 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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \

-exp 'output.::SampleVolume (uL):: = (step.::Final Diluted Concentration (ng/uL):: * output.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; if (step.::Enrichment Plexity:: == 1) {output.::Final Volume (uL):: = 7.5} else {output.::Final Volume (uL):: = 2.5} ; step.::RSB (uL):: = output.::Final Volume (uL):: - output.::SampleVolume (uL)::' \

-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 4: Hybridize Probes

Step Type: Standard

Input = Diluted Libraries

Output = Hybridized Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537

Illumina Exome Panel

Illumina

20020183

Respiratory Virus Panel

Illumina

20042472

TruSight RNA Fusion Panel

Illumina

  

TruSight RNA PanCancer Panel

Illumina

  
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• HYB
 

Multiline Text Fields

Thermal Cycler Program Notes

Multiline Text

Read Only

Default

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 25 µl u 95°C for 5 minutes.
• 18 cycles of 1 minute each: u 94°C for the first cycle.
• Decrease 2°C per subsequent cycle.
• 58°C for 90 minutes.
• Hold at 58°C for ≤ 24 hours.

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 5: Capture Hybidized Probes

Step Type: Standard

Input = Hybridized Libraries

Output = Enriched Libraries

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric
• Calculated = Total Samples + 1

Default

• 0

 

 

EEL (µl)

Numeric
• Decimal Places Displayed = 0
• Calculated = 28.5 * Total Samples
   

HP3 (µl)

Numeric
• Decimal Places Displayed = 0
• Calculated = 1.5 * Total Samples
 

Multiline Text Fields

Instruction Notes

Multiline Text

Read Only

Default

• Add 23 µl Elution Master Mix to each well.
   

Thermal Cycler Program Notes

Multiline Text

Read Only

Default

• Choose the preheat lid option and set to 70°C.
• Reaction volume is 100 µl.
• Hold at 58°C.

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate Elution Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::EEL (uL):: = 28.5 * step.::Total Samples:: ; step.::HP3 (uL):: = 1.5 * step.::Total Samples::' \

-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 6: Amplify Enriched Library

Step Type: No Output

Input = Enriched Libraries

Output = Amplified enriched libraries

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• AMP
 

Multiline Text Fields

Thermal Cycler Program Notes

Multiline Text

Default

Read Only

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 50 µl u 98°C for 30 seconds.
• 14 cycles as follows:
– 98°C for 10 seconds.
– 60°C for 30 seconds.
– 72°C for 30 seconds.
– 72°C for 5 minutes.
– Hold at 10°C.

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

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 7: Clean Up Enriched Library

Step Type: Standard

Input = Amplified enriched libraries

Output = Cleaned up enriched libraries

Reagent Kit

Kit Name

Supplier

Cat. #

Illumina RNA Prep, Tagmentation (L) with Enrichment

Illumina

16 - 20040536, 96 - 20040537

Agencourt AMPure XP, 5 ml

Beckman Coulter

A63880
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Sub-section

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Prep Date

Date

  

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}"

End of WoRKFLOW