The Illumina DNA PCR-Free Library Prep Manual workflow includes the following functionality.
Preset Illumina DNA PCR-Free Library Prep Manual protocols that support the preparation of up to 96 dual-indexed paired-end single-stranded libraries from DNA using the Illumina DNA PCR-Free Library Prep workflow.
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.
Master Step Name = Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Step Type = Add Labels
Derived Sample Generation = Fixed, 1
The version of Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.
Automations
Calculate Dilution for HP3 & Copy Desired DNA Input
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Set Next Step - Clean Up Libraries
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Add Labels
Label Groups
IDT for Illumina Nextera DNA UD Indexes Set A for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
IDT for Illumina Nextera DNA UD Indexes Set A-D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
Record Details
Step Data (Master Step Fields)
Step 5: Clean Up Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Master Step Name = Clean Up Libraries - Thermal Cycler v1.0.2
Step Type = Standard
Derived Sample Generation = Fixed, 1
The version of Clean Up Libraries - Thermal Cycler master step name may be different depending on the version of IPP installed.
Automations
Copy Desired DNA Input
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Set Next Step-Remove & Prep Input Type
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Routing - Quantify & Pool Libraries
Trigger Location = Step
Trigger Style = Automatic upon exit
Set Next Step - Advance
Trigger Location = Not Used
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Step Data (Master Step Fields)
Step 6: Clean Up Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Master Step Name = Clean Up Libraries - Thermal Cycler v1.0.2
Step Type = Standard
Derived Sample Generation = Fixed, 1
The version of Clean Up Libraries - Thermal Cycler master step name may be different depending on the version of IPP installed.
Automations
Copy Desired DNA Input
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Set Next Step-Remove & Prep Input Type
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Routing - Quantify & Pool Libraries
Trigger Location = Step
Trigger Style = Automatic upon exit
Set Next Step - Advance
Trigger Location = Not Used
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Step Data (Master Step Fields)
Protocol 4: Quantify and Pool Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Protocol Type = Library Prep
Next Steps Configuration
Step 1: Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Master Step Name = Pool Samples (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Step Type = Pooling
Aliquot Generation = Fixed, 1
The version of Pool Samples (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.
Automations
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Pooling
Label Uniqueness = On
Defaults
Sample Grouping = Group by Containers
Record Details
Step Data (Master Step Fields)
Step 2: Qubit (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Master Step Name = Qubit (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Step Type = Standard QC
Measurement Generation = Fixed, 1
The version of Qubit (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.
Automations
Assign QC flags
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step-Remove and Copy Concentration
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Step Data (Master Step Fields)
Protocol 5: Quantify and Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Master Step Name = Dilute Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Automations
Copy Concentration (nM)
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Calculate Dilution Volumess
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Step Data (Master Step Fields)
Step 5: Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Master Step Name = Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Step Type = Pooling
Aliquot Generation = Fixed, 1
Automations
Copy Final Concentration (nM)
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Pooling
Label Uniqueness = On
Defaults
Sample Grouping = Group by Containers
Record Details
Step Data (Master Step Fields)
ℹ The version of the nextStep step names may be different depending on the version of IPP installed.
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
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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
Default = Concentration
Criteria 1 - Threshold Value
Numeric
Default = 300
Range = 300 To 2000
Criteria 2 - Operator
Text
Default = <=
Criteria 2 - Source Data Field
Text
Default = Concentration
Criteria 2 - Threshold Value
Numeric
Default = 2000
Range = 300 To 2000
Instruction Notes
Multiline Text
Read Only
Default =
1. Upload or enter Concentration and Conc. Units.
2. Set Threshold Values then click on Assign QC Flags.
Step File Placeholders
Log - Automatically attached
QC Log File - Automatically attached
QC Result File - Automatically attached
Upload File - Manually uploaded
Sample Table
Enable QC Flags = Yes
Sample Display Default = Expand
Well Sort Order = Column
File Column Options
File Column Display = Show
File Attachment Method = Manual
Table Columns - Global Fields
Naming Convention = {InputItemName}
ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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
Default = Concentration
Criteria 1 - Threshold Value
Numeric
Default = 25
Criteria 2 - Operator
Text
Default = <=
Criteria 2 - Source Data Field
Text
Default = Concentration
Criteria 2 - Threshold Value
Numeric
Default = 2000
Instruction Notes
Multiline Text
Read Only
Default =
1. Upload or enter Concentration and Conc. Units.
2. Set Threshold Values then click on Assign QC Flags.
Step File Placeholders
Log - Automatically attached
QC Log File - Automatically attached
QC Result File - Automatically attached
Upload File - Manually uploaded
Sample Table
Enable QC Flags = Yes
Sample Display Default = Expand
Well Sort Order = Column
File Column Options
File Column Display = Show
File Attachment Method = Manual
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina Lysis Reagent Kit
Supplier = Illumina
Catalog Number = 20042221
Website =
ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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
Placement Pattern = Column
Destination Containers
96 well plate
Default =
1. Combine MLB, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube.
2. Add 280 µl of lysis master mix to a new 2 mL tube.
3. Add 20 uL to each 2 mL tube, mix by pipetting then vortex briefly and centrifuge.
4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly.
5. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix.
6. Incubate at RT for 5 mins.
7. Place tube on magnetic stand for 5 mins then remove and discard supernatant.
8. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash.
9. Air dry on magnetic stand for 5 mins.
10. Add 35 uL of RSB to each tube and vortex to mix.
11. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
MLB (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Nuclease-free water (uL)
Numeric
Read Only
Decimal Places Displayed = 0
PK1 (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Total Samples
Numeric
Read Only
Default = 0
Decimal Places Displayed = 0
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina Lysis Reagent Kit
Supplier = Illumina
Catalog Number = 20042221
Website =
ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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
Placement Pattern = Column
Destination Containers
96 well plate
Instruction Notes
Multiline Text
Read Only
Default =
1. Combine 10x Lysis Buffer, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube.
2. Add 300 uL of lysis master mix to a new 2 mL tube.
3. Add 6 x 3 mm² punches to a each 2 ml tube, mix by pipetting then vortex briefly and centrifuge.
4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly.
5. Without removing the punches, transfer all supernatant from each tube to a new 2 ml tube.
6. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix.
7. Incubate at RT for 5 mins.
8. Place tube on magnetic stand for 5 mins then remove and discard supernatant.
9. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash.
10. Air dry on magnetic stand for 5 mins.
11. Add 35 uL of RSB to each tube and vortex to mix.
12. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear.
13. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
Nuclease-free water (uL)
Numeric
Read Only
Decimal Places Displayed = 0
PK1 (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Total Samples
Numeric
Read Only
Default = 0
Decimal Places Displayed = 0
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina Lysis Reagent Kit
Supplier = Illumina
Catalog Number = 20042221
Website =
ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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
Placement Pattern = Column
Destination Containers
96 well plate
Default =
1. Incubate saliva overnight at 50°C in a water bath or air incubator.
2. Add 250 uL of Nuclease-Free Water to a new 2 mL tube.
3. Invert each heat-treated saliva collection tube 5 times to mix, add 50 uL of Saliva to each 2 mL tube, pipette to mix, vortex briefly and then centrifuge.
4. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix.
5. Incubate at RT for 5 mins.
6. Place tube on magnetic stand for 5 mins then remove and discard supernatant.
7. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash.
8. Air dry on magnetic stand for 5 mins.
9. Add 35 uL of RSB to each tube and vortex to mix.
10. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear.
11. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina DNA PCR-Free Library Prep
Supplier = Illumina
Catalog Number = 24 - 20041794; 96 - 20041795
Website =
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
Placement Pattern = Column
Destination Containers
96 well plate
Range = 300 To 2000
Decimal Places Displayed = 0
Instruction Notes
Multiline Text
Read Only
Default =
1. Label a new 96 well MIDI plate with LP1 and add the calculated DNA and RSB volumes of each sample to a new well on the plate.
2. Add 10 uL of TB1 to each well.
3. Add 15 uL of BLT-PF to each well.
4. Seal and shake at 1800 rpm for 1 minute.
5. Incubate in pre-heated Hybex for 8 minutes.
IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
Illumina DNA-RNA UD Indexes Set A B C D Tagmentation
Illumina DNA-RNA UD Indexes Set A Tagmentation
Illumina DNA-RNA UD Indexes Set B Tagmentation
Illumina DNA-RNA UD Indexes Set C Tagmentation
Illumina DNA-RNA UD Indexes Set D Tagmentation
Default =
1. Remove and discard all supernatant.
2. Add 45 µl ELM to each well.
3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min.
4. Incubate in the preheated Hybex for 8 mins.
5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
6. Remove and discard all supernatant from each well.
7. Add 75 µl TWB onto the beads in each well then seal and shake at 1800 rpm for 1 minute.
8. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
9. Remove and discard all supernatant from each well.
10. Without disturbing the bead pellet, use a 20 µl pipette to remove and discard residual TWB from each well.
11. Add 45 µl diluted HP3 to each well then seal and shake at 1800 rpm for 1 minute.
Nuclease-free water (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Total Samples
Numeric
Read Only
Default = 0
Decimal Places Displayed = 0
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina DNA PCR-Free Library Prep
Supplier = Illumina
Catalog Number = 24 - 20041794; 96 - 20041795
Website =
ℹ The actual version of the workflows and steps in the routing automation script may be different depending on the version of IPP installed.
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
Default =
1. Add 36 µl IPB to each well then seal and shake at 1800 rpm for 1 minute.
2. Incubate at room temperature for 2 minutes.
3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
4. Label a new 96-well MIDI plate LP2.
5. Add 42 µl IPB to each well of LP2.
6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of LP2 then seal and shake at 1800 rpm for 1 minute.
7. Discard LP1 and incubate LP2 at room temperature for 2 minutes.
8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
9. Without disturbing the bead pellet, remove and discard all supernatant from each well.
10. Wash beads 2x with 180 uL of fresh 80% EtOH for each wash.
11. Using a 20 µl pipette, remove residual EtOH from each well.
12. Air-dry on the magnetic stand (~4 minutes).
13. Add 22 µl of RSB onto the beads in each well then seal and shake at 1800 rpm for 1 minute.
14. Incubate at room temperature for 2 minutes.
15. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
16. Label a new PCR plate FLP.
17. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Row
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina DNA PCR-Free Library Prep
Supplier = Illumina
Catalog Number = 24 - 20041794; 96 - 20041795
Website =
ℹ The actual version of the nextStep step names may be different depending on the version of IPP installed.
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
Placement Pattern = Column
Destination Containers
96 well plate
Range = 100 To 2000
Instruction Notes
Multiline Text
Read Only
Default =
1. Label a new 96 well PCR plate LP1.
2. Add the calculated DNA, and RSB volumes to the LP1 plate and then pipette to mix.
3. Add the calculated BLT-PF to each well and pipette to mix.
4. Add 10 uL of TB1 to each well, pipette to mix and then seal.
5. Place the LP1 plate on the thermo cycler and run the TAG program.
Thermal Cycler Program
Text
Default = TAG
Thermal Cycler Program Notes
Multiline Text
Read Only
Default =
1. Choose the preheat lid option and set to 100°C
2. Set the reaction volume to 50 µl u 41°C for 5 minutes.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina DNA PCR-Free Library Prep
Supplier = Illumina
Catalog Number = 24 - 20041794; 96 - 20041795
Website =
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
Placement Pattern = Column
Destination Containers
96 well plate
Decimal Places Displayed = 0
Desired DNA Input (ng)
Numeric
Required Field
Default = 25
Range = 25 To 99</li
Instruction Notes
Multiline Text
Read Only
Default =
1. Label a new 96 well MIDI plate LP1.
2. Combine the calculated BLT-PF and TB1 volumes into a 1.5 mL tube to prepare the Tagmentation Master Mix.
3. Add the calculated DNA, and RSB volumes to the LP1 plate and then pipette to mix.
4. Add 20 µl Tagmentation Master Mix to each well, pipette to mix and then seal.
5. Place the LP1 plate on the thermo cycler and run the TAG program.
TB1 (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Thermal Cycler Program
Text
Default = TAG
Thermal Cycler Program Notes
Multiline Text
Read Only
Default =
1. Choose the preheat lid option and set to 100°C
2. Set the reaction volume to 50 µl u 41°C for 5 minutes.
IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
Illumina DNA-RNA UD Indexes Set A B C D Tagmentation
Illumina DNA-RNA UD Indexes Set A Tagmentation
Illumina DNA-RNA UD Indexes Set B Tagmentation
Illumina DNA-RNA UD Indexes Set C Tagmentation
Illumina DNA-RNA UD Indexes Set D Tagmentation
Default =
1. Remove and discard all supernatant.
2. Add 45 µl ELM to each well and pipette to mix.
3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min.
4. Place on the preprogrammed thermal cycler and run the ELM program.
5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
6. Remove and discard all supernatant from each well.
7. Add 75 µl TWB onto the beads in each well then pipette to mix.
8. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
9. Remove and discard all supernatant from each well.
10. Seal and centrifuge at 280 x g for 10 seconds and then place on magnetic stand.
11. Without disturbing the bead pellet, remove and discard residual TWB from each well.
12. Remove the plate from the magnetic stand.
13. Add 45 µl diluted HP3 to each well, pipette to mix and then incubate at RT for 2 mins.
Nuclease-free water (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Thermal Cycler Program
Text
Default = ELM
Thermal Cycler Program Notes
Multiline Text
Read Only
Default =
ELM Program
1. Choose the preheat lid option and set to 100°C
2. Set the reaction volume to 50 μl
3. Run for 37°C for 5 minutes
4. Run for 50°C for 5 minutes
Total Samples
Numeric
Read Only
Default = 0
Decimal Places Displayed = 0
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina DNA PCR-Free Library Prep
Supplier = Illumina
Catalog Number = 24 - 20041794; 96 - 20041795
Website =
ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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
Default =
1. Add 36 µl IPB to each well containing BLT-PF beads and pipette to mix.
2. Incubate at room temperature for 2 minutes.
3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
4. Label a new 96-well PCR plate LP2.
5. Add 42 µl IPB to each well of LP2.
6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of the LP2, pipette to mix and remove LP1 from the magnetic stand, and then discard.
7. Incubate LP2 at room temperature for 2 minutes.
8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
9. Without disturbing the bead pellet, remove and discard all supernatant from each well.
10. Wash beads 2x with 180 µl fresh 80% ethanol per well for each wash.
11. Seal and then centrifuge 280 x g for 10 seconds.
12. Place on the magnetic stand, and then wait 10 seconds.
13. Remove residual EtOH from each well, air-dry on the magnetic stand (~2 minutes) then remove from the magnetic stand.
14. Add 22 µl RSB onto the beads in each well and pipette to mix.
15. Incubate at room temperature for 2 minutes.
16. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
17. Label a new PCR plate FLP.
18. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina DNA PCR-Free Library Prep
Supplier = Illumina
Catalog Number = 24 - 20041794; 96 - 20041795
Website =
ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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
Default =
1. Add 81 µl IPB to each well and pipette to mix.
2. Incubate at room temperature for 5 minutes.
3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
4. Remove and discard all supernatant from each well.
5. Wash beads 2x with 180 uL of fresh 80% EtOH for each wash.
6. Seal plate then centrifuge 280 x g for 10 seconds.
7. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
8. Remove residual EtOH from each well and air-dry on the magnetic stand (~2 minutes).
9. Remove the plate from the magnetic stand.
10. Add 15 µl RSB onto the beads in each well and pipette to resuspend.
11. Incubate at room temperature for 2 minutes.
12. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
13. Transfer 14 µl supernatant from each well of the plate to the corresponding well of a new PCR plate.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {PoolName}
Reagent Kits
Illumina DNA PCR-Free Library Prep
Supplier = Illumina
Catalog Number = 24 - 20041794; 96 - 20041795
Website =
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
Well Sort Order = Column
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
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
Default = Concentration
Criteria 1 - Threshold Value
Numeric
Criteria 2 - Operator
Text
Default = <=
Criteria 2 - Source Data Field
Text
Default = Concentration
Criteria 2 - Threshold Value
Numeric
Step File Placeholders
Log - Automatically attached
QC Log File - Automatically attached
QC Result File - Automatically attached
Upload File - Manually uploaded
Sample Table
Enable QC Flags = Yes
Sample Display Default = Expand
Well Sort Order = Column
File Column Options
File Column Display = Hide
File Attachment Method = Auto
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
0.05% Tween® 20
10 mM Tris-HCl, pH 8.0 – 8.5 (25°C)
Control Types
KAPA DNA Standard 1
Supplier = Roche
Catalog Number = KK4828-07960166001
Website =
Conc. = 20 pM
Control Use
Single step only = No
KAPA DNA Standard 2
Supplier = Roche
Catalog Number = KK4828-07960166001
KAPA DNA Standard 3
Supplier = Roche
Catalog Number = KK4828-07960166001
KAPA DNA Standard 4
Supplier = Roche
Catalog Number = KK4828-07960166001
KAPA DNA Standard 5
Supplier = Roche
Catalog Number = KK4828-07960166001
KAPA DNA Standard 6
Supplier = Roche
Catalog Number = KK4828-07960166001
KAPA Library Quantification Dilution Control
Supplier = Roche
Catalog Number = KK4906
KAPA NTC
Control Use
Single step only = No
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
Default =
- Prepare the appropriate library dilutions (using DNA dilution buffer).
- Depending on the expected concentration of the library, 1:1,000 – 1:100,000 dilutions may be appropriate.
Default =
1. Set reaction volume and adding Rox? then click on calculate qPCR Master Mix and Sample Volume button.
2. Combine KAPA SYBR FAST qPCR Master Mix + Primer Premix, ROX and PCR-grade water to produce the qPCR Master Mix.
3. Add the qPCR Master Mix and Sample volume to a new 96 well PCR plate, seal the plate, vortex briefly to mix and then centrifuge.
4. Place the plate on the qPCR instrument.
5. Select Absolute Quantification and run the following cycling protocol:
- Initial denaturation Temp. at 95C fro 5 mins for 1 cycle
- Denaturation Temp. at 95C for 30 secs for 35 cycles
- Annealing/Extension/Data acquisition Temp. at 95C for 45 sec for 35 cycles
- Melt curve analysis (C) between 65C - 95C
6. Upload Cq values
7. Set Cq Thresholds then click on Assign QC Flags button.
8. Review Cq values and remove outliers.
9. Click on Calculate Average Cq.
KAPA SYBR FAST qPCR Master Mix + Primer Premix (uL)
Numeric
Read Only
Decimal Places Displayed = 2
PCR-grade water (uL)
Numeric
Read Only
Decimal Places Displayed = 2
qPCR Master Mix with Primer Prep Date
Date
Reaction Volume (uL)
Numeric Dropdown
Presets
20
10
ROX (uL)
Numeric
Read Only
Decimal Places Displayed = 2
Total Samples
Numeric
Read Only
Default = 0
Step File Placeholders
Log - Automatically attached
QC Log File - Automatically attached
QC Result File - Automatically attached
Upload File - Manually uploaded
Sample Table
Enable QC Flags = Yes
Sample Display Default = Expand
Well Sort Order = Column
File Column Options
File Column Display = Hide
File Attachment Method = Auto
Table Columns - Global Fields
Naming Convention = {InputItemName}
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
Default = Concentration (nM)
Criteria 1 - Threshold Value
Numeric
Default = 25
Criteria 2 - Operator
Text
Default = <=
Criteria 2 - Source Data Field
Text
Default = Concentration (nM)
Criteria 2 - Threshold Value
Numeric
Default = 99
Instruction Notes
Multiline Text
Read Only
Default =
1. Enter the Slope and the Y-Intercept for your standard curve then click on the Calculate Concentration button.
2. Set your Concentration Threshold then click on the Assign QC Flogs button.
Library Length (bp)
Numeric
Default = 450
Library Length Ratio
Numeric
Read Only
Decimal Places Displayed = 0
Standard Curve Slope
Numeric
Standard Curve Y-Intercept
Numeric
Step File Placeholders
Log - Automatically attached
QC Log File - Automatically attached
QC Result File - Manually uploaded
Upload File - Manually uploaded
Sample Table
Enable QC Flags = Yes
Sample Display Default = Expand
Well Sort Order = Column
File Column Options
File Column Display = Show
File Attachment Method = Manual
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Resuspension Buffer (RSB)
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
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {PoolName}
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
Well Sort Order = Column
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default = Select a sample type and protocol type for each sample.
Field Name
Field Type
Options
Additional Options and Dropdown Items
Criteria 1 - Operator
Text
Default = >=
Criteria 1 - Source Data Field
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Criteria 1 - Operator
Text
Default = >=
Criteria 1 - Source Data Field
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
10x Lysis Buffer (uL)
Numeric
Read Only
Decimal Places Displayed = 0
80% EtOH Prep Date
Date
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Desired DNA Input (ng)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default =
1. Add 10 µl ST2 to each well then seal and shake at 1800 rpm for 1 minute.
2. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
3. Without disturbing the bead pellet, remove and discard all supernatant from each well.
5. Add 150 μl TWB to each well then seal and shake at 1800 rpm for 1 minute.
6. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
Field Name
Field Type
Options
Additional Options and Dropdown Items
HP3 (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Instruction Notes for Hybex Protocol
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Desired DNA Input (ng)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
BLT-PF (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default =
1. Add 10 µl ST2 to each well, seal and then shake at 1800 rpm for 1 minute.
2. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
3. Without disturbing the bead pellet, remove and discard all supernatant from each well.
4. Add 150 μl TWB to each well, seal and shake at 1800 rpm for 1 minute.
5. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
Field Name
Field Type
Options
Additional Options and Dropdown Items
HP3 (uL)
Numeric
Read Only
Decimal Places Displayed = 0
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Instruction Notes for Standard Input
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Instruction Notes for Low Input
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default =
1. Combine 9 µl of each library in a 1.5 or 1.7 ml microcentrifuge tube.
2. Vortex to mix, and then centrifuge at 280 x g for 1 minute.
Field Name
Field Type
Options
Additional Options and Dropdown Items
Criteria 1 - Operator
Text
Default = >=
Criteria 1 - Source Data Field
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
DNA Dilution Buffer Prep Date
Date
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Adding Rox?
Text Dropdown
Presets
Yes
No
Default = Yes
Criteria 1 - Operator
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Criteria 1 - Operator
Text
Default = >=
Criteria 1 - Source Data Field
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Concentration (nM)
Numeric
Default = 2
Final Volume (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default =
1. Combine libraries equimolarly to a 2 nM final concentration.
2. Vortex to mix, and then centrifuge at 280 x g for 1 minute.
Sample Volume (uL)
Numeric
Read Only
Read Only
Read Only
Read Only
Required Field
Read Only
Read Only
Read Only
Read Only
Read Only
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 (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::gDNA:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Qubit - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::gDNA:: && input.::Protocol Type:: == ::Hybex::) {nextStep = ::Qubit - Hybex (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Whole Blood:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Dried Blood Spot:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type for DNA PCR-Free Lib Prep:: == ::Saliva:: && input.::Protocol Type:: == ::Thermal Cycler::) {nextStep = ::Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::}' \
-log {compoundOutputFileLuid0}"
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 (output.name.contains(::KAPA DNA Standard 1::)) {output.::Desired DNA Input (ng):: = 0.005952} ; \
if (output.name.contains(::KAPA DNA Standard 2::)) {output.::Desired DNA Input (ng):: = 0.000595} ; \
if (output.name.contains(::KAPA DNA Standard 3::)) {output.::Desired DNA Input (ng):: = 0.00006} ; \
if (output.name.contains(::KAPA DNA Standard 4::)) {output.::Desired DNA Input (ng):: = 0.000006} ; \
if (output.name.contains(::KAPA DNA Standard 5::)) {output.::Desired DNA Input (ng):: = 0.0000006} ; \
if (output.name.contains(::KAPA DNA Standard 6::)) {output.::Desired DNA Input (ng):: = 0.00000006} ; \
if (output.name.contains(::KAPA Library Quantification Dilution Control::)) {output.::Desired DNA Input (ng):: = 0.059524} ; \
if (output.name.contains(::KAPA NTC::)) {output.::Desired DNA Input (ng):: = 0} ; \
if (!output.name.contains(::KAPA::)) {output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::}' \
-log {compoundOutputFileLuid0}"
