Illumina Preset Protocol - Infinium HTS Assay Manual

The Infinium HTS Assay Manual workflow includes the following functionality.

• Preconfigured Infinium HTS Assay Manual protocols.
• 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 HTS Assay Manual)

Protocol Type: Sample Prep

Master Step 1: Select Infinium Kit-Infinium HTS

Step Type: No Output

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

HTS Infinium Kits

Text Dropdown

Default list of HTS Infinium kits

Step File Placeholders

  

File Name

Log

Auto

Sample Table

Table Columns

Infinium Kit Name

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from step field
Automation

Automation Name

Setting

Value

Specification

Set Infinium HTS Kit Name

Trigger Location

Step

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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:: = step.::Infinium HTS Kits::' -log {compoundOutputFileLuid0}"

Set Next Step

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

 
     
     

 
Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Manual

Must Move Forward

Optional:

• Quantify DNA (Infinium HTS Assay Manual).
• Amplify DNA (Infinium HTS Assay Manual).

Master Step 2: Quantify DNA-Infinium PicoGreen

Step Type: Standard QC

Reagent Kits

Kit Name

Supplier

1X TE

 

Lambda DNA

 

PicoGreen dsDNA quantification reagent

 
Placement

Setting

Value

Destination Containers

96 well plate
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Stock Lambda DNA (ng/µl)

Numeric

  
   

Stock Lambda DNA for A1 (µl)

Numeric

Calculated - (233.3 * 75)/Stock Lambda DNA (ng/µl)
   

1X TE for A1 (µl)

Numeric

Calculated - 233.3 - Stock Lambda DNA for A1 (µl)
   

No. of QDNA Plates

Numeric Dropdown

1,2,3
   

PicoGreen (µl)

Numeric

Calculated - If No. of QDNA Plates = 1 then 115; if No. of QDNA Plates = 2 then 215; if No. of QDNA Plates = 3 then 315
   

1X TE (ml)

Numeric

Calculated - If No. of QDNA Plates = 1 then 23; if No. of QDNA Plates = 2 then 43; if No. of QDNA Plates = 3 then 63

Step Data

Multiline Text Fields

Lambda serial dilution instruction notes

Multiline Text

Default

Read Only

• Add 66.7 µl 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.
   

Standard QDNA Plate instruction notes

Multiline Text

Default

Read Only

• 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.
   

Sample QDNA Plate instruction notes

Multiline Text

Default

Read Only

• 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.

Step File Placeholders

  

File Name

Log

Auto
   

File Name

PicoGreen Results File

Manual

Sample Table

Table Columns

LIMS ID

Sample Name

Container Name

Well

Project Name

 

Defaults
   

Infinium Kit Name

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from master step field
Automation

Automation Name

Setting

Value

Specification

Calculate Stock Lambda DNA and PicoGreen Dilution

Trigger Location

Record Details

  
 

Trigger Style

Manual button

 
     

Command line

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

 
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

  

Master Step 3: Amplify DNA-Infinium HTS

Step Type: Standard

Reagent Kits

Kit Name

Supplier

MA1 - Infinium

Illumina

MA2 - Infinium

Illumina

MSM - Infinium

Illumina

NaOH

 
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column

Placement Pattern

Same Shape
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

HTS Instruction Notes

Multiline Text

Default

Read Only

• Add 20 µl MA1 to each well of the plate.
• Transfer 4 µl DNA sample from the DNA plate to the corresponding wells of the plate.
• Add 4 µl 0.1N NaOH into each sample well of the plate.
• Add 34 μl MA2 into each sample well of the plate.
• Add 38 μl MSM into each sample well of the plate.
• Vortex at 1600 rpm for 1 minute then pulse centrifuge at 280 × g.
   

Comment

Multiline Text

 

Step File Placeholders

  

File Name

Log

Auto

Sample Table

Table Columns

LIMS ID

Sample Name

Container Name

Well

Project Name

  

Defaults
   

Infinium Kit Name

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from previous step
   

DNA Plate Barcode

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from previous step
Automation

Automation Name

Setting

Value

Specification

Validate MSA3 Plate Barcode and Set Sample Placement Information

Trigger Location

Placement

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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 'if (!output.container.name.matches( ::[A-Z]{2}[0-9]{7}-MSA3:: ) ) {fail ( ::Invalid MSA3 Plate Barcode. Please verify and try again.:: ) } ; if (output.container.name.matches( ::[A-Z]{2}[0-9]{7}-MSA3:: ) ) {output.::Sample_Plate:: = output.container.name ; output.::Sample_Well:: = output.well ; output.::DNA Plate Barcode:: = input.container.name}' -log {compoundOutputFileLuid0}"

     

Calculation

Regular expression for MSA3 barcode 2 letters, 7 number - MSA3.

Saves for future use:

• The DNA source plate barcode in the derived sample DNA Plate Barcode.
• The MSA3 Barcode in Sample_Plate.
• The well placement Sample_Well.

Copy Infinium Kit Name

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

 
     

Command line

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

 
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must Move Forward

  

Master Step 4: Incubate DNA-Infinium

Step Type: No Outputs

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Incubate the plate in the Illumina Hybridization Oven for 20– 24 hours at 37°C.
   

Comment

Multiline Text

 

Step File Placeholders

  

File Name

Log

Auto

Sample Table

Table Columns

Infinium Kit Name

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from step field
 

 

DNA Plate Barcode

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from previous step
 

 

Sample_Plate

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from previous step
 

 

Sample_Well

  
• Global field - derived sample
• Text
• Required
• Calculated - copied from previous step
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

  

Master Step 5: Fragment DNA-Infinium

Step Type: No Outputs

Reagent Kits

Kit Name

Supplier

FMS - Infinium

Illumina
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Add 25 μl FMS to each well of the plate.
• Vortex at 1600 rpm for 1 minute then centrifuge at 50 × g at 22°C for 1 minute.
   

Comment

Multiline Text

 

Step File Placeholders

  

File Name

Log

Auto

Sample Table

Table Columns

Infinium Kit Name

  

Global field - derived sample
   

DNA Plate Barcode

  

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

  

Master Step 6: Precipitate DNA-Infinium

Step Type: No Outputs

Reagent Kits

Kit Name

Supplier

PM1-Infinium

Illumina

100% 2-propanol

 
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Blue Pellet Presence

Checkbox

  
   

100% 2-propanol Prep Date

Date Selection

  

 

Multiline Text Fields

Instruction notes

Multiline Text

Default

• Add 50 μl PM1 to each well of the plate
• Add 155 μl 100% 2-propanol to each sample well
   

Comment

Multiline Text

 

Step File Placeholders

  

File Name

Log

Auto

Sample Table

Table Columns

Infinium Kit Name

  

Global field - derived sample
   

DNA Plate Barcode

  

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

  

Master Step 7: Resuspend DNA-Infinium

Step Type: No Outputs

Reagent Kits

Kit Name

Supplier

RA1-Infinium

Illumina
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes (HTS)

Multiline Text

Default

• Add 23 µl RA1 to each well of the plate.
   

Comment

Multiline Text

 

Step File Placeholders

  

File Name

Log

Auto

Sample Table

Table Columns

Infinium Kit Information

  

Global field - derived sample
   

DNA Plate Barcode

  

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

  

Protocol 2: BeadChip Processing (Infinium HTS Assay Manual)

Protocol Type: Sample Prep, Other

Master Step 1: Hybridize DNA to BeadChip - Infinium

Step Type: Standard

Reagent Kits

Kit Name

Supplier

PB2-Infinium

Illumina

XC4-Infinium

Illumina
Placement

Setting

Value

Specification

Destination Containers
• 24x1 HTS BeadChip
• 24x1 HTS BeadChip Multichannel
• Hybrid Pattern_96 well plate_24x1 HTS BeadChip.tsv
• Hybrid Pattern_96 well plate_24x1 HTS BeadChip Multichannel.tsv

Well Sort Order

Row

Configured on master step

Placement Pattern

Row

Configured on master step

Placement Screen

 

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Rescan the BeadChips as they are removed from the incubator.
   

Comment

Multiline Text

 

Step File Placeholders

  

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Copy Sample Information

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

 
     

Command line

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:: ; output.::DNA Plate Barcode = input.::DNA Plate Barcode::' ; output.::Sample_Plate:: = input.::Sample_Plate:: ; output.::Sample_Well:: = input.::Sample_Well:: ; output.::BeadChip Barcode:: = output.container.name ; output.::BeadChip Position:: = output.well' -log {compoundOutputFileLuid0}

     

Copies over the derived sample information from previous step for use in future step.

• Infinium Kit Name
• Sample Plate
• Sample Well
• DNA Plate Barcode
• Sample Group

Generate Sample Placement for InfiniumBeadChip

Trigger Location

Placement

  
 

Trigger Style

Automatic upon entry

 
     

Command line

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/infinium/placementpatterns"

     

Generates the auto-placement of the samples from the plate to the BeadChip using a PlacementHelper java script and placement pattern file. The placement pattern file describes exactly how the 96 samples map to the BeadChip. Placement pattern file must be named Hybrid Pattern_96_well plate_[container type for the Beadchip].csv.

Validate BeadChip Barcodes

Trigger Location

Placement

  
 

Trigger Style

Automatic upon exit

 
     

Command line

/usr/bin/python /opt/gls/clarity/extensions/conf/infinium/verifyBeadChipIDs.py -u {username} -p {password} -s {stepURI}

     

Checks for uniqueness of the barcode and possibly for the actual barcode.

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must Move Forward

  

Master Step 2: Wash BeadChips-Infinium

Step Type: No Outputs

Reagent Kits

Kit Name

Supplier

PB1-Infinium

Illumina
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Fill two wash dishes with 200 ml PB1 each. Label the wash dishes Wash 1 and Wash 2.
   

Comment

Multiline Text

 

Step File Placeholders

  

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must Move Forward

  

Master Step 3: Extend and Stain BeadChip- Infinium

Step Type: No Outputs

Reagent Kits

Kit Name

Supplier

ATM-Infinium

Illumina

PB1-Infinium

Illumina

SML-Infinium

Illumina

LX1-Infinium

Illumina

LX2-Infinium

Illumina

XC3-Infinium

Illumina

XC4-Infinium

Illumina

EML-Infinium

Illumina

RA1-Infinium

Illumina

70% EtOH

 
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

EDTA Prep Date

Date Selector

  
   

20% EthOH Prep Data

Date Selector

  

 

Multiline Text Fields

Extend Instruction Notes

Multiline Text

Default

Without allowing pipette tips to touch BeadChip surfaces, fill the reservoir of each flow-through chamber as follows:

• 150 µl RA1. Incubate for 30 seconds. Repeat 5 times.
• 225 µl LX1. Incubate for 10 minutes. Repeat one time.
• 225 µl LX2. Incubate for 10 minutes. Repeat one time.
• 300 µl EML. Incubate for 15 minutes.
• 250 µl 95% formamide/1 mM EDTA. Incubate for 1 minute. Repeat twice.
• Incubate 5 minutes.
• Set the chamber rack temperature to the temperature indicated on the SML tube.
• 250 µl XC3. Incubate for 1 minute. Repeat twice.
   

Stain Instruction Notes

Multiline Text

Default

Fill the reservoir of each flow-through chamber as follows:

• 250 µl SML. Incubate for 10 minutes.
• 250 µl XC3. Incubate for 1 minute. Repeat twice.
• Wait 5 minutes.
• 250 µl ATM. Incubate for 10 minutes.
• 250 µl XC3. Incubate for 1 minute. Repeat twice.
• Wait 5 minutes. g 250 µl SML. Incubate for 10 minutes.
• 250 µl XC3. Incubate for 1 minute. Repeat twice.
• Wait 5 minutes.
• 250 µl ATM. Incubate for 10 minutes.
• 250 µl XC3. Incubate for 1 minute. Repeat twice.
• Wait 5 minutes.
• 250 µl SML. Incubate for 10 minutes.
• 250 µl XC3. Incubate for 1 minute. Repeat twice.
• Wait 5 minutes.
   

Wash and Coat BeadChips Instruction Notes

Multiline Text

Default

• Add 310 ml PB1 to the PB1 wash dish.
• Add 310 ml XC4 to the XC4 wash dish.

Step File Placeholders

  

File Name

Log

Auto

Sample Table

Table Columns

Array Instrument

    
   

Infinium Kit Name

    
   

DNA Plate Barcode

    
   

BeadChip Barcode

    
   

BeadChip Position

    
Automation

Automation Name

Setting

Value

Specification

Set Array Instrument

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

 
     

Command line

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 'input.::Array Instrument:: = ::iScan::' -log {compoundOutputFileLuid0}"

     

Needed because the default setting is not always honored.

Routing Script -iScan

Trigger Location

Step

  
 

Trigger Style

Automatic upon exit

 
     

Command line

bash -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 'Array Instrument' \

--FIELD_VALUE 'iScan' \

--WORKFLOW 'iScan v1.0' \

--STEP 'Image Infinium BeadChip (iScan v1.0)' \

--INPUTS_OR_OUTPUTS 'INPUTS'"

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

 
     

Command line

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}"

End of Workflow