TruSeq DNA Exome v2.0

The TruSeq DNA Exome v2.0 includes the following functionality:

• Preconfigured TruSeq DNA Exome v2.0 protocol that supports the preparation of 96 indexed, paired-end libraries, followed by enrichment using reagents provided in an Illumina® TruSeq® Exome Kit.
• 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.
• A routing script that allows sequencing of libraries to any Illumina sequencing instrument.

Protocol 1: Library Prep (TruSeq DNA Exome v2.0)

Master Step 1: Normalize DNA (TruSeq DNA Exome v2.0)

Step Type: Standard

Input = 100 ng gDNA

Output = normalized gDNA

Reagent Kits

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• Tube
– 96 well platecolumn

Record Details

• Placeholder
• Step Data
• Master Step Fields
• RSB (mL)
• Numeric
– Preset = 5
– Read only
– EDTA (uL) Numeric
– Preset = 10
– Read only
– DNA Amount (ng) Numeric
– Preset = 100
– Read only
– Total Volume (uL) Numeric
– Preset = 50
– Read only
– Multiline Text Fields
– Notes
– Multiline text
– Preset = DNA Amout (ng) and Total Volume (uL) are both per sample.
• Step Field Placeholders
• Log
• Auto
• Table Columns
• Concentration Derived Sample
– Numeric
– Info from Qubit
– Conc. Units Derived Sample
– Single-line text
– Infor from Qubit
– Sample Volume (uL) Derived Sample
– Numeric
– Calculated = DNA Amount (ng) / Concentration
– Shearing Buffer Mix (uL) Derived Sample
– Numeric
– Calculated = Total Volume - Sample Volume

Automation - Script Description

• Calculate Sample Volume and Shearing Buffer MixTrigger Location: Record Details
– Trigger Style: Manual

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.::Concentration:: = input.::Concentration:: ; \output.::Conc. Units:: = input.::Conc. Units:: ; \output.::Sample Volume (uL):: = step.::DNA Amount (ng):: / output.::Concentration:: ; \output.::Shearing Buffer Mix (uL):: = step.::Total Volume (uL):: - output.::Sample Volume (uL)::' \-log {compoundOutputFileLuid0}["]

– Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Automatic

Assign Next Step - Automatic

Must proceed to next step. Do not stop.

Master Step 2: Fragment DNA (TruSeq Exome v1.0)

Step Type: Standard

Input = Normalized gDNA

Output = Fragment DNA

Reagent Kits

TruSeq Exome Kit Supplier - Illumina

– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step 96 well plateauto placement
– Tube

Record Details

• Placeholder Log
• Step Data
• Master Step Fields
• Covaris Duty Factor (%)Numeric Dropdown
– Preset = see table
– Covaris IntensityNumeric Dropdown
– Preset = see table
– Covaris Peak/Displayed Power (W)Numeric
– Preset = see table
– Covaris Cycles/BurstNumeric
– Preset = see table
– Covaris Duration (seconds)Numeric
– Preset = see table
– Covaris Temperature (oC)Numeric
– Preset = see table
– Covaris Water LevelNumeric
– Preset = see table
– Covaris IntensifierText
– Preset = see table
– Multiline Text Fields
– Comment
– Multiline text
• Step Field Placeholders
• Log
• Auto
Covaris Preset based on instrument (M220, S2, S220, E220 and LE220 - rack or LE220 - tube)

Covaris Setting

M220

S2

S220

E220

LE220 - rack

LE220 - tube

Duty Factor (%)

20

10

10

10

30

30

Intensity

-

5

-

-

-

-

Peak Power (W)

50

-

175

175

450

450

Cycles/Burst

200

200

200

200

200

200

Duration (seconds)

375

280

280

280

360

420

Temperature (oC)

20

7

7

7

7

7

Water Level

-

12

12

6

6

6

Intensifier

-

-

-

Yes

-

-

Automation - Script Description

• Set Next Step  - Advance
• Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Automatic

Assign Next Step - Automatic

Must proceed to next step. Do not stop.

Master Step 3: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Fragmented DNA

Output = Cleaned Up Fragmented DNA

Reagent Kits

• TruSeq Exome Kit Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step96 well plate
– Tube

Record Details =

• Step Data
• Master Step Fields
• Step Custom Field80% EtOH Prep Date
• Date
• Step File Placeholders
• Log
• Auto

Automation - Script Description

• Set Next Step  - Advance
• Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 4: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Cleaned Up Fragmented DNA

Output = Repaired DNA and Library Size Selected

Reagent Kits

• TruSeq Exome Kit Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• 96 well plate
– Tube

Record Details

• Step Data
• Master Step Fields
– Step Custom Field
– Thermal Cycler Program
– Preset = ERP
– 80% EtOH Prep Date
– Date
• Step File Placeholders
• Log
• Auto

Automation - Script Description

• Set Next Step  - Advance
• Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 5: Ligate Adapters (TruSeq DNA Exome v2.0)

Step Type: Add Labels

Input = Adenylated DNA

Output = Ligated DNA

Reagent Kits

• TruSeq Exome Kit Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• 96 well plate
– Tube

Add Labels

• TruSeq DNA Single Indexes Set A
• TruSeq DNA Single Indexes Set B
• TruSeq Exome HT (aka TruSeq DNA Combinatorial Dual Indexes)
• IDT-ILMN TruSeq DNA-RNA UD 24 Indexes Plate
• IDT-ILMN TruSeq DNA-RNA UD 96 Indexes  PlateRecord Details

Record Details

• Step Data
• Master Step Fields
• Step Custom Field
• Thermal Cycler Program
– Preset = LIG
– 80% EtOH Prep Date
– Date
• Step File Placeholders
• Log
• Auto

Automation - Script Description

• Set Next Step  - Advance
• Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 6: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Ligated DNA

Output = Enriched DNA Fragments

Reagent Kits

• TruSeq Exome Kit Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• 96 well plate
– Tube

Record Details

• Step Data
• Master Step Fields
• Thermal Cycler Program
• Text
– Preset = PCRNano
– PCR CyclesNumeric
– 80% EtOH Prep Date
– Date
• Step File Placeholders
• Log
– Auto

Automation - Script Description

• Set Next Step  - Advance
• Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 7: Qubit DNA QC (TruSeq Exome v1.0)Step Type: Standard QC

Input = Enriched DNA Fragments

Output = Quantified Libraries

Placement

• Master Step
• None
• Step Tube
• Placeholder

Record Details

• Step Data
• Master Step Fields
– Criteria 1 - Source Data Field numeric
– Preset = Concentration
– Criteria 1 - Operator
– Text
– Preset: >=
– Criteria 1 - Threshold Value numeric
– Criteria 2 - Source Data Field
– Numeric
– Preset = Concentration
– Criteria 2 - Operator
– Text
– Preset: <=
– Criteria 2 - Threshold Value numeric
• Step File Placeholders
• Log
• Auto
– QC Log File auto
– QC Result File auto
– Upload File
• Manual
• Table Columns
• Concentration Derived Sample and Measurement
– Numeric
– Conc. Units Derived Sample and Measurement
– Text

Automation - Script description

• Average Concentration and Assign QC flagsTrigger Location: Record Details
• Trigger Style: Manual

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 'Concentration' -dest 'Concentration' -log {compoundOutputFileLuid3} && /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}  && /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 'input.::Concentration:: = output.::Concentration::' -log {compoundOutputFileLuid3}["]

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 8: Bioanalyzer QC (Library Validation) v1.0

Step Type: Standard QC

Input = Enriched DNA Fragments

Output = Result File

Record Details =

• Step Data
• Master Step Fields
• Criteria 1 - Source Data Fieldnumeric
– Preset: Peak 2 Size - bp
– Criteria 1 - Operatortext
– Preset: >=
– Criteria 1 - Thresholdnumeric
– Preset:150
– Criteria 2 - Source Data Fieldnumeric
– Preset:Peak 2 Size - bp
– Criteria 2 - Operatortext
– Preset: <=
– Criteria 2 - Threshold Valuenumeri
– Preset:1000
• Preset:TruSeq Exome Library Validation
• Step File Placeholders
• 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 Fileauto
– QC Assignment Logauto
– QC Assignment Reportauto
• Table Columns
• ConcentrationSample Measurement
– numeric
– Conc. UnitsSample Measurement
– numeric
– Molarity (nM)Sample Measurement
– numeric
– Region 1 Average Size - bpSample Measurement
– numeric
– Region 1 Conc.Sample Measurement
– numeric
– Region 1 MolaritySample Measurement
– numeric

Automation - Script description

• Generate Bioanalyzer Input File Trigger location: Record Details
– Trigger Style: automatic upon entry

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/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

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}"

• Set Next Step - Output PASS/FAIL
• Trigger location: Record Details
– Trigger Style: automatic upon exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 9: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Enriched DNA Fragments

Output = Hybridized Probes

Reagent Kits =

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• Tube

Record Details

• Step Data
• Master Step Fields
• Thermal Cycler Program
• Preset = TE HYB
• Step File Placeholders
• Log File
• Auto

Automation Description

• Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Automatic

Assign Next Step - Automatic

Must move forward

Master Step 10: TruSeq DNA Exome v2.0

Step Type: No Output

Input = Hybridized Probes

Output = Captured Hybridized Probes

Reagent Kits =

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Record Details

• Step File Placeholders
• Log File
• Auto

Automation - Script Description

• Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Automatic

Assign Next Step - Automatic

Must proceed to next step. Do not stop.

Master Step 11: TruSeq DNA Exome v2.0

Step Type: No Output

Input = Captured Hybridized Probes

Output = Second Hybridization

Reagent Kits =

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Record Details

• Step Data
• Master Step Fields
• Thermal Cycler Program
• Preset = TE HYB
• Step File Placeholders
• Log File
• Auto

Automation - Script Description

• Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Automatic

Assign Next Step - Automatic

Must proceed to next step. Do not stop.

Master Step 12: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Second Hybridization

Output = Second capture

Reagent Kits =

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• 96 well plate
– Column

Record Details

• Step File Placeholders
• Log File
• Auto

Automation - Script Description

• Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Automatic

Assign Next Step - Automatic

Must proceed to next step. Do not stop.

Master Step 13: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Second Hybridization

Output = Cleaned Up Captured Library

Reagent Kits =

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• 96 well plate
– Auto placement

Record Details

• Step Data
• Master Step Fields
– 80% EtOH Prep Date
– Date
• Step File Placeholders
• Log File
• Auto

Automation - Script Description

• Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 14: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Cleaned Up Captured Library

Output = Amplified Enriched Library

Reagent Kits =

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• 96 well plate
– Auto placement

Record Details

• Step Data
• Master Step Fields
– Thermal Cycler Program
– Text
– Preset = AMP8
– PCR Cycles
– Nnumeric
• Step File Placeholders
• Log File
• Auto

Automation - Script Description

• Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 15: TruSeq DNA Exome v2.0

Step Type: Standard

Input = Amplified Enriched Library Cleaned Up Captured Library

Output = Cleaned Up Amplified Enriched Library

Placement = 96 well plate or tube

Reagent Kits =

• TruSeq Exome Kit
– Supplier - Illumina
– Cat. # - 24 - 20020614; 96 - 20020615
– Website - www.illumina.com

Placement

• Master Step
• None
• Step
• 96 well plate
– Row

Record Details

• Step Data
• Master Step Fields
– 80% EtOH Prep Date
– Date
• Step File Placeholders
• Log File
• Auto

Automation - Script Description

• Set Next Step  - Advance
– Trigger Location: Record Details
– Trigger Style: Automatic on Exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 16: Bioanalyzer QC (Library Validation) v1.0

Step Type: Standard QC

Input = Purified DNA Libraries

Output = Result File

Placement

• Master Step
• None
• Step
• Bioanalyzer DNA High Sensitivity Chip
– Row

Record Details =

• Step Data
• Master Step Fields - the manual did not give any preset values this can be set by each lab
• Criteria 1 - Source Data Field
• Numeric
– Preset: Peak 2 Size - bp
– Criteria 1 - Operatortext
– Preset: >=
– Criteria 1 - Thresholdnumeric
– Preset: 150
– Criteria 2 - Source Data Field
– Numeric
– Preset: Peak 2 Size - bp
– Criteria 2 - Operatortext
– Preset: <=
– Criteria 2 - Threshold Valuenumeri
– Preset: 1000
– Preset:
• TruSeq Exome Library Validation
• Step File Placeholders
• Bioanalyzer Input File
• Auto
– Bioanalyzer Input File Generation Log FileAuto
– Bioanalyzer XML Result File (required) manual
– Result File (optional)manual
– PDF Summary File (optional)manual
– Bioanalyzer XML Parsing Log Fileauto
– QC Assignment Logauto
– QC Assignment Reportauto
• Table Columns
• Concentration
• Sample Measurement
– Numeric
– Conc. Units
– Sample Measurement
– Numeric
– Molarity (nM)
– Sample Measurement
– Numeric
– Region 1 Average Size - bp
– Sample Measurement
– Numeric
– Region 1 Conc.Sample Measurement
– Numeric
– Region 1 MolaritySample Measurement
– Numeric

Automation - Script description

• Generate Bioanalyzer Input file
• Trigger location: Record Details
– Trigger Style: Automatic upon entry

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/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, Copy nM and Assign QC flags
• Trigger location: Record Details
– Trigger Style: manual button

bash -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' && /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.::Conc. Units::.contains(::pg::)) {output.::Molarity (nM):: = output.::Region 1 Molarity:: / 1000} else {output.::Molarity (nM):: = output.::Region 1 Molarity::} ; (input.::Molarity (nM):: = output.::Molarity (nM)::) ' -log {compoundOutputFileLuid8} && /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 {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}"

• Set Next Step - Output PASS/FAIL
• Trigger location: Record Details
– Trigger Style: Automatic upon exit

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

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 17: Normalize Libraries 1 v1.0

Step Type: Standard

Input = Final Libraries Plate/ Tube

Output = Normalized Libraries

Placement

• Master Step
• None
• Step
• 96 well plate
– Auto placement

Record Details

• Step Data
• Master Step Fields
• Final Volume (ul)
• Numeric
– Mandatory
– Target Normalization (nM)
– Numeric
– Preset: 2
– Multiline Text Fields
– Comments
– Multiline text
• Step File Placeholders
• Log File
• Auto
• Table Columns
• Molarity (nM) copied in from input
– Numeric
– Derived Sample
• Sample Volume (ul)
– Numeric
– Derived Sample
– Copied down from step UDF Sample Volume (ul)
• Buffer Volume (ul)
– Numeric
– Derived Sample
– Calculated in the EPP trigger
• Normalized Molarity (nM)
– Numeric
– Mandatory
• Sequencing Instrument
– Text
– Mandatory
– Presets =MiSeq
– NextSeq
– HiSeq2500
– HiSeq3000/4000
– NovaSeq 2.0
– HiSeqX
– NovaSeq 3.0

Automation - Script description

• Normalization Calculations - Option 1
• Trigger location: Record Details
– Trigger Style: Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'output.::Molarity (nM):: = input.::Molarity (nM):: ; if (output.::Molarity (nM):: <= step.::Target Normalization (nM)::) {output.::Sample Volume (ul):: = step.::Final Volume (ul):: ; output.::Buffer Volume (ul):: = 0 ; output.::Normalized Molarity (nM):: = output.::Molarity (nM)::} else {output.::Sample Volume (ul):: = (step.::Target Normalization (nM):: * step.::Final Volume (ul):: ) / input.::Molarity (nM):: ; output.::Buffer Volume (ul):: = step.::Final Volume (ul):: - output.::Sample Volume (ul):: ; output.::Normalized Molarity (nM):: = step.::Target Normalization (nM)::}' -log {compoundOutputFileLuid0}"

• Set Next Step script to remove
• Trigger location: Record Details
– Trigger Style: Automatic upon exit

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

• Routing script - Normalize Libraries
• Trigger location: Step
– Trigger Style: Automatic upon exit

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 ' Sequencing Instrument ' \--FIELD_VALUE 'MiSeq' \--WORKFLOW 'MiSeq Sequencing v1.0' \--STEP 'Library Pooling (MiSeq v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' \\--FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'NextSeq' \--WORKFLOW 'NextSeq 500/550 Sequencing v1.0' \--STEP 'Library Pooling (NextSeq 500/550 v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' \\--FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'HiSeq2500' \--WORKFLOW 'HiSeq 2500 Sequencing v1.0' \--STEP 'Library Pooling (HiSeq 2500 v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' \\--FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'HiSeq3000/4000' \--WORKFLOW 'HiSeq 3000/4000 Sequencing v1.0' \--STEP 'Library Pooling (HiSeq 3000/4000 v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' \\--FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'HiSeqX' \--WORKFLOW 'HiSeq X Sequencing v1.0' \--STEP 'Library Pooling (HiSeq X v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' \\--FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'NovaSeq' \--WORKFLOW 'NovaSeq 5000/6000 v2.0' \--STEP 'Define Run Format (NovaSeq 5000/6000 v2.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS'

\ --FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'NovaSeq v3.0' \--WORKFLOW 'NovaSeq 5000/6000 v3.0' \--STEP 'Define Run Format (NovaSeq 5000/6000 v3.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS'

End of Protocol