TruSeq Stranded mRNA v2.0

TruSeq Stranded mRNA v2.0 includes the following functionality:

• Preconfigured TruSeq Stranded mRNA v2.0.
• Protocol explaining how to convert the mRNA in total RNA into a library of template molecules of known strand origin. The library is suitable for subsequent cluster generation and DNA sequencing.
• Automated calculation of sample and buffer volumes.
• Automated calculation or display of reagents at every step in the protocol.
• Automatic step transition when required.
• Automatic placement of samples (when necessary).
• Automated assignment of QC Pass/Fail, based on user-selected threshold values. A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

Protocol 1: TruSeq Stranded mRNA v2.0

Master Step 1: Purify v1.0

Step Type: Standard

Input = 0.1-4 µg  Total RNA

Output = Cleaved RNA fragments

Reagent Kits =

• TruSeq Stranded mRNA Sample Prep Kit Box 1 of 2 -for RPBHT (part # 15032624)
– LT (part # 15027078)
• TruSeq Stranded mRNA Sample Prep Kit Box 2 of 2 -for BBB, BWB, ELB, FPFHT (part # 15032623)
– LT (part # 15032614)
• TruSeq Stranded mRNA Sample Prep Kit - Set A, B or Core Box -

for RSB HTCore Part # 15032620

LT part # of kit depends on which was ordered: Set A Part # 15032612

– Set B Part # 15032613

Placement

• Master Stepnone
• Step96 well plate
– row

Record Details

• STEP DATAMASTER STEP FIELDS

Thermal Cycler Program 1 text

– Preset: mRNA Denaturation

Thermal Cycler Program 2 text

– Preset: mRNA Elution 1

Thermal Cycler Program 3 text

– Preset: Elution 2 - Frag - Prime

MULTILINE TEXT FIELDS CommentsMulti-line text

STEP FILE PLACEHOLDERS Log FileAuto

Automation - Script description =

• Set Next Step - Advance Trigger location: Record Details
– Trigger Style: Automatic upon exitbash -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 2: First Strand cDNA Synthesis v1.0

Step Type: No Outputs

Input = Cleaved RNA fragments

Output = no output

Reagent Kits =

• TruSeq Stranded mRNA cDNA Synthesis PCR BoxHT (part # 15032621)
– LT (part # 15032611)

Record Details

• STEP DATAMASTER STEP FIELDS

Thermal Cycler Program text

– Preset: Synthesize 1st Strand

MULTILINE TEXT FIELDS CommentsMulti-line text

STEP FILE PLACEHOLDERS Log FileAuto

Automation - Script description

• Set Next Step - Advance 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 = ::ADVANCE::'  -log { compoundOutputFileLuid0 }["]

Start Next Step - Automatic

Assign Next Step - Automatic

Must move forward.

Master Step 3: Second Strand cDNA Synthesis v1.0

Step Type: Standard

Input = First strand cDNA

Output = Blunt-ended cDNA

Reagent Kits =

• TruSeq Stranded mRNA Sample Prep Kit - Set A, B or Core Box - for CTE, RSBsupplier: Illumina
– cat #: HT (Core Part # 15032620), LT part # Set A Part # 15032612, Set B Part # 15032613
– website: www.illumina.com
• TruSeq Stranded mRNA cDNA Synthesis PCR Box - SMMHT (part # 15032621)
– LT (part # 15032611)
• AMPure XP Beadssupplier: Beckman Coulter Genomics
– cat #: A63881
– website: https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm

Placement

• Master Stepnone
• Step96 well plate
– auto placement

Record Details

• STEP DATAMASTER STEP FIELDS

80% EtOH Prep Date date

MULTILINE TEXT FIELDS CommentsMulti-line text

STEP FILE PLACEHOLDERS Log FileAuto

Automation - Script description

• Set Next Step - Advance Trigger location: Record Details
– Trigger Style: Automatic upon exitbash -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: Adenylate 3' Ends v1.0

Step Type: No Outputs

Input = Blunt-ended cDNA

Output = no output

Reagent Kits =

• TruSeq Stranded mRNA Sample Prep Kit - Set A, B or Core Box - for CTA, ATL and RSB HTCore Part # 15032620

LT part # of kit depends on which was ordered: Set A Part # 15032612

– Set B Part # 15032613

Record Details

• STEP DATAMASTER STEP FIELDS

Thermal Cycler Program text

– Preset: ATAIL70

MULTILINE TEXT FIELDS CommentsMulti-line text

STEP FILE PLACEHOLDERS Log FileAuto

Automation - Script description =

• Set Next Step - Advance Trigger location: Record Details
– Trigger Style: Automatic upon exitbash -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 5: Ligate Adapters v2.0Step Type: Standard

Input = Adenylated DNA

Output = DNA Fragments

Reagent Kits =

• TruSeq Stranded mRNA Sample Prep Kit - Set A, B or Core Box - for CTL, LIG, STL and RSB HTCore Part # 15032620

LT part # of kit depends on which was ordered: Set A Part # 15032612

– Set B Part # 15032613
• AMPure XP Beadssupplier: Beckman Coulter Genomics
– cat #: A63881
– website: https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm

Placement

• Master Stepnone
• Step96 well plate
– auto placement

Add Labels

• TruSeq Stranded mRNA HT.xlsx
• TruSeq Stranded mRNA LT.xlsx

Record Details

• STEP DATAMASTER STEP FIELDS

80% EtOH Prep Date Date

– Thermal Cycler Programtext
– preset = LIG

MULTILINE TEXT FIELDS CommentsMulti-line text

STEP FILE PLACEHOLDERS Log FileAuto

Automation - Script description

• Set Next Step - Advance 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 = ::ADVANCE::' -log { compoundOutputFileLuid0 }["]

Start Next Step - Manual

Assign Next Step - Automatic

Safe Stopping Point

Master Step 6: Amplification v1.0Step Type: Standard

Input = DNA fragments

Output = DNA libraries

Reagent Kits =

• TruSeq Stranded mRNA Sample Prep Kit - Set A, B or Core Box - for RSB HTCore Part # 15032620

LT part # of kit depends on which was ordered: Set A Part # 15032612

– Set B Part # 15032613
• TruSeq Stranded mRNA cDNA Synthesis PCR Box - for PMM and PPCHT (part # 15032621)
– LT (part # 15032611)
• AMPure XP Beadssupplier: Beckman Coulter Genomics
– cat #: A63881
– website: https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm

Placement

• Master Stepnone
• Step96 well plate
– auto placement

Record Details

• STEP DATAMASTER STEP FIELDS

70% EtOH Prep Date Date

– Thermal Cycler Programtext
– preset = PCR

MULTILINE TEXT FIELDS CommentsMulti-line text

STEP FILE PLACEHOLDERS Log FileAuto

Automation - Script description

• Set Next Step - Advance Trigger location: Record Details
– Trigger Style: Automatic upon exitbash -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: Bioanalyzer QC (Library Validation) v1.0

Step Type: Standard QC

Input = DNA Libraries

Output = Result File

Placement

• Master Stepnone
• StepBioanalyzer DNA High Sensitivity Chip
– row

Record Details =

• STEP DATAMASTER STEP FIELDS
• Criteria 1 - Source Data Fieldnumeric
– Preset: Region 1 Average Size - bp
– Criteria 1 - Operatortext
– Preset: >=
– Criteria 1 - Thresholdnumeric
– Preset: 250
– Criteria 2 - Source Data Fieldnumeric
– Preset: Region 1 Average Size - bp
– Criteria 2 - Operatortext
– Preset: <=
– Criteria 2 - Threshold Valuenumeri
– Preset: 275
• Preset:TruSeq Stranded mRNA Library Validation

STEP FILE PLACEHOLDERS Bioanalyzer Input FileAuto

– 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 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

• Parse Bioanalyzer XML, Calculate nM and assign QC flags Trigger location: Record Details
– Trigger Style: manual buttonbash -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}"
• Set Next Step - Output PASS/FAIL

Trigger location: Record Details

– Trigger Style: Automatic upon exitbash -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 8:Normalize Libraries 2 v1.0

Step Type: Standard

Input = DNA Libraries

Output = Normalized Libraries

Placement

• Master Stepnone
• Step96 well plate
– auto placement

Record Details

• STEP DATAMASTER STEP FIELDS
• Sample Volume (ul)numeric
– Preset: 10
– Target Normalization (nM)numeric
– Preset: 10

MULTILINE TEXT FIELDS CommentsMulti-line text

STEP FILE PLACEHOLDERS Log FileAuto

TABLE COLUMNS Molarity (nM)copied in from input

– numeric
– derived sample
– Sample Volume (ul)copied down from step UDF Sample Volume (ul)
– numeric
– derived sample
– Buffer Volume (ul)calculated
– numeric
– derived sample
– Normalized Molarity (nM)copied down from the step UDF Target Normalization (nM). This will be the UDF to move into pooling.
– Mandatory
– numeric
– derived sample
– Sequencing Instrument

text dropdown

– Mandatory
– Presets =MiSeq
– NextSeq
– HiSeq2500
– HiSeq3000/4000
– NovaSeq
– HiSeqX

Automation - Script description

• Place Samples = Add Automatic Sample Placement (For 4.x ONLY) bash -l -c ["]/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/PlacementHelper.jar script:place_samples -i {stepURI:v2} -u {username} -p {password} -d /opt/gls/clarity/ customextensions /96_to_96 -log {compoundOutputFileLuid0}["]
• Normalization Calculations - Option 2 Trigger location: Record Details
– Trigger Style: manual buttonbash -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.::Sample Volume (ul):: ; output.::Buffer Volume (ul):: = 0 ; output.::Normalized Molarity (nM):: = output.::Molarity (nM)::} else {output.::Sample Volume (ul):: = step.::Sample Volume (ul):: ; output.::Buffer Volume (ul):: = ((output.::Molarity (nM):: * step.::Sample Volume (ul)::) / step.::Target Normalization (nM)::) - step.::Sample Volume (ul):: ; output.::Normalized Molarity (nM):: = step.::Target Normalization (nM)::}' -log {compoundOutputFileLuid0}"

Set Next Step - 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 Step Trigger location: Step
– Trigger Style: Automatic upon exitbash -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'["]