TruSeq Small RNA v1.0
Overview
TruSeq Small RNA v1.0 includes the following functionality:
| • | Preconfigured TruSeq Small RNA v1.0 protocol that explains how to prepare total RNA or purified small RNA using a Illumina® TruSeq® Small RNA Library Prep Kit. |
| • | Automated calculation of sample and buffer volumes. |
| • | Automated calculation or display of reagents at every step in the protocol. |
| • | Automatic step transition when required. |
| • | Automatic placement of samples when necessary. |
| • | Automated assignment of QC Pass/Fail, based on user-selected threshold values. |
| • | A routing script that allows sequencing of libraries using any Illumina sequencing instrument. |
It is not required to have the total RNA samples quantified prior to starting this protocol, however it is highly recommended to ensure the starting number of samples.
Protocol 1: TruSeq Small RNA v1.0
Step 1: Ligate Adapters (TruSeq Small RNA v1.0)
Master Step 1: Ligate Adapters (TruSeq Small RNA v1.0)
Process Type 1: Ligate Adapters (TruSeq Small RNA v1.0)
Step Type: No Outputs
Input = 1 µg total RNA
Output = 200 µl tube
Reagent Kits =
| • | TruSeq Small RNA Library Prep Kit, Core Solutions - for ATP, HML, RNase Inhibitor, STP, T4 RNA Ligase and Ultrapure Water Indexes 1–12 Catalog # RS-200-0012 |
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
| • | TruSeq Small RNA Library Prep Kit, indexes A, B, C, or D |
- for RA3 and RA5 Indexes 1–12 Catalog # RS-200-0012
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
| • | T4 RNA Ligase 2, Deletion Mutant |
Catalog # LR2D1132K
| – | Catalog # LR2D11310K |
Control =
| • | Human Brain Total RNA |
- Thermo Fisher Scientific Catalog # AM7962
Record Details
| • | STEP DATAMASTER STEP FIELDS |
Total samples numeric
| – | Preset = 0 |
| – | calculated |
HML Volume (µl) numeric
RNase Inhibitor Volume (µl) numeric
| – | calculated |
| – | T4 RNA Ligase 2, Depletion Mutant Volume (µl) numeric |
| – | calculated |
RA5 Volume (µl) numeric
| – | calculated |
10 mM ATP Volume (µl) numeric
| – | calculated |
T4 RNA Ligase Volume (µl) numeric
| – | calculated |
MULTILINE TEXT FIELDS CommentsMulti-line text
STEP FILE PLACEHOLDERS Log FileAuto
Automation - Script description
| • | Calculate Master Mix 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/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp '(step.::Total samples:: = step.::Total samples:: + 1)' -log {compoundOutputFileLuid0} && /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 '(step.:: HML Volume (ul) :: = step.::Total samples:: * 2.2) ; (step.:: RNase Inhibitor Volume (ul) :: = step.::Total samples:: * 1.1) ; (step.::T4 RNA Ligase 2, Depletion Mutant Volume (ul):: = step.::Total samples:: * 1.1) ; (step.:: RA5 Volume (ul) :: = step.::Total samples:: * 1.21) ; (step.:: 10mM ATP Volume (ul) :: = step.::Total samples:: * 1.21) ; (step.::T4 RNA Ligase Volume (ul):: = step.::Total samples:: * 1.21)' -log {compoundOutputFileLuid1}["]
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.
Step 2: Perform Reverse Transcription (TruSeq Small RNA v1.0)
Master Step 2: Perform Reverse Transcription (TruSeq Small RNA v1.0)
Process Type 2: Perform Reverse Transcription (TruSeq Small RNA v1.0)
Step Type: No Outputs
Input = Tube
Output = Tube
Reagent Kits =
| • | TruSeq Small RNA Library Prep Kit, Core Solutions - for dNTP Mix, PML, RNAse Inhibitor, Ultrapure Water Indexes 1–12 Catalog # RS-200-0012 |
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
| • | TruSeq Small RNA Library Prep Kit, indexes A, B, C, or D |
- for RP1, RNA PCR Primer Index and RTP Indexes 1–12 Catalog # RS-200-0012
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
| • | SuperScript II Reverse Transcriptase |
Life Technologies
| – | Catalog # 18064-014 |
Record Details
| • | STEP DATAMASTER STEP FIELDS |
Total samples numeric
| – | Preset = 0 |
| – | calculated |
Dilute 25 mM dNTP mix to 12.5 mM (per library): 0.5ul of 25 mM dNTP Mix and 0.5ul of ultrapure water. Checkbox
| – | 5X First Strand Buffer Volume (ul) numeric |
| – | calculated |
| – | 12.5 mM dNTP Mix Volume (ul) |
numeric
| – | calculated |
| – | 100 mM DTT Volume (ul) |
numeric
| – | calculated |
| – | RNase Inhibitor Volume (ul) |
numeric
| – | calculated |
| – | SuperScript II Reverse Transcriptase Volume (ul) numeric |
| – | calculated |
MULTILINE TEXT FIELDS CommentsMulti-line text
Automation - Script description
| • | Calculate Master Mix 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/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp '(step.::Total samples:: = step.::Total samples:: + 1)' -log {compoundOutputFileLuid0} && /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 '(step.:: 5X First Strand Buffer Volume (ul) :: = step.::Total samples:: * 2.2) ; (step.:: 12.5 mM dNTP Mix Volume (ul) :: = step.::Total samples:: * 0.55) ; (step.:: 100mM DTT Volume (ul) :: = step.::Total samples:: * 1.1) ; (step.::RNase Inhibitor Volume (ul):: = step.::Total samples:: * 1.1) ; (step.:: SuperScript II Reverse Transcriptase Volume (ul) :: = step.::Total samples:: * 1.1)' -log {compoundOutputFileLuid1}["] |
| • | 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.
Step 3: Amplify Libraries (TruSeq Small RNA v1.0)
Master Step 3: Amplify Libraries (TruSeq Small RNA v1.0)
Process Type 3: Amplify Libraries (TruSeq Small RNA v1.0)
Step Type: Add Labels
Input = Tube
Output = Tube
Reagent Kits =
| • | TruSeq Small RNA Library Prep Kit, Core Solutions - for dNTP Mix, PML, RNAse Inhibitor, Ultrapure Water Indexes 1–12 Catalog # RS-200-0012 |
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
| • | TruSeq Small RNA Library Prep Kit, indexes A, B, C, or D |
- for RP1, RNA PCR Primer Index and RTP Indexes 1–12 Catalog # RS-200-0012
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
Placement
| • | Master Step none |
| • | Step tube plate |
| – | row |
Add Labels.
| • | TruSeq Small RNA.xml |
| • | TruSeq Small RNA.xlsx |
Record Details
| • | STEP DATA |
MASTER STEP FIELDS
Total samples Numeric
| – | Preset = 0 |
| – | calculated |
Add 2ul of chosen RPIX to each sample. Checkbox
| – | Ultrapure water Volume (ul) Numeric |
| – | calculated |
| – | PML Volume (ul) |
Numeric
| – | calculated |
| – | RP1 Volume (ul) Numeric |
| – | calculated |
MULTILINE TEXT FIELDS CommentsMulti-line text
STEP FILE PLACEHOLDERS Log FileAuto
Automation - Script description
| • | Calculate Master Mix 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 '(step.::Total samples:: = step.::Total samples:: + 1)' -log {compoundOutputFileLuid0} && /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 '(step.::Ultrapure water Volume (ul):: = step.::Total samples:: * 9.35) ; (step.::PML Volume (ul):: = step.::Total samples:: * 27.5) ; (step.::RP1 Volume (ul):: = (step.::Total samples:: * 2.2).round(2)) ' -log {compoundOutputFileLuid1}["]
Optional next step
| • | Bioanalyzer QC (Library Validation) v1.0 |
| • | Run Gel Electrophoresis and Recover Purified Construct (TruSeq Small RNA v1.0) |
Start Next Step - Manual.
Assign Next Step - Manual.
Safe Stopping Point
Step 4: Bioanalyzer QC (Library Validation) v1.0
OPTIONAL Master Step 4: Bioanalyzer QC (Library Validation) v1.0
Process Type 4: Bioanalyzer QC (Library Validation) v1.0
Step Type: Standard QC
Input = Amplified Libraries Tube
Output = Result File
Placement
| • | Master Step none |
| • | StepBioanalyzer DNA High Sensitivity Chip |
| – | row |
Record Details =
| • | STEP DATAMASTER STEP FIELDS |
| • | Criteria 1 - Source Data Field numeric |
| – | Preset: Region 1 Average Size - bp |
| – | Criteria 1 - Operator text |
| – | Preset: >= |
| – | Criteria 1 - Threshold numeric |
| – | Criteria 2 - Source Data Field numeric |
| – | Preset: Region 1 Average Size - bp |
| – | Criteria 2 - Operator text |
| – | Preset: <= |
| – | Criteria 2 - Threshold Value numeric |
| – | Use strict match for Bioanalyzer results. |
Toggle switch.
| • | 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 File auto |
| – | QC Assignment Log auto |
| – | QC Assignment Report auto |
TABLE COLUMNS ConcentrationSample Measurement
| – | numeric |
| – | Conc. UnitsSample Measurement |
| – | numeric |
| – | Molarity (nM) Sample Measurement |
| – | numeric |
| – | Region 1 Average Size - bpSample Measurement |
| – | numeric |
| – | Region 1 Conc. Sample Measurement |
| – | numeric |
| – | Region 1 MolaritySample Measurement |
| – | numeric |
Automation - Script description
Generate Bioanalyzer Input file Trigger location: Record Details
| – | Trigger Style: automatic upon entry bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar script:driver_file_generator -i {processURI:v2} -u {username} -p {password} -t /opt/gls/clarity/extensions/ngs-common/v5/EPP/conf/readonly/bioA_driver_file_template.csv -o {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:addBlankLines -i {stepURI:v2} -u {username} -p {password} -f {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} -sep COMMA -b ', False,' -h 1 -c LIMSID -pre 'Sample '" |
| • | Parse Bioanalyzer XML and assign QC flags Trigger location: Record Details |
| – | Trigger Style: manual button |
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' script:assignQC -log {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}"
Set Next Step script - Output PASS/FAIL Trigger location: Record Details
| – | Trigger Style: Automatic upon exit |
bash -l -c ["]/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -excludeControls true -exp 'if (output.QC == true) { nextStep = ::ADVANCE:: } else { nextStep = ::ESCALATE:: }' -log {compoundOutputFileLuid0}["]
Start Next Step - Manual.
Assign Next Step - Automatic.
Safe Stopping Point
Step 5: Run Gel Electrophoresis and Recover Purified Construct (TruSeq Small RNA v1.0)
Master Step 5: Run Gel Electrophoresis and Recover Purified Construct (TruSeq Small RNA v1.0)
Process Type 5: Run Gel Electrophoresis and Recover Purified Construct (TruSeq Small RNA v1.0)
Step Type: No Outputs
Input = Tube
Output = Purified cDNA construct Tube.
Reagent Kits =
| • | TruSeq Small RNA Library Prep Kit, Core Solutions - for CRL, HRL, and Ultrapure Water Indexes 1–12 Catalog # RS-200-0012 |
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
| • | Novex TBE gels, 6%, 10 well |
Life Technologies, catalog # EC6265BOX
| • | Novex TBE running buffer (5X) |
Invitrogen, catalog # LC6675
STEP FILE PLACEHOLDERS Gel Imagemanual
Record Details
| • | STEP DATAMASTER STEP FIELDS |
70% EtOH Prep Date date
MULTILINE TEXT FIELDS CommentsMulti-line text
Optional next steps
| • | Concentrate Final Library (TruSeq Small RNA v1.0) |
| • | Bioanalyzer QC (Library Validation) v1.0 |
Start Next Step - Manual.
Assign Next Step - manual.
Safe Stopping Point
Step 6: Concentrate Final Library (TruSeq Small RNA v1.0)
OPTIONAL Master Step 6: Concentrate Final Library (TruSeq Small RNA v1.0)
Process Type 6: Concentrate Final Library (TruSeq Small RNA v1.0)
Step Type: Standard
Input = Tube
Output = Purified cDNA construct Tube.
Reagent Kits =
| • | TruSeq Small RNA Library Prep Kit, Core Solutions - for CRL, HRL, and Ultrapure Water Indexes 1–12 Catalog # RS-200-0012 |
| – | Indexes 13–24 Catalog # RS-200-0024 |
| – | Indexes 25–36 Catalog # RS-200-0036 |
| – | Indexes 37–48 Catalog # RS-200-0048 |
Placement
| • | Master Step none |
| • | Step tube |
| – | auto placement |
Record Details
| • | STEP DATAMASTER STEP FIELDS |
| • | Numeric: |
Total samples Preset = 0
| – | Date: 70% EtOH Prep Date |
| – | Numeric: Optional - 1X Pellet Paint NF Co-Precipitant Volume (ul) |
| – | Numeric: Optional - Ultrapure water Volume (ul) |
| – | Numeric: |
Glycogen Volume (ul) Preset: 2
| – | Numeric: |
3M NaOAc Volume (ul) Preset: 30
| – | Numeric: 0.1X Pellet Paint Volume (ul) |
| – | Single-line text: Is Pellet Paint being used? |
Preset: Yes
| – | No |
MULTILINE TEXT FIELDS CommentsMulti-line text
STEP FILE PLACEHOLDERS Log FileAuto
Automation - Script description
| • | Calculate Master Mix for Pellet Paint 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 '(step.::Total samples:: = step.::Total samples:: + 1)' -log {compoundOutputFileLuid0} && /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 (step.::Is Pellet Paint being used?:: == ::Yes:: ) {(step.::Optional - Ultrapure water Volume (ul):: = step.::Total samples:: * 1.98) ; (step.::Optional - 1X Pellet Paint NF Co-Precipitant Volume (ul):: = step.::Total samples:: * 0.22) ; (step.::0.1X Pellet Paint Volume (ul):: = 2)}' -log {compoundOutputFileLuid1}["]
| • | 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
Step 7: Bioanalyzer QC (Library Validation) v1.0
Master Step 7: Bioanalyzer QC (Library Validation) v1.0
Process Type 7: Bioanalyzer QC (Library Validation) v1.0
Step Type: Standard QC
Input = Purified cDNA construct Tube.
Output = Result File
Placement
| • | Master Step none |
| • | StepBioanalyzer DNA High Sensitivity Chip |
| – | row |
Record Details =
| • | STEP DATAMASTER STEP FIELDS |
| • | Criteria 1 - Source Data Field numeric |
| – | Preset: Region 1 Average Size - bp |
| – | Criteria 1 - Operator text |
| – | Preset: >= |
| – | Criteria 1 - Threshold numeric |
| – | Preset: 100 |
| – | Criteria 2 - Source Data Field numeric |
| – | Preset: Region 1 Average Size - bp |
| – | Criteria 2 - Operator text |
| – | Preset: <= |
| – | Criteria 2 - Threshold Value numeric |
| – | Preset: 200 |
| – | Use strict match for Bioanalyzer results. |
Toggle switch.
| • | 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 File auto |
| – | QC Assignment Log auto |
| – | QC Assignment Report auto |
TABLE COLUMNS ConcentrationSample Measurement
| – | numeric |
| – | Conc. UnitsSample Measurement |
| – | numeric |
| – | Molarity (nM) Sample Measurement |
| – | numeric |
| – | Region 1 Average Size - bpSample Measurement |
| – | numeric |
| – | Region 1 Conc. Sample Measurement |
| – | numeric |
| – | Region 1 MolaritySample Measurement |
| – | numeric |
Automation - Script description
| • | Generate Bioanalyzer Input file Trigger location: Record Details |
| – | Trigger Style: automatic upon entry |
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar script:driver_file_generator -i {processURI:v2} -u {username} -p {password} -t /opt/gls/clarity/extensions/ngs-common/v5/EPP/conf/readonly/bioA_driver_file_template.csv -o {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:addBlankLines -i {stepURI:v2} -u {username} -p {password} -f {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} -sep COMMA -b ',False,' -h 1 -c LIMSID -pre 'Sample '"
| • | Parse Bioanalyzer XML, Copy nM and Assign QC flags Trigger location: Record Details |
| – | Trigger Style: manual button |
bash -c ["]/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'if (output.::Conc. Units::.contains(::pg::)) {output.::Molarity (nM):: = output.::Region 1 Molarity:: / 1000} else {output.::Molarity (nM):: = output.::Region 1 Molarity::} ; (input.::Molarity (nM):: = output.::Molarity (nM)::) ' -log {compoundOutputFileLuid8} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:assignQC -log {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}["]
Set Next Step - Output PASS/FAIL Trigger location: Record Details
| – | Trigger Style: automatic upon exit bash -l -c ["]/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -excludeControls true -exp 'if (output.QC == true) { nextStep = ::ADVANCE:: } else { nextStep = ::ESCALATE:: }' -log {compoundOutputFileLuid0}["] |
Start Next Step - Manual.
Assign Next Step - Automatic.
Safe Stopping Point
Step 8: Normalize Libraries (TruSeq Small RNA v1.0)
Master Step 8: Normalize Libraries 1 v1.0
Process Type 8: Normalize Libraries (TruSeq Small RNA v1.0)
Step Type: Standard
Input = Purified cDNA construct Tube.
Output = Normalized libraries Tube
Placement
| • | Master Step none |
| • | Step tube |
| – | auto placement |
Record Details
| • | STEP DATAMASTER STEP FIELDS |
| • | Final Volume (ul) numeric |
| – | mandatory |
| – | Target Normalization (nM) numeric |
| – | Preset: 2 |
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), is 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
| • | Normalization Calculations - Option 1 Trigger location: Record Details |
| – | Trigger Style: manual button |
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'output.::Molarity (nM):: = input.::Molarity (nM):: ; if (output.::Molarity (nM):: <= step.::Target Normalization (nM)::) {output.::Sample Volume (ul):: = step.::Final Volume (ul):: ; output.::Buffer Volume (ul):: = 0 ; output.::Normalized Molarity (nM):: = output.::Molarity (nM)::} else {output.::Sample Volume (ul):: = (step.::Target Normalization (nM):: * step.::Final Volume (ul):: ) / input.::Molarity (nM):: ; output.::Buffer Volume (ul):: = step.::Final Volume (ul):: - output.::Sample Volume (ul):: ; output.::Normalized Molarity (nM):: = step.::Target Normalization (nM)::}' -log {compoundOutputFileLuid0}"
| • | Set Next Step script to REMOVE Trigger location: Record Details |
| – | Trigger Style: Automatic upon exit |
bash -l -c ["]/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::REMOVE::' -log {compoundOutputFileLuid0} ["]
Routing script - Normalize Libraries Trigger location: Step
| – | Trigger Style: Automatic upon 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'
\ --FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'NovaSeq 3.0' \--WORKFLOW 'NovaSeq 5000/6000 v3.0' \--STEP 'Define Run Format (NovaSeq 5000/6000 v3.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS'["]
