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Infinium HD Methylation Assay Manual v1.2

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Last updated 8 days ago

Overview

The Infinium HD Methylation Assay Manual workflow includes the following functionality.

Preconfigured protocols to prepare 96 DNA samples for use with the 8x1 HD BeadChip products.
Automated calculation of sample and buffer volumes.
Automatic placement of samples when necessary.
Automatic step transition when required.
Automatic calculation or display of reagents at every step in the protocol.

Protocol 1: Prep Sample (Infinium HD Methylation Assay Manual v1.2)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Select Infinium Kit (Infinium HD Methylation Assay Manual v1.2)

Master Step Name = Select Infinium Kit-Infinium HD Methylation v1.2
Step Type = No Outputs

Automations

Set Infinium HD Methylation Kit Name

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 'input.::Infinium Kit Name:: = step.::Infinium HD Methylation Kits::' -log {compoundOutputFileLuid0}"

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
Project
Project Name
Built-in

Record Details

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
Infinium HD Methylation Kits
Text
Required Field
Read Only
Default = Infinium MethylationEPIC Kit
Step File Placeholders
- Log - Automatically attached
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
  Well
  Built-in
  Derived Sample
  Infinium Kit Name
  Text
  Read Only
  Derived Sample
  Sample Name
  Built-in
  Project
  Project Name
  Built-in

Step 2: Quantify DNA (Infinium HD Methylation Assay Manual v1.2)

Master Step Name = Quantify DNA-Infinium PicoGreen v1.2
Step Type = Standard QC
Measurement Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- 1X TE
- Lambda DNA
- PicoGreen dsDNA quantification reagent

Automations

Calculate Stock Lambda DNA and PicoGreen Dilution

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.::Stock Lambda DNA for A1 (uL):: = (233.3 * 75) / step.::Stock Lambda DNA (ng/uL):: ; step.::1X TE for A1 (uL):: = 233.3 - step.::Stock Lambda DNA for A1 (uL):: ; if (step.::No. of QDNA Plates:: == 1) {step.::PicoGreen (uL):: = 115 ; step.::1X TE (mL):: = 23} ; if (step.::No. of QDNA Plates:: == 2) {step.::PicoGreen (uL):: = 215 ; step.::1X TE (mL):: = 43} ; if (step.::No. of QDNA Plates:: == 3) {step.::PicoGreen (uL):: =  315 ; step.::1X TE (mL):: = 63}' -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}"

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
Derived Sample
Infinium Kit Name
Text
Read Only

Placement = Enabled

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
1X TE (mL)
Numeric
Read Only
1X TE for A1 (uL)
Numeric
Read Only
Lambda serial dilution instruction notes
Multiline Text
Read Only
Default = Add 66.7 uL 1X TE to well B1. Add 100 µl 1X TE to the remaining wells of column 1. Transfer 133.3 µl Lambda DNA from well A1 to well B1. Pipette up and down several times to mix. Transfer 100 µl from well B1 to well C1. Pipette up and down several times to mix. Repeat the sequential transfer of 100 µl for wells D1, E1, F1, and G1. Do not transfer from well G1 to H1. Well H1 serves as the blank 0 ng/µl Lambda DNA.
No. of QDNA Plates
Numeric Dropdown
Presets
1
2
3
Default = 1
PicoGreen (uL)
Numeric
Read Only
Sample QDNA Plate instruction notes
Multiline Text
Read Only
Default = Transfer 195 μl PicoGreen/1xTE dilution to each sample well of the Sample QDNA plate. Add 2 µl DNA sample to each well containing PicoGreen/1xTE
Standard QDNA Plate instruction notes
Multiline Text
Read Only
Default = Transfer 195 µl PicoGreen/1X TE dilution from the Standard DNA plate to columns 1 and 2 of the Standard QDNA plate. Transfer 2 µl stock Lambda DNA dilution from the Standard DNA plate to columns 1 and 2 of the Standard QDNA plate.
Stock Lambda DNA (ng/uL)
Numeric
Stock Lambda DNA for A1 (uL)
Numeric
Read Only
Step File Placeholders
- Log - Automatically attached
- Results File - Manually uploaded
Sample Table
- Enable QC Flags = Yes
- Sample Display Default = Collapse
- Well Sort Order = Row
- File Column Options
  - File Column Display = Show
  - File Attachment Method = Manual
- Table Columns - Global Fields
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Derived Sample
  Infinium Kit Name
  Text
  Read Only
  Derived Sample
  Sample Name
  Built-in

Step 3: Bisulfite Conversion (Infinium HD Methylation Assay Manual v1.2)

Master Step Name = Bisulfite Conversion-Infinium v1.2
Step Type = No Outputs
Reagent Kits
- EZ DNA Methylation Kit
  - Supplier = Zymo Research
  - Website = https://www.zymoresearch.com/collections/ez-dna-methylation-kits

Automations

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}"

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
Infinium Kit Name
Text
Read Only
Derived Sample
Sample Name
Built-in
Derived Sample
Waiting
Built-in
Project
Project Name
Built-in

Record Details

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
Cleanup Conversion Reagent Instruction Notes
Multiline Text
Read Only
Default = Use the instructions in the Zymo EZ DNA Methylation Kit to do the following. Clean the samples using the provided spin columns or filter plate. Wash off the remaining conversion reagent. Centrifuge the plate at 3000–5000 × g. Desulphonate the column or the plate with desulphonation buffer. Incubate at room temperature for 15 minutes. Clean the samples and wash to remove the desulphonation buffer. Repeat one time. Add 12 µl elution buffer from 500 ng gDNA. Centrifuge to elute at 3000–5000 × g for 5 minutes
Denature DNA instruction Notes
Multiline Text
Read Only
Default = Follow the instructions in the Zymo EZ DNA Methylation Kit to denature the genomic DNA and add conversion reagent.
Incubate DNA Instruction Notes
Multiline Text
Read Only
Default = Incubate in a thermal cycler using the following settings for 16 cycles: 95°C for 30 seconds 50°C for 1 hour Hold DNA at 4°C for 10 minutes in the thermal cycler until cleanup
Step File Placeholders
- Log - Automatically attached
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
  Infinium Kit Name
  Text
  Read Only
  Derived Sample
  Sample Name
  Built-in
  Project
  Project Name
  Built-in

Step 4: Create BCD Plate (Infinium HD Methylation Assay Manual v1.2)

Master Step Name = Create BCD Plate-Infinium v1.2
Step Type = Standard
Reagent Kits
- EZ DNA Methylation Kit
  - Supplier = Zymo Research
  - Website = https://www.zymoresearch.com/collections/ez-dna-methylation-kits

Automations

Set DNA Plate Barcode

Trigger Location = Placement
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 -exp 'output.::DNA Plate Barcode:: = input.container.name' -log {compoundOutputFileLuid0}"

Copy Infinium Kit Name

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 'output.::Infinium Kit Name:: = input.::Infinium Kit Name::' -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}"

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

Record Details

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default = Apply a BCD barcode label Transfer the BCD to the plate as follows: Midi plate: 20 µl BCD sample to each well (requires ≥ 1000 ng input in bisulfite conversion) TCY plate: 10 µl BCD sample to each well (requires ≥ 500 ng input in bisulfite conversion
Step File Placeholders
- Log - Automatically attached
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
  DNA Plate Barcode
  Text
  Read Only
  Derived Sample
  Infinium Kit Name
  Text
  Read Only
  Derived Sample
  Sample Name
  Built-in
  Project
  Project Name
  Built-in

Step 5: Amplify DNA (Infinium HD Methylation Assay Manual v1.2)

Master Step Name = Amplify DNA-Infinium HD Methylation v1.2
Step Type = Standard