Enrichment of ThruPLEX libraries with Agilent SureSelect platforms is easily performed. The chart below details the reagents necessary for this SureSelectXT protocol. The modules marked in red are not required when integrating with ThruPLEX kits. This target enrichment protocol is compatible with all ThruPLEX DNA-Seq, ThruPLEX Plasma-Seq, and ThruPLEX Tag-seq kits.
Integration of SureSelectXT with ThruPLEX kits
Additional reagents
Primers
Required
Illumina P5 and P7 primers
Blocking oligos
Requred
xGen Universal Blocking Oligos (TS HT-i5 and TS HT-i7)
Agilent Herculase II Fusion DNA Polymerase
Required
Agilent SureSelectXT Reagent Kit
SureSelectXT Library Prep Kit ILM
Not used
Replace with a ThruPLEX kit.
Contact Agilent to purchase a Reagent Kit without this component.
SureSelect Target Enrichment Box #1
Required
The following components are not used:
SureSelect Elution Buffer
SureSelect Neutralization Buffer
SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box #2
Required
The following components are not used:
SureSelect ILM Indexing Pre Capture Reverse PCR Primer
SureSelect ILM Indexing Post Capture Forward PCR Primer
Materials required
Reagents
A ThruPLEX library preparation kit (choose from the ThruPLEX DNA-Seq kits, ThruPLEX Plasma-Seq kits, and ThruPLEX Tag-seq kits listed in the Related Products section at the bottom of this page)
SureSelectXT reagents: Refer to the "Required Reagents" section of the Agilent SureSelectXT Protocol
Equipment
As specified in the "Required Equipment" section of the Agilent SureSelectXT Protocol.
NOTE: When integrating ThruPLEX kits with the SureSelectXT library capture system, all components of the SureSelectXT Reagent Kit are used except the following:
SureSelectXT Library Prep Kit ILM*
SureSelect ILM Indexing Pre Capture Reverse PCR Primer
SureSelect ILM Indexing Post Capture Forward PCR Primer
*Contact Agilent to order a SureSelectXT Reagent Kit without the SureSelectXT Library Prep Kit ILM.
Protocol
ThruPLEX Library Preparation
Prepare ThruPLEX libraries according to the ThruPLEX DNA-Seq, Plasma-Seq, or Tag-seq kit user manual.
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.
Follow the Agilent SureSelectXT Protocol starting at the beginning of Chapter 4 and continuing to the end of Chapter 5 with the following modifications:
Chapter 4, Step 1. Hybridize DNA Samples to the Capture Library:
Depending on sample type, quality, fragment size, and thermal cycler used, ThruPLEX library preparation may not yield 750 ng as called for in the SureSelectXT Protocol. If this is the case, use the entire volume of library for concentration.
In addition to the reagents included in the SureSelect Block Mix in Table 32 on page 66, add:
1 µl of xGen Universal Blocking Oligo - TS HT-i5
1 µl of xGen Universal Blocking Oligo - TS HT-i7
NOTE: This results in a volume of 7.6 µl per reaction instead of 5.6 µl as stated in the SureSelectXT Protocol.
Chapter 5, Step 1A. Amplify the Capture Libraries with Indexing Primers: Modify the Post-Capture PCR Reaction Mix (Table 38, page 71) to the following:
Reagent
Volume per rxn
Nuclease-free water
19.5 µl
5x Herculase Rxn Buffer (clear cap)
10.0 µl
Herculase II Fusion DNA Polymerase (red cap)
1.0 µl
100 mM dNTP Mix (green cap)
0.5 µl
10 µM Illumina P5 Primer
2.5 µl
10 µM Illumina P7 Primer
2.5 µl
Total
36.0 µl
NOTE: This protocol was developed using the SureSelectXT Human All Exon v5 Capture Library.
Related Products
User-generated protocols
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