TruSeq Methyl Capture EPIC v1.0

Master Step 1: Fragment DNA (TruSeq Methyl Capture EPIC v1.0)

Step Type: Standard

Input = gDNA - 10 ng/µL or 500 ng total

Output = dsDNA fragments

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for EDTA and RSB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Controls (Multi step controls) =

•

Normal SamplesHCC1187 normal (BL) (TruSeq Methyl EPIC v1.0) (ATCC, catalog # CRL2323-D)

–

NA12878 (TruSeq Methyl EPIC v1.0)  (Coriell Institute, catalog # NA12878)

•

Cancer samples:HCC1187 breast cancer tumor (TruSeq Methyl EPIC v1.0)  (ATCC, catalog # CRL2322)

–

HeLa (TruSeq Methyl EPIC v1.0)  (Biochain, catalog # D1255811)

–

Jurkat (TruSeq Methyl EPIC v1.0)  (Biochain, catalog # D1255815)

Placement

•

Master Step

•

None

•

Step

•

1x8 strip tube

Record Details

•

Step Data

•

Master Step Fields

•

Duty Factor (%)

–

Numeric

–

Preset: 10

–

Peak/Displayed Power (W)

–

Numeric

–

Preset: 175

–

Cycles/Burst

–

Numeric

–

Preset: 200

–

Duration (seconds)

–

Numeric

–

Preset: 280

–

Temperature (C)

–

Numeric

–

Preset: 7

–

Water Level

–

Numeric

–

Preset: 12

–

Control Concentration (ng/ul)

–

Numeric

–

Required to be filled in

–

Total Samples

–

Preset = 0

–

Calculated

–

Numeric

–

RSB (mL)

–

Numeric

–

Decimal Places = 2

–

Calculated

–

0.5 M EDTA (uL)

–

Numeric

–

Decimal Places = 2

–

Calculated

–

Control Type

–

Text dropdown

–

Presets =HCC1187 normal (BL) (TruSeq Methyl EPIC v1.0) (ATCC, catalog # CRL2323-D)

–

NA12878 (TruSeq Methyl EPIC v1.0)  (Coriell Institute, catalog # NA12878)

–

HCC1187 breast cancer tumor (TruSeq Methyl EPIC v1.0)  (ATCC, catalog # CRL2322)

–

HeLa (TruSeq Methyl EPIC v1.0)  (Biochain, catalog # D1255811)

–

Jurkat (TruSeq Methyl EPIC v1.0)  (Biochain, catalog # D1255815)

–

Multiline Text Fields

–

Comments

–

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

•

TABLE COLUMNS

•

Buffer Volume (ul)

–

Numeric

–

Derived Sample

–

Calculated

–

Sample Volume (ul)Numeric

–

Derived Sample

–

Calculated

–

ConcentrationNumeric

–

Derived Sample

–

Calculated

–

Conc. UnitText

–

Derived Sample

–

Calculated

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.::RSB (mL):: = step.::Total Samples:: * 5 * 1.1 ; step.::0.5 M EDTA (uL):: = step.::Total Samples:: * 10 * 1.1 ; if (! input.name.contains(::TruSeq Methyl EPIC v1.0::)) {output.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units::}' -log {compoundOutputFileLuid1}["]

•

Normalize gDNA

•

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 'if (input.name.contains( ::(TruSeq Methyl EPIC v1.0)::) ) { output.::Concentration:: = step.::Control Concentration (ng/ul):: ; output.::Conc. Units:: = ::ng/uL:: ; output.::Sample Volume (ul):: = 500 / step.::Control Concentration (ng/ul):: ; output.::Buffer Volume (ul):: = 50 - output.::Sample Volume (ul):: } else {output.::Sample Volume (ul):: = 500 / input.::Concentration:: ; output.::Buffer Volume (ul):: = 50 - output.::Sample Volume (ul)::}' -log {compoundOutputFileLuid0}["]

•

[Optional] Next Steps

•

Bioanalyzer QC (D NA) 5.0

•

Clean Up Fragment DNA (TruSeq Methyl Capture EPIC v1.0)

Start Next Step - Manual

Assign Next Step - manual

Safe Stopping Point

Master Step 2: Bioanalyzer QC (DNA) 5.0

Step Type: Standard QC

Input = dsDNA fragments

Output = Result File

Placement

•

Master Step

•

None

•

Step

•

Bioanalyzer DNA High Sensitivity Chip

–

Row

Record Details =

•

Step Data

•

Master Step Fields

•

Criteria 1 - Source Data Field

•

Numeric

–

Preset: Peak Size 2 - bp

–

Criteria 1 - Operator

–

Text

–

Preset: >=

–

Criteria 1 - Threshold

–

Numeric

–

Preset: 100

–

Criteria 2 - Source Data Field

–

Numeric

–

Preset: Peak Size 2 - bp

–

Criteria 2 - Operator

–

Text

–

Preset: <=

–

Criteria 2 - Threshold Value

–

Numeric

–

Preset: 300

•

Preset: TruSeq Methyl Capture EPIC

•

Step File Placeholders

•

Bioanalyzer Input File

–

Auto

–

Bioanalyzer Input File Generation

–

Log File

–

Auto

–

Bioanalyzer XML Result File (required)

–

Manual

–

Result File (optional)

–

Manual

–

PDF Summary File (optional)

–

Manual

–

Bioanalyzer XML Parsing Log File

–

Auto

–

QC Assignment Log

–

Auto

–

QC Assignment Report

–

Auto

•

TABLE COLUMNS

•

Concentration

•

Sample Measurement

–

Numeric

–

Conc. Units

–

Sample Measurement

–

Numeric

–

Molarity (nM)

–

Sample Measurement

–

Numeric

–

Region 1 Average Size - bp

–

Sample Measurement

–

Numeric

–

Region 1 Conc.

–

Sample Measurement

–

numeric

–

Region 1 Molarity

–

Sample 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 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 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 - 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 3: Clean Up Fragmented DNA (TruSeq Methyl Capture EPIC v1.0)

Step Type: No Outputs

Input = dsDNA fragments

Output = no output

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

–

for RSB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 2 - SPB Reagents

–

for SPB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Date

•

Date

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

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 4: Repair Ends (TruSeq Methyl Capture EPIC v1.0)

Step Type: No Outputs

Input = Cleaned up DNA fragments

Output = no output

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for RSB and ERP3 LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 2 - SPB Reagents

- for SPM LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Date

•

Date

–

Thermal Cycler Program text

–

default = ERP

•

Multiline Text Fields

•

CommentsMulti-line text

•

Step File Placeholders

•

Log File

•

Auto

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 5: Adenylate 3' Ends v1.0

Step Type: No Outputs

Input = 5' phosphorylated DNA fragments

Output = no output

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for RSB and ATL2 LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Datedate

–

Thermal Cycler Program text

–

default = ATAIL70

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placholders

•

Log File

•

Auto

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 6: Ligate Adapters v1.0

Step Type: Add Labels

Input = Adenylated fragments

Output = Ligated DNA fragments

Reagent Kits =

•

TruSeq Methyl Capture Epic HT/LT - Box 3- Adapters - for DNA adapters

•

LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for LIG2, RSB, and STL LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Placement

•

Master Step

•

None

•

Step

•

96 well plate

Add Labels

•

TruSeq Methyl Capture EPIC.xlsx

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Date

•

Date

–

Thermal Cycler Program text

–

preset = LIG

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

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 7: Clean Up and Pool v1.0

Step Type: Pool

Input = DNA fragments with indexed adapters

Output = pooled DNA libraries

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 2 - SPB Reagents

- for SPM LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for RSB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Placement

•

Master Step

•

None

•

Step

•

Tube

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Date

•

Date

•

Multiline Text Fields

•

Comments

•

Multi-line text

•

Step File Placeholders

•

L og File

•

Auto

Automation - Script description

Record Details =

•

Step UDF

•

Multiline text: Comment

–

Date: 80% EtOH Prep Date

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 8: Hybridize Probes v1.0

Step Type: Standard

Input = DNA libraries

Output = DNA libraries with capture probes

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 2 - SPB Reagents

- for EHB2 and SPM LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for CT4 and BLR LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 5 - Probes

- for EPIC LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Placement

•

Master Step

•

None

•

Step

•

96 well plate

–

Auto placement

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Date date

–

Thermal Cycler Program text

–

Default = MC HYB1

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

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 9: Capture Hybridized Probes v1.0

Step Type:

Input = DNA libraries with capture probes

Output =  Enriched libraries

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 2 - SPB Reagents

- for ET2 and SMB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for ET1, EWS and HP3 LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Record Details

•

Step Data

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

Automation - Script description

•

Place Samples = Add Automatic

•

Sample Placement into strip tubes (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/Strip_tubes -log {compoundOutputFileLuid0}["]

•

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 10: Perform Second Hybridization v1.0

Step Type: No Outputs

Input = Enriched libraries

Output =   no output

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for CT3 LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 5 - Probes

- for EPIC LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Date date

–

Thermal Cycler Program text

–

default = MC HYB2

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

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 11: Perform Second Capture (TruSeq Methyl Capture EPIC v1.0)

Step Type:

Input = Enriched DNA with capture probes

Output =  Enriched libraries

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 2 - SPB Reagents

- for ET2 and SMB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for ET1, EWS and HP3 LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Record Details

•

Step Data

•

Master Step Fields

•

Bisulfite Conversion? toggle switchTrue

–

False

–

Mandatory

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placehoders

•

Log File

•

Auto

Automation - Script description

•

Place Samples = Add Automatic Sample Placement into strip tubes (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/customextensions/Strip_tubes -log {compoundOutputFileLuid0}["]

•

Set Next Step - IF/ELSE

•

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 'if (step.::Bisulfite Conversion?:: == ::True:: ) {nextStep = ::Bisulfite Conversion (TruSeq Methyl Capture EPIC v1.0):: } else {nextStep = ::Amplify Enriched Library (TruSeq Methyl Capture EPIC v1.0):: }'  -log {compoundOutputFileLuid0}"

Start Next Step - Automatic

Assign Next Step - Automatic

Must move forward.

Master Step 12: Bisulfite Conversion (TruSeq Methyl Capture EPIC v1.0)

Step Type:

Input = Enriched libraries

Output =  Bisulfite treated libraries

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 1 - Bisulphite Conversion Reagents

- for Lightning Conversion Reagent, MagBinding Beads, M-Wash Buffer, M-Binding Buffer, L-Desulphonation Buffer LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for RSB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Record Details

•

Step Data

•

Master Step Fields

•

Thermal Cycler Program text

–

Preset: BSF CON

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

Automation - Script description

•

Place Samples = Add Automatic Sample Placement into strip tubes (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/Strip_tubes -log {compoundOutputFileLuid0}["]

•

Set Next Steps - 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 13: Amplify Enriched Library v1.0

Step Type: No Outputs

Input = Enriched libraries or Bisulfite treated libraries depending on previous step

Output =  no output

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for PPC LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

KAPA HiFi HotStart Uracil+ Ready Mix (2X)Catalog # KK2801

Record Details

•

Step Data

•

Master Step Fields

•

Thermal Cycler Program text

–

Preset: AMP MC

–

PCR Program text dropdown

–

Preset: Eleven-cycle for four-plex

–

Preset: Twelve-cycle for two-plex

–

Preset: Twelve-cycle for three-plex

–

Preset: Thirteen-cycle for one-plex

–

Use first preset value

•

Multiline Text Fields

•

Comments

•

Multiline text

•

Step File Placeholders

•

Log File

•

Auto

Automation - Script description

•

Next Step = 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 14: Clean Up v1.0

Step Type:

Input = Amplified libraries

Output =  Purified enriched libraries

Reagent Kits =

•

TruSeq Methyl Capture EPIC HT/LT - Box 2 - SPB Reagents

- for SPB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

•

TruSeq Methyl Capture EPIC HT/LT - Box 4 - Core Reagents

- for RSB LT Catalog # - FC-151-1002

–

HT Catalog # - FC-151-1003

Placement

•

Master Step

•

None

•

Step

•

1x8 strip tube

–

Auto placement

Record Details

•

Step Data

•

Master Step Fields

•

80% EtOH Prep Date

•

Date

–

Multiline Text Fields

–

Comments

–

Multi-line text

•

Step File Placeholders

•

Log File

•

Auto

Automation - Script description

•

Place Samples = Add Automatic Sample Placement into strip tubes (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/Strip_tubes -log {compoundOutputFileLuid0}["]

•

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}"

Master Step 15: Bioanalyzer QC (Library Validation) v1.0

Step Type: Standard QC

Input = DNA Libraries

Output = Result File

Record Details =

•

Step Data

•

Master Step Fields

•

Criteria 1 - Source Data Field

•

Preset: Peak 2 Size - bp

–

Criteria 1 - Operator

–

Preset: >=

–

Criteria 1 - Threshold

–

Preset: 200

–

Criteria 2 - Source Data Field

–

Preset: Peak 2 Size - bp

–

Criteria 2 - Operator

–

Preset: <=

–

Criteria 2 - Threshold Value

–

Preset: 300

•

Preset: TruSeq Methyl Capture EPIC Library Validation

•

Table Columns

•

Concentration

•

Sample Measurement

–

Numeric

–

Conc. UnitsSample Measurement

–

Text

–

Molarity (nM)Sample Measurement

–

Numeric

–

Region 1 Average Size - bp

–

Sample Measurement

–

Numeric

–

Region 1 Conc.

–

Sample Measurement

–

Numeric

–

Region 1 Molarity

–

Sample Measurement

–

Numeric

Automatin - 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 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 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, Calculate nM 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' && /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 - 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 16: Normalize Libraries 1 v1.0

Step Type: Standard

Input = Final Libraries Plate/ Tube

Output = Normalized Libraries

Placement

•

Master Step

•

None

•

Step

•

1x 8 tube strip

Record Details

•

Step Data

•

Master Step Fields

•

Target Normalization (nM)

•

Preset: 2

–

Mandatory

–

Numeric

–

Final Volume (ul)

–

Mandatory

–

Numeric

•

Multiline Text Fields

•

Comments

•

Multiline text

•

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 in the EPP trigger

–

Numeric

–

Derived Sample

Normalized Molarity (nM) Mandatory

–

Numeric

–

Derived Sample

–

Sequencing Instrument

Single-line Text

–

Mandatory

–

Presets =MiSeq

–

NextSeq

–

HiSeq2500

–

HiSeq3000/4000

–

NovaSeq

–

HiSeqX

Automation - Script description

•

Place Samples = Add Automatic Sample Placement (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/extensions/conf/v5/96_to_96 -log {compoundOutputFileLuid0}["]

•

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 - 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 exit

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 {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'["]

End of Protocol