> For the complete documentation index, see [llms.txt](https://help.connected.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.connected.illumina.com/clarity-lims/library-prep/truseq/truseq-rna-access-v2.0.md). # TruSeq RNA Access v2.0 ## Overview The TruSeq RNA Access v2.0 workflow includes the following functionality: * Preconfigured TruSeq RNA Access v2.0 protocol that supports the conversion of total RNA or FFPE samples into a library of template molecules suitable for subsequent cluster generation and sequencing. * Automated calculation of sample and buffer volumes. * Automated calculation or display of reagents at every step in the workflow. * Automatic step transition when required. * Automatic placement of samples when necessary. * Automated generation of Bioanalyzer input file that the user may upload to the Bioanalyzer instrument’s Expert Software and use to set up a run. * Automated parsing of a Bioanalyzer instrument's XML result file into the LIMS, and storing of QC measurement records for the completed run. * Automated assignment of QC Pass/Fail, based on user-selected threshold values. * Routing script that allows sequencing of libraries using any Illumina sequencing instrument. ## Protocol 1: TruSeq RNA Access v2.0 Protocol Type = Library Prep **Next Steps Configuration**

### Step 1: Sort Samples (TruSeq RNA Access v2.0) * Master Step Name = Sort Samples (TruSeq RNA Access v2.0.10) * Step Type = No Outputs {% hint style="info" %} The version of Sort Samples master step name may be different depending on the version of IPP installed. {% endhint %} #### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | * Sample Table * Sample Display Default = Collapse * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 2: Fragment RNA (TruSeq RNA Access v2.0) * Master Step Name = Fragment RNA v2.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {SubmittedSampleName} * Reagent Kits * TruSeq RNA Access - 12 Index Set A or B, Box 2 * Supplier = Illumina * Catalog Number = Set A Part # 15052311; Set B Part # 15052312 * Website = #### Automations

Dilute Total RNA

Set Next Step - Advance

* Trigger Location = Record Details * Trigger Style = Automatic upon exit {% code overflow="wrap" %} ```markup 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}" ``` {% endcode %}

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Placement * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Placement Pattern = Row * Destination Containers * 96 well plate #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | Thermal Cycler Program | Text | | Default = Elution 2 - Frag - Prime | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Buffer Volume (ul) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Sample Volume (ul) | Numeric | | Decimal Places Displayed = 2 | | Project | Project Name | Built-in | | | ### Step 3: First Strand cDNA Synthesis (TruSeq RNA Access v2.0) * Master Step Name = First Strand cDNA Synthesis v2.0 * Step Type = No Outputs * Reagent Kits * TruSeq RNA Access - cDNA Synthesis-PCR, Box 1 * Supplier = Illumina * Catalog Number = 15052309 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------- | -------------- | -------------- | ----------------------------------------------------------------------------------------------------------------------------- | | Comment | Multiline Text | | | | Thermal Cycler Program | Text Dropdown | Custom Entries |

Presets
- Synthesize 1st Strand
- 1stSS
Default = Synthesize 1st Strand

| * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 4: Second Strand cDNA Synthesis (TruSeq RNA Access v2.0) * Master Step Name = Second Strand cDNA Synthesis v2.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {SubmittedSampleName} * Reagent Kits * TruSeq RNA Access - cDNA Synthesis-PCR, Box 1 * Supplier = Illumina * Catalog Number = 15052309 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------ | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | 80% EtOH Prep Date | Date | | | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 5: Adenylate 3' Ends (TruSeq RNA Access v2.0) * Master Step Name = Adenylate 3' Ends v2.0 * Step Type = No Outputs * Reagent Kits * TruSeq RNA Access - 12 Index Set A or B, Box 2 * Supplier = Illumina * Catalog Number = Set A Part # 15052311; Set B Part # 15052312 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 6: Ligate Adapters (TruSeq RNA Access v2.0) * Master Step Name = Ligate Adapters v2.0 * Step Type = Add Labels * Derived Sample Generation = Fixed, 1 * Naming Convention = {SubmittedSampleName} * Reagent Kits * TruSeq RNA Access - 12 Index Set A or B, Box 2 * Supplier = Illumina * Catalog Number = Set A Part # 15052311; Set B Part # 15052312 * Website = #### Automations

Set Next Step - Advance

Copy to Output

* Trigger Location = Not Used {% code overflow="wrap" %} ```markup 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.::Target Insert Size (bp):: = input.::Target Insert Size (bp)::' -log {compoundOutputFileLuid0}" ``` {% endcode %}

Set Next Step & Copy to Input

* Trigger Location = Not Used {% code overflow="wrap" %} ```markup 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:: ; output.::Target Insert Size (bp):: = input.::Target Insert Size (bp)::' -log {compoundOutputFileLuid0}" ``` {% endcode %}

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Add Labels * Label Groups * TruSeq RNA Access #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------ | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | 80% EtOH Prep Date | Date | | | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Reagent Name | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 7: First PCR Amplification (TruSeq RNA Access v2.0) * Master Step Name = First PCR Amplification (TruSeq RNA Access v2.0.10) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {SubmittedSampleName} * Reagent Kits * TruSeq RNA Access - cDNA Synthesis-PCR, Box 1 * Supplier = Illumina * Catalog Number = 15052309 * Website = {% hint style="info" %} The version of First PCR Amplification master step name may be different depending on the version of IPP installed. {% endhint %} #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | 80% EtOH Prep Date | Date | | | | Thermal Cycler Program | Text | | Default = PCR | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 8: Bioanalyzer QC (Library Validation) (TruSeq RNA Access v2.0) * Master Step Name = Bioanalyzer QC (Library Validation) v2.0 * Step Type = Standard QC * Measurement Generation = Fixed, 1 * Naming Convention = {InputItemName} Bioanalyzer #### Automations

Generate Bioanalyzer Input file

* Trigger Location = Record Details * Trigger Style = Automatic upon entry {% code overflow="wrap" %} ```markup 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 '" ``` {% endcode %}

Parse Bioanalyzer XML and assign QC flags

Parse Bioanalyzer XML, Assign QC flags, and Copy Concentrations

* Trigger Location = Record Details * Trigger Style = Manual button {% code overflow="wrap" %} ```markup 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} && /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:: = output.::Region 1 Conc.:: ; input.::Concentration:: = output.::Concentration:: ; output.::Conc. Units:: = ::ng/ul:: ; input.::Conc. Units:: = output.::Conc. Units::' -log {compoundOutputFileLuid8}" ``` {% endcode %}

Set Next Step - Output PASS/FAIL

* Trigger Location = Record Details * Trigger Style = Automatic upon exit {% code overflow="wrap" %} ```markup 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}" ``` {% endcode %}

Parse Bioanalyzer XML, Calculate nM and assign QC flags

* Trigger Location = Not Used {% code overflow="wrap" %} ```markup 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' && /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:: = output.::Region 1 Conc.:: ; output.::Molarity (nM):: = (output.::Concentration:: * 1000000) / (660 * output.::Region 1 Average Size - bp::) ; input.::Molarity (nM):: = output.::Molarity (nM):: ; output.::Conc. Units:: = ::ng/ul::' -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}" ``` {% endcode %}

Parse Bioanalyzer XML, Copy nM and Assign QC flags

* Trigger Location = Not Used {% code overflow="wrap" %} ```markup 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' && /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}" ``` {% endcode %}

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Placement = Enabled * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Placement Pattern = Column * Destination Containers * BioAnalyzer DNA High Sensitivity Chip

* BioAnalyzer DNA 1000 Chip

#### Record Details **Group of Defaults**

Nextera DNA Flex Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Peak 2 Size - bp * Criteria 1 - Threshold Value = 150.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Peak 2 Size - bp * Criteria 2 - Threshold Value = 1,500.00

Nextera Mate Pair Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Region 1 Average Size - bp * Criteria 1 - Threshold Value = 150.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Region 1 Average Size - bp * Criteria 2 - Threshold Value = 400.00

Nextera XT DNA Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Peak 2 Size - bp * Criteria 1 - Threshold Value = 250.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Peak 2 Size - bp * Criteria 2 - Threshold Value = 1,000.00

NRCC Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Peak 2 Size - bp * Criteria 1 - Threshold Value = 350.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Peak 2 Size - bp * Criteria 2 - Threshold Value = 1,000.00

TruSeq ChIP-Seq Library Validation

TruSeq Exome Library Validation

TruSeq Methyl Capture EPIC Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Peak 2 Size - bp * Criteria 1 - Threshold Value = 200.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Peak 2 Size - bp * Criteria 2 - Threshold Value = 300.00

TruSeq Rapid Exome Library Validation

TruSeq RNA Access Library Validation

TruSeq RNA Exome Library Validation

TruSeq Small RNA Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Region 1 Average Size - bp * Criteria 1 - Threshold Value = 100.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Region 1 Average Size - bp * Criteria 2 - Threshold Value = 200.00

TruSeq Stranded mRNA Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Region 1 Average Size - bp * Criteria 1 - Threshold Value = 250.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Region 1 Average Size - bp * Criteria 2 - Threshold Value = 275.00

TruSeq Stranded Total RNA Library Validation

TruSeq Targeted RNA Expression Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Peak 2 Size - bp * Criteria 1 - Threshold Value = 100.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Peak 2 Size - bp * Criteria 2 - Threshold Value = 300.00

TruSight Myeloid Library Validation

TruSight RNA Fusion Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Region 1 Average Size - bp * Criteria 1 - Threshold Value = 160.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Region 1 Size - bp * Criteria 2 - Threshold Value = 700.00

TSCA Library Validation

* Criteria 1 - Operator = >= * Criteria 1 - Source Data Field = Region 1 Average Size - bp * Criteria 1 - Threshold Value = 300.00 * Criteria 2 - Operator = <= * Criteria 2 - Source Data Field = Region 1 Size - bp * Criteria 2 - Threshold Value = 400.00

* Step Data * Group of Defaults = TruSeq RNA Access Library Validation * Master Step Fields | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------------------------------- | -------------- | -------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Criteria 1 - Operator | Text Dropdown | Custom Entries |

Presets

Presets

Concentration
Conc. Units
Number of Peaks found
Peak 1 Size - bp
Peak 1 Conc.
Peak 1 Molarity
Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity
Peak 3 Size - bp
Peak 3 Conc.
Peak 3 Molarity
Peak 4 Size - bp
Peak 4 Conc.
Peak 4 Molarity
Peak 5 Size - bp
Peak 5 Conc.
Peak 5 Molarity
Number of Regions found
Region 1 Average Size - bp
Region 1 Conc.
Region 1 Molarity
Region 2 Average Size - bp
Region 2 Conc.
Region 2 Molarity
Region 3 Average Size - bp
Region 3 Conc.
Region 3 Molarity
Region 4 Average Size - bp
Region 4 Conc.
Region 4 Molarity
Region 5 Average Size - bp
Region 5 Conc.
Region 5 Molarity

Presets

Presets

Concentration
Conc. Units
Number of Peaks found
Peak 1 Size - bp
Peak 1 Conc.
Peak 1 Molarity
Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity
Peak 3 Size - bp
Peak 3 Conc.
Peak 3 Molarity
Peak 4 Size - bp
Peak 4 Conc.
Peak 4 Molarity
Peak 5 Size - bp
Peak 5 Conc.
Peak 5 Molarity
Number of Regions found
Region 1 Average Size - bp
Region 1 Conc.
Region 1 Molarity
Region 2 Average Size - bp
Region 2 Conc.
Region 2 Molarity
Region 3 Average Size - bp
Region 3 Conc.
Region 3 Molarity
Region 4 Average Size - bp
Region 4 Conc.
Region 4 Molarity
Region 5 Average Size - bp
Region 5 Conc.
Region 5 Molarity

| | Criteria 2 - Threshold Value | Numeric | | Decimal Places Displayed = 2 | | Use strict matching for Bioanalyzer results | Toggle Switch | | Default = None Set | * Step File Placeholders * Bioanalyzer Input File - Automatically attached * Bioanalyzer Input File Generation Log File - Automatically attached * Bioanalyzer XML Result File (required) - Manually uploaded * Result File (optional) - Manually uploaded * PDF Summary File (optional) - Manually uploaded * Bioanalyzer XML Parsing Log File - Automatically attached * QC Assignment Log File - Automatically attached * QC Assignment Report - Automatically attached * Sample Table * Enable QC Flags = Yes * Sample Display Default = Expand * Well Sort Order = Column * File Column Options * File Column Display = Hide * File Attachment Method = Auto * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------------------- | -------------- | ----------- | ----------------------------------------- | | Derived Sample | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Measurement | BA Sample Name | Text | | | | Measurement | Concentration | Numeric | | Decimal Places Displayed = 2 | | Measurement | Conc. Units | Text | | | | Measurement | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Measurement | Number of Peaks found | Numeric | | Decimal Places Displayed = 0 | | Measurement | Number of Regions found | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 1 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 1 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 1 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 2 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 2 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 2 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 3 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 3 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 3 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 4 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 4 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 4 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 5 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 5 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 5 Size - bp | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 1 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 1 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 1 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 2 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 2 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 2 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 3 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 3 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 3 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 4 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 4 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 4 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 5 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 5 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 5 Molarity | Numeric | | Decimal Places Displayed = 2 | ### Step 9: First Hybridization (TruSeq RNA Access v2.0) * Master Step Name = Hybridize with Pooling v2.0 * Step Type = Pooling * Aliquot Generation = Fixed, 1 * Naming Convention = {PoolName} * Reagent Kits * TruSeq RNA Access - Coding Transcriptome, Box 4 * Supplier = Illumina * Catalog Number = 15052314 * Website = #### Automations

Set Next Step - Advance

Presets

RNA HYB
NRC HYB

| * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 10: First Capture (TruSeq RNA Access v2.0) * Master Step Name = Capture Hybridized Probes v2.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * TruSeq RNA Access - Coding Capture Reagents, Box 3 * Supplier = Illumina * Catalog Number = 15052313 * Website = * TruSeq RNA Access - Coding Transcriptome, Box 4 * Supplier = Illumina * Catalog Number = 15052314 * Website = #### Automations

Calculate Master Mix

* Trigger Location = Record Details * Trigger Style = Automatic upon entry {% code overflow="wrap" %} ```markup 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 Number of Samples:: = step.::Total Number of Samples:: + 1' -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 false -h false -exp 'step.::Enrichment Elution Buffer 1 (ul):: = 28.5 * step.::Total Number of Samples:: ; step.::2N NaOH (ul):: = 1.5 * step.::Total Number of Samples::' -log {compoundOutputFileLuid0}" ``` {% endcode %}

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------------------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | Enrichment Elution Buffer 1 (ul) | Numeric | | Decimal Places Displayed = 2 | | 2N NaOH (ul) | Numeric | | Decimal Places Displayed = 2 | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 11: Second Hybridization (TruSeq RNA Access v2.0) * Master Step Name = Perform Second Hybridization v2.0 * Step Type = No Outputs * Reagent Kits * TruSeq RNA Access - 12 Index Set A or B, Box 2 * Supplier = Illumina * Catalog Number = Set A Part # 15052311; Set B Part # 15052312 * Website = * TruSeq RNA Access - Coding Transcriptome, Box 4 * Supplier = Illumina * Catalog Number = 15052314 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | Thermal Cycler Program | Text | | Default = NRC HYB | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 12: Second Capture (TruSeq RNA Access v2.0) * Master Step Name = Perform Second Capture v2.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * TruSeq RNA Access - Coding Capture Reagents, Box 3 * Supplier = Illumina * Catalog Number = 15052313 * Website = * TruSeq RNA Access - Coding Transcriptome, Box 4 * Supplier = Illumina * Catalog Number = 15052314 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 13: Capture Sample Clean Up (TruSeq RNA Access v2.0) * Master Step Name = Clean Up v2.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * AMPure XP Beads * Supplier = Beckman Coulter Genomics * Catalog Number = A63881 * Website = * TruSeq RNA Access - 12 Index Set A or B, Box 2 * Supplier = Illumina * Catalog Number = Set A Part # 15052311; Set B Part # 15052312 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------ | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | 80% EtOH Prep Date | Date | | | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 14: Second PCR Amplification (TruSeq RNA Access v2.0) * Master Step Name = Second PCR Amplification v1.0 * Step Type = No Outputs * Reagent Kits * TruSeq RNA Access - cDNA Synthesis-PCR, Box 1 * Supplier = Illumina * Catalog Number = 15052309 * Website = * TruSeq RNA Access - Coding Transcriptome, Box 4 * Supplier = Illumina * Catalog Number = 15052314 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------- | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | Thermal Cycler Program | Text | | Default = EPM AMP | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 15: Second PCR Clean Up (TruSeq RNA Access v2.0) * Master Step Name = Clean Up v2.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * AMPure XP Beads * Supplier = Beckman Coulter Genomics * Catalog Number = A63881 * Website = * TruSeq RNA Access - 12 Index Set A or B, Box 2 * Supplier = Illumina * Catalog Number = Set A Part # 15052311; Set B Part # 15052312 * Website = #### Automations

Set Next Step - Advance

#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Sample Table * Column Headers | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | * Expanded View Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------ | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | LIMS ID (Container) | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------ | -------------- | ----------- | ----------------------------------------- | | Comment | Multiline Text | | | | 80% EtOH Prep Date | Date | | | * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 16: Bioanalyzer QC (Library Validation) (TruSeq RNA Access v2.0) * Master Step Name = Bioanalyzer QC (Library Validation) v2.0 * Step Type = Standard QC * Measurement Generation = Fixed, 1 * Naming Convention = {InputItemName} Bioanalyzer #### Automations

Generate Bioanalyzer Input file

Parse Bioanalyzer XML, Assign QC flags, and Copy Concentrations

Parse Bioanalyzer XML, Calculate nM and assign QC flags

* Trigger Location = Record Details * Trigger Style = Manual button {% code overflow="wrap" %} ```markup 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' && /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:: = output.::Region 1 Conc.:: ; output.::Molarity (nM):: = (output.::Concentration:: * 1000000) / (660 * output.::Region 1 Average Size - bp::) ; input.::Molarity (nM):: = output.::Molarity (nM):: ; output.::Conc. Units:: = ::ng/ul::' -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}" ``` {% endcode %}

Set Next Step - Output PASS/FAIL

* Trigger Location = Record Details * Trigger Style = Automatic upon exit {% code overflow="wrap" %} ```markup 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}" ``` {% endcode %}

Parse Bioanalyzer XML and assign QC flags

Parse Bioanalyzer XML, Copy nM and Assign QC flags

* Trigger Location = Not Used {% code overflow="wrap" %} ```markup 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' && /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}" ``` {% endcode %}

* BioAnalyzer DNA 1000 Chip

#### Record Details **Group of Defaults**

Nextera DNA Flex Library Validation

Nextera Mate Pair Library Validation

Nextera XT DNA Library Validation

NRCC Library Validation

TruSeq ChIP-Seq Library Validation

TruSeq Exome Library Validation

TruSeq Methyl Capture EPIC Library Validation

TruSeq Rapid Exome Library Validation

TruSeq RNA Access Library Validation

TruSeq RNA Exome Library Validation

TruSeq Small RNA Library Validation

TruSeq Stranded mRNA Library Validation

TruSeq Stranded Total RNA Library Validation

TruSeq Targeted RNA Expression Library Validation

TruSight Myeloid Library Validation

TruSight RNA Fusion Library Validation

TSCA Library Validation

Presets

Presets

Concentration
Conc. Units
Number of Peaks found
Peak 1 Size - bp
Peak 1 Conc.
Peak 1 Molarity
Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity
Peak 3 Size - bp
Peak 3 Conc.
Peak 3 Molarity
Peak 4 Size - bp
Peak 4 Conc.
Peak 4 Molarity
Peak 5 Size - bp
Peak 5 Conc.
Peak 5 Molarity
Number of Regions found
Region 1 Average Size - bp
Region 1 Conc.
Region 1 Molarity
Region 2 Average Size - bp
Region 2 Conc.
Region 2 Molarity
Region 3 Average Size - bp
Region 3 Conc.
Region 3 Molarity
Region 4 Average Size - bp
Region 4 Conc.
Region 4 Molarity
Region 5 Average Size - bp
Region 5 Conc.
Region 5 Molarity

Presets

Presets

Concentration
Conc. Units
Number of Peaks found
Peak 1 Size - bp
Peak 1 Conc.
Peak 1 Molarity
Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity
Peak 3 Size - bp
Peak 3 Conc.
Peak 3 Molarity
Peak 4 Size - bp
Peak 4 Conc.
Peak 4 Molarity
Peak 5 Size - bp
Peak 5 Conc.
Peak 5 Molarity
Number of Regions found
Region 1 Average Size - bp
Region 1 Conc.
Region 1 Molarity
Region 2 Average Size - bp
Region 2 Conc.
Region 2 Molarity
Region 3 Average Size - bp
Region 3 Conc.
Region 3 Molarity
Region 4 Average Size - bp
Region 4 Conc.
Region 4 Molarity
Region 5 Average Size - bp
Region 5 Conc.
Region 5 Molarity

| | Criteria 2 - Threshold Value | Numeric | | Decimal Places Displayed = 2 | | Use strict matching for Bioanalyzer results | Toggle Switch | | Default = None Set | * Step File Placeholders * Bioanalyzer Input File - Automatically attached * Bioanalyzer Input File Generation Log File - Automatically attached * Bioanalyzer XML Result File (required) - Manually uploaded * Result File (optional) - Manually uploaded * PDF Summary File (optional) - Manually uploaded * Bioanalyzer XML Parsing Log File - Automatically attached * QC Assignment Log File - Automatically attached * QC Assignment Report - Automatically attached * Sample Table * Enable QC Flags = Yes * Sample Display Default = Expand * Well Sort Order = Column * File Column Options * File Column Display = Hide * File Attachment Method = Auto * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------------------- | -------------- | ----------- | ----------------------------------------- | | Derived Sample | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Measurement | BA Sample Name | Text | | | | Measurement | Concentration | Numeric | | Decimal Places Displayed = 2 | | Measurement | Conc. Units | Text | | | | Measurement | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Measurement | Number of Peaks found | Numeric | | Decimal Places Displayed = 0 | | Measurement | Number of Regions found | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 1 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 1 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 1 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 2 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 2 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 2 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 3 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 3 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 3 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 4 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 4 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 4 Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Peak 5 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 5 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Peak 5 Size - bp | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 1 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 1 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 1 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 2 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 2 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 2 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 3 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 3 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 3 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 4 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 4 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 4 Molarity | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 5 Average Size - bp | Numeric | | Decimal Places Displayed = 0 | | Measurement | Region 5 Conc. | Numeric | | Decimal Places Displayed = 2 | | Measurement | Region 5 Molarity | Numeric | | Decimal Places Displayed = 2 | ### Step 17: Normalize Libraries (TruSeq RNA Access v2.0) * Master Step Name = Normalize Libraries 1 v2.0.10 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} {% hint style="info" %} The version of Normalized Libraries 1 master step name may be different depending on the version of IPP installed. {% endhint %} #### Automations

Normalization Calculations - Option 1

* Trigger Location = Record Details * Trigger Style = Manual button {% code overflow="wrap" %} ```markup 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}" ``` {% endcode %}

Set Next Step - Remove

* Trigger Location = Record Details * Trigger Style = Automatic upon exit {% code overflow="wrap" %} ```markup 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}" ``` {% endcode %}

Routing script - Normalize Libraries

* Trigger Location = Step * Trigger Style = Automatic upon exit {% code overflow="wrap" %} ```markup 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 v3.2' \ --STEP 'Library Pooling (MiSeq v3.2)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Sequencing Instrument' \ --FIELD_VALUE 'NextSeq' \ --WORKFLOW 'NextSeq 500/550 Sequencing v1.2' \ --STEP 'Library Pooling (NextSeq 500/550 v1.2)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Sequencing Instrument' \ --FIELD_VALUE 'NovaSeq 2.0' \ --WORKFLOW 'NovaSeq 6000 v2.3' \ --STEP 'Define Run Format (NovaSeq 6000 v2.3)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Sequencing Instrument' \ --FIELD_VALUE 'NovaSeq 3.0' \ --WORKFLOW 'NovaSeq 6000 v3.8' \ --STEP 'Define Run Format (NovaSeq 6000 v3.8)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Sequencing Instrument' \ --FIELD_VALUE 'NovaSeqDx' \ --WORKFLOW 'NovaSeqDx v1.2' \ --STEP 'Define Run Format (NovaSeqDx v1.2)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Sequencing Instrument' \ --FIELD_VALUE 'NextSeq 1000/2000' \ --WORKFLOW 'NextSeq 1000/2000 Sequencing v2.4' \ --STEP 'Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Sequencing Instrument' \ --FIELD_VALUE 'NovaSeq X Series' \ --WORKFLOW 'NovaSeq X Series v1.1' \ --STEP 'Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Sequencing Instrument' \ --FIELD_VALUE 'NextSeq 1000/2000 On-Prem' \ --WORKFLOW 'NextSeq 1000/2000 On-Prem Sequencing v1.0' \ --STEP 'Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS'" ``` {% endcode %} > ℹ The field value and actual version of the workflows and steps in the routing automation script may be different depending on the version of IPP installed.

Decimal Places Displayed = 2

Default = 2
Decimal Places Displayed = 2

| * Step File Placeholders * Log File - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Row * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------------ | -------------- | -------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Buffer Volume (ul) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Normalized Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Sample Volume (ul) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sequencing Instrument | Text Dropdown | Required Field |

Presets

MiSeq
NextSeq
NextSeq 1000/2000
NextSeq 1000/2000 On-Prem
NovaSeq 2.0
NovaSeq 3.0
NovaSeq X Series
NovaSeqDx

| | Project | Project Name | Built-in | | | > ℹ The preset options for Derived Sample Sequencing Instrument may vary depending on the version of the IPP. --- # Agent Instructions This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com. ## Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter, and the optional `goal` query parameter: ``` GET https://help.connected.illumina.com/clarity-lims/library-prep/truseq/truseq-rna-access-v2.0.md?ask=&goal= ``` `ask` is the immediate question: it should be specific, self-contained, and written in natural language. `goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.