Enrichment of ThruPLEX libraries with Agilent SureSelect platforms is easily performed. The module marked in red is not required when integrating with ThruPLEX kits. This target enrichment protocol is compatible with the ThruPLEX DNA-Seq Kit, ThruPLEX DNA-Seq FLEX, and ThruPLEX Tag-Seq FLEX.
Integration of SureSelectQXT with ThruPLEX kits
Additional reagents
Primers
Required
Illumina P5 and P7 primers
Blocking oligos
Required
IDT xGen Universal Blocking Oligos (TS HT-i5 and TS HT-i7)
Agilent Herculase II Fusion DNA Polymerase
Required
Agilent Cat. # 600677, 600679 (with dNTPs)
Agilent SureSelectQXT Reagent Kit
SureSelectQXT Library Prep Kit, ILM, Box #2
Not used
Replace with ThruPLEX DNA-Seq FLEX, ThruPLEX Tag-Seq FLEX, or a ThruPLEX DNA-Seq Kit
SureSelectQXT Target Enrichment Kit, ILM (Hyb module, Box #1)
Required
The following reagent is not used: SureSelectQXT Stop Solution
SureSelectQXT Target Enrichment Kit, ILM (Hyb module, Box # 2)
Required
The following reagent is not used: SureSelectQXT Primer Mix
Materials required
Reagents
ThruPLEX DNA-Seq Kit, ThruPLEX DNA-Seq FLEX, or ThruPLEX Tag-Seq FLEX for library preparation
SureSelectQXT reagents: Refer to the "Required Reagents" section of the Agilent SureSelectQXT protocol.
NOTE: The following item may be required for the post-capture amplification step: Herculase II Fusion DNA Polymerase with dNTPs (Agilent Technologies, Cat. # 600677 or 600679)
Equipment
As specified in the "Required Equipment" section of the Agilent SureSelectQXT protocol.
NOTE: When integrating ThruPLEX kits with the SureSelectQXT library capture system, all components of the SureSelectQXT Reagent Kit are used except the following:
SureSelectQXT Buffer
SureSelectQXT Enzyme Mix ILM
DMSO
SureSelectQXT Read Primer 1
SureSelectQXT Read Primer 2
SureSelectQXT Index Read Primer
SureSelectQXT P7 dual indexing primers
SureSelectQXT P5 dual indexing primers
SureSelectQXT Stop Solution
SureSelectQXT Primer Mix
Contact Agilent to order a SureSelectQXT Reagent Kit without the SureSelectQXT Library Prep Kit ILM, Box 2.
Protocol
ThruPLEX library preparation
Prepare ThruPLEX libraries according to the ThruPLEX user manual for your kit.
Perform library purification using AMPure XP beads as described in the appropriate ThruPLEX user manual.
CAUTION: For the final elution, DNA must be eluted by resuspending the beads in 30 µl of PCR grade water, not TE buffer.
ThruPLEX library capture
Resuspend xGen Universal Blocking Oligos to 1 µl per reaction (or 1 nmol/µl) in nuclease-free water.
Using a narrow gauge needle, poke hole(s) in the lid of each tube containing a ThruPLEX library to be used for capture.
Concentrate the ThruPLEX library using a vacuum concentrator held at ≤45°C to reduce the volume in the tube to <10 µl. Do not completely dry the mixture.
Bring the volume to 10 µl with nuclease-free water.
To each resuspended library add:
1 µl xGen Universal Blocking Oligo - TS HT-i5
1 µl xGen Universal Blocking Oligo - TS HT-i7
Follow procedures in the Agilent SureSelectQXT Protocol starting at Chapter 3, Step 2 through the end of Chapter 4, Step 5 with the following modification: At Chapter 4, Step 1. Amplify the Captured Libraries, modify the Post-Capture PCR Reaction Mix to the following:
Reagent
Volume per rxn
Nuclease-free water
10.5 µl
5x Herculase Rxn Buffer
10.0 µl
Herculase II Fusion DNA Polymerase
1.0 µl
100 mM dNTP Mix
0.5 µl
10 µM Illumina P5 Primer
2.5 µl
10 µM Illumina P7 Primer
2.5 µl
Total
27.0 µl
The ThruPLEX libraries are already indexed, so do not use the SureSelectQXT indexing primers.
NOTE: This protocol was developed using the SureSelectXT Human All Exon v5 Capture Library.
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