Enrichment of ThruPLEX libraries with Agilent SureSelect platforms is easily performed. The chart below details the reagents necessary for this SureSelect^XT2 protocol. The modules marked in red are not required when integrating with ThruPLEX kits. This target enrichment protocol is compatible with ThruPLEX DNA-Seq FLEX, ThruPLEX Tag-Seq FLEX, and the ThruPLEX DNA-Seq Kit.

Reagents

• ThruPLEX DNA-Seq FLEX, ThruPLEX Tag-Seq FLEX, or a ThruPLEX DNA-Seq Kit for library preparation
• Two blocking oligos (both required):
  • xGen Universal Blocking Oligo - TS HT-i5 (Integrated DNA Technologies; IDT)
  • xGen Universal Blocking Oligo - TS HT-i7 (IDT)
• SureSelect^XT2 reagents:
  • Refer to the "Required Reagents" section of the Agilent SureSelect^XT2 Protocol
  • A SureSelect^XT2 Capture Library (e.g. SureSelect^XT2 Human All Exon V5, 16; Agilent Technologies, Cat. # 5190-6208)

Equipment

• As specified in the "Required Equipment" section of the Agilent SureSelect^XT2 Protocol.

ThruPLEX Library Preparation

1. Prepare ThruPLEX libraries according to the kit user manual.
2. Perform library purification using AMPure XP beads as described in the appropriate ThruPLEX user manual.

ThruPLEX library capture

1. Resuspend xGen Universal Blocking Oligos to 1 µl per reaction (or 1 nmol/µl) in nuclease-free water.
2. Pool ThruPLEX libraries for hybridization by adding equal amounts of each library to obtain 1.5 µg of DNA.
   Depending on capture library size, equal amounts of 8 or 16 libraries are pooled. For example, the SureSelect^XT2 protocol recommends pooling of 8 libraries with different indexes (187.5 ng of each) when using the Human All Exon v5 Capture Library.
3. In a 1.5 ml microcentrifuge tube combine:
   • 1.5 µg pooled ThruPLEX libraries
   • 1 µl xGen Universal Blocking Oligo - TS HT-i5
   • 1 µl xGen Universal Blocking Oligo - TS HT-i7
4. Concentrate the ThruPLEX libraries/xGen Universal Blocking Oligo mixture using a vacuum concentrator held at ≤45°C to reduce the volume in the tube to <7 μl. Do not completely dry the mixture.
5. Bring the volume to 7 μl with nuclease-free water.
6. Vortex the tube vigorously for 30 sec and centrifuge to bring contents to the bottom of the tube.
7. Add 9 μl SureSelect^XT2 Blocking Mix; pipette up and down to mix.
8. Transfer the contents to a 0.2-ml PCR tube or to one well of a 96-well plate.
9. Proceed with the SureSelect^XT2 Protocol starting at Chapter 4, Step 2, #2 (“Cap the wells…”) to the end of Chapter 5, Step 3.