Library Validation QC 5.1.2

Overview

The Library Validation QC protocol contains steps to track quality control data from commonly used QC instruments. Through data integrations, QC data can be added to Clarity LIMS directly from the instrument outputs.

Highlights of this protocol include:

• Data integrations for Bioanalyzer 2100 and Tapestation 2200, including creation of the input file and parsing of data output.

• Possible integrations with qPCR instruments: TaqMan 7900 or ViiA7.

• Assignment of pass/fail QC based on chosen criteria.

• Aggregation of data from multiple QC steps.

Protocol 1: Library Validation QC 5.1.2

• Protocol Type = QC

• Capacity = 50

• QC Filters

  • Bioanalyzer QC (DNA) 5.1.2

    • Did not pass - Bioanalyzer QC (DNA) 5.1.2

  • qPCR QC 5.1.2

    • Did not pass - qPCR QC 5.1.2

  • Tapestation QC (DNA) 5.1.2

    • Did not pass - Tapestation QC (DNA) 5.1.2

  • Aggregate QC (Library Validation) 5.1.2

    • Passed - Bioanalyzer QC (DNA) 5.1.2 OR

    • Passed - qPCR QC 5.1.2 OR

    • Passed - Tapestation QC (DNA) 5.1.2

Step: Bioanalyzer QC (DNA) 5.1.2

• Master Step Name = Bioanalyzer QC (DNA) 5.1.2

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName} Bioanalyzer

Automations

Generate Bioanalyzer driver 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 -u {username} -p {password} \
      script:driver_file_generator \
        -i {processURI:v2} \
        -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 -u {username} -p {password} \
      script:addBlankLines \
        -i {stepURI:v2} \
        -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 -u {username} -p {password} -i {processURI:v2} \
      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 - Advance

• 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 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Set Next Step - Output PASS/FAIL

• 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 -excludeControls true -exp 'if (output.QC == true) { nextStep = ::ADVANCE:: } else { nextStep = ::ESCALATE:: }' -log {compoundOutputFileLuid0}"

Set Next Step and Copy to Input

• 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 'nextStep = ::ADVANCE:: ; input.::Concentration (ng/ul):: = output.::Concentration::' -log {compoundOutputFileLuid7}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Previous Steps

  • 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
    Derived Sample	Workflow	Text
    Project	Project Name	Built-in
    Submitted Sample	Application	Text Dropdown	Custom Entries	Presets TruSeq mRNA sequencing TruSeq DNA sequencing (large genome de novo) TruSeq DNA sequencing (large genome re-seq) TruSeq DNA sequencing (small genome de novo) TruSeq DNA sequencing (small genome re-seq) Nextera DNA sequencing TruSeq Custom Amplicon sequencing ChIP-sequencing Exome sequencing Mate pair sequencing Small RNA Sequencing
    Submitted Sample	Pooling	Text Dropdown	Custom Entries	Presets Yes No
    Submitted Sample	Read Length	Text
    Submitted Sample	Sample Type	Text
    Submitted Sample	Sequencing Coverage	Text
    Submitted Sample	Sequencing Method	Text Dropdown	Custom Entries	Presets Single Read Paired End Read Indexed Single Read Indexed Paired End Read

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

  • Placement Pattern = Column

• Destination Containers

  • BioAnalyzer DNA High Sensitivity Chip

  • BioAnalyzer DNA 1000 Chip

Record Details

Group of Defaults

NRCC

• 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 = 1,000.00

• Use strict matching for Bioanalyzer results = No

Peak 2 Size Thresholds

• 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 = 1,000.00

• Use strict matching for Bioanalyzer results = No

TruSeq ChIP-Seq

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Molarity

• Criteria 1 - Threshold Value = 5.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Molarity

• Criteria 2 - Threshold Value = 10.00

• Use strict matching for Bioanalyzer results = No

TruSeq Methyl Capture EPIC

• 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

TruSeq Rapid Exome

• 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 = 1,000.00

• Use strict matching for Bioanalyzer results = No

• Step Data

  • Step Data Heading = Begin by uploading the required Bioanalyzer XML result file

  • Group of Defaults = Peak 2 Size Thresholds

  • Master Step Fields

    Field Name	Field Type	Options	Additional Options and Dropdown Items
    Criteria 1 - Operator	Text Dropdown		Presets >= <= = !=
    Criteria 1 - Source Data Field	Text Dropdown		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 1 - Threshold Value	Numeric		Decimal Places Displayed = 2
    Criteria 2 - Operator	Text Dropdown		Presets >= <= = !=
    Criteria 2 - Source Data Field	Text Dropdown		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 Driver File - Automatically attached

  • Bioanalyzer Driver 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 = Row

  • 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
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Measurement	BA Sample Name	Text
    Measurement	Concentration	Numeric		Decimal Places Displayed = 2
    Measurement	Conc. Units	Text
    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
    Measurement	Sample Comment	Text
    Project	Project Name	Built-in

Step: qPCR QC 5.1.2

• Master Step Name = qPCR QC 5.1.2

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName} qPCR

• Control Types

  • Endogenous Positive Control

  • Exogenous Positive Control

  • No Amplification Control

    • Supplier = In house

  • No Reverse Transcriptase Control

    • Supplier = In house

  • No Template Control

    • Supplier = In house

Automations

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 -u {username} -p {password} \
      script:assignQC \
        -i {processURI:v2} \
        -log {compoundOutputFileLuid1} \
        -qcResult {compoundOutputFileLuid2}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Previous Steps

  • 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
    Derived Sample	Workflow	Text
    Project	Project Name	Built-in
    Submitted Sample	Application	Text Dropdown	Custom Entries	Presets TruSeq mRNA sequencing TruSeq DNA sequencing (large genome de novo) TruSeq DNA sequencing (large genome re-seq) TruSeq DNA sequencing (small genome de novo) TruSeq DNA sequencing (small genome re-seq) Nextera DNA sequencing TruSeq Custom Amplicon sequencing ChIP-sequencing Exome sequencing Mate pair sequencing Small RNA Sequencing
    Submitted Sample	Pooling	Text Dropdown	Custom Entries	Presets Yes No
    Submitted Sample	Read Length	Text
    Submitted Sample	Sample Type	Text
    Submitted Sample	Sequencing Coverage	Text
    Submitted Sample	Sequencing Method	Text Dropdown	Custom Entries	Presets Single Read Paired End Read Indexed Single Read Indexed Paired End Read

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

  • Placement Pattern = Column

• Destination Containers

  • 96 well plate

  • 384 well plate

Record Details

Group of Defaults

Concentration Thresholds

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Concentration

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Concentration

• Step Data

  • Step Data Heading = Begin by completing the Sample Details table

  • Group of Defaults = Concentration Thresholds

  • Master Step Fields

    Field Name	Field Type	Options	Additional Options and Dropdown Items
    Criteria 1 - Operator	Text Dropdown		Presets >= <= = !=
    Criteria 1 - Source Data Field	Text Dropdown		Presets Concentration Conc. Units
    Criteria 1 - Threshold Value	Numeric		Decimal Places Displayed = 2
    Criteria 2 - Operator	Text Dropdown		Presets >= <= = !=
    Criteria 2 - Source Data Field	Text Dropdown		Presets Concentration Conc. Units
    Criteria 2 - Threshold Value	Numeric		Decimal Places Displayed = 2

• Step File Placeholders

  • qPCR Result File - Manually uploaded

  • QC Assignment Log File - Automatically attached

  • QC Assignment Report - Automatically attached

• Sample Table

  • Enable QC Flags = Yes

  • Sample Display Default = Collapse

  • Well Sort Order = Row

  • 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
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Measurement	Concentration	Numeric		Decimal Places Displayed = 2
    Measurement	Conc. Units	Text
    Project	Project Name	Built-in

Step: Tapestation QC (DNA) 5.1.2

• Master Step Name = Tapestation QC (DNA) 5.1.2

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName} Tapestation

Automations

Generate Tapestation Input Sampletable CSV

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry