Enrichment is a sample preparation strategy used to isolate and sequence only those genes of interest, reducing cost and improving informatics efficiency. Our target enrichment protocols are compatible with all SMARTer ThruPLEX DNA-Seq, SMARTer ThruPLEX Plasma-Seq, and SMARTer ThruPLEX Tag-seq kits.
A SMARTer ThruPLEX library preparation kit (choose from the SMARTer ThruPLEX DNA-Seq kits, SMARTer ThruPLEX Plasma-Seq kits, and SMARTer ThruPLEX Tag-seq kits listed in the Related Products section at the bottom of this page)
SeqCap EZ Accessory Kit V2 (Roche NimbleGen; Cat. # 07145594001 or 06776345001)
SeqCap EZ Hybridization and Wash Kit (Roche NimbleGen, Cat. # 05634261001)
SeqCap Pure Capture Bead Kit (Roche NimbleGen; Cat. # 06977952001)
Other consumables as specified in the the SeqCap EZ Library SR User's Guide under "Consumables Purchased from Other Vendors"
As specified in the "Laboratory Equipment" section of the SeqCap EZ Library SR User's Guide
SMARTer ThruPLEX library preparation
Prepare SMARTer ThruPLEX libraries according to the SMARTer ThruPLEX DNA-Seq, Plasma-Seq, or Tag-seq kit user manual.
Perform library purification using AMPure XP beads as described in the appropriate SMARTer 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.
SMARTer ThruPLEX library capture
Resuspend xGen Panel to 4.5 µl per hybridization in nuclease-free water and aliquot for single use into 0.2-ml tubes.
Resuspend xGen Universal Blocking Oligos to 1 µl per reaction (or 1 nmol/µl) in nuclease-free water.
Pool SMARTer ThruPLEX libraries for hybridization by adding equal amounts of each library to obtain 1 µg of DNA. For example, to hybridize four SMARTer ThruPLEX libraries with different indexes, 250 ng of each library would be added; or if pooling 10 uniquely indexed libraries, 100 ng of each library would be added.
In a 1.5-ml microcentrifuge tube combine:
5 µl of COT Human DNA (1 mg/ml) from the SeqCap EZ Accessory Kit v2
1 µg pooled SMARTer ThruPLEX libraries
1 µl xGen Universal Blocking Oligo - TS HT-i5
1 µl xGen Universal Blocking Oligo - TS HT-i7
Follow the SeqCap EZ Library SR User’s Guide (Version 5.0) starting at Chapter 5, Step 5, #4 (“Close the tube’s lid….”) and continue to the end of Chapter 7 with the following modification at Chapter 5, Step 5, #12: transfer the cocktail to the 4.5 µl aliquot of xGen Lockdown Panel in a 0.2-ml tube prepared in Step 1 above.
NOTE: This protocol was developed using the Roche NimbleGen SeqCap EZ System and the IDT xGen Acute Myeloid Leukemia (AML) Cancer Panel v1.0.