Enrichment is a sample preparation strategy used to isolate and sequence only those genes of interest, reducing cost and improving informatics efficiency. Our enrichment protocols are compatible with ThruPLEX DNA-Seq FLEX, ThruPLEX Tag-Seq FLEX, and the ThruPLEX DNA-Seq Kit.
User-generated protocol
Targeted capture of ThruPLEX libraries with IDT xGen panels
Introduction
Materials required
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)
- Enrichment panel: An IDT xGen Pel
- SeqCap reagents:
- 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"
Equipment
- As specified in the "Laboratory Equipment" section of the SeqCap EZ Library SR User's Guide
Protocol
ThruPLEX library preparation
- Prepare ThruPLEX libraries according to the 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 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 ThruPLEX libraries for hybridization by adding equal amounts of each library to obtain 1 µg of DNA.
For example, to hybridize four 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 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.
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User-generated protocols
User-generated protocols are based on internal proof-of-concept experiments, customer collaborations, and published literature. In some cases, relevant results are discussed in our research news BioView blog articles. While we expect these protocols to be successful in your hands, they may not be fully reviewed or optimized. We encourage you to contact us or refer to the published literature for more information about these user-generated and -reported protocols.
If you are looking for a product-specific, fully optimized User Manual or Protocol-At-A-Glance, please visit the product's product page, open the item's product details row in the price table, and click Documents. More detailed instructions for locating documents are available on our website FAQs page.
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