Enrichment is a sample preparation strategy used to isolate and sequence only those genes of interest (in this case the entire exome), 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 Kits.
User-generated protocol
Exome capture of ThruPLEX libraries with Illumina Nextera® Rapid Capture
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)
- Illumina capture reagents (one of the following required):
- Nextera Rapid Capture Exome Enrichment Kit (FC-140-1000; FC-140-1001; FC-140-1002; FC-140-1003; FC-140-1083; FC-140-1086; FC-140-1089)
- Nextera Rapid Capture Expanded Exome Enrichment Kit (FC-140-1004; FC-140-1005; FC-140-1006)
Other consumables and equipment
Refer to Appendix A "Consumables and Equipment" (page 44–46) in the Nextera Rapid Capture Enrichment Reference Guide, Cat. # FC-140-9001DOC, Part # 15037436 Rev. J.
Protocol
ThruPLEX library preparation
- Prepare ThruPLEX libraries according to the kit user manual.
- Perform library purification using AMPure XP beads as described in the 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
- Combine 500 ng of each uniquely indexed ThruPLEX library.
NOTE: The number of libraries that may be pooled is determined by the kit configuration of the Nextera Rapid Capture Exome Enrichment kit purchased (1-plex to 12-plex).
- If necessary, adjust the volume of the pooled libraries to 38 μl.
- If the total volume of libraries is >38 μl, use a vacuum concentrator without heating to reduce volume to 38 μl.
- If the total volume is <38 μl, increase the volume to 38 μl with nuclease-free water.
- In a 0.2-ml PCR tube combine:
- 38 μl DNA library or sample pool
- 1 µl xGen Universal Blocking Oligo - TS HT-i5
- 1 µl xGen Universal Blocking Oligo - TS HT-i7
- 50 μl buffer EHB (from Nextera kit)
- 10 μl capture oligos CEX, EEX, or RCO (from Nextera kit)
- Continue with Procedure Step 2, page 18 of the probe hybridization per the Nextera Rapid Capture Enrichment Reference Guide, Catalog # FC-140-9001DOC, Part #15037436 Rev. J, "Shake at 1200 rpm...."
- For Procedure Step 1 of the Second Hybridization (page 22) add the following to each well containing 25 μl of product from the first capture:
- 13 μl buffer RSB (from Nextera kit)
- 1 μl xGen Universal Blocking Oligo - TS HT-i5
- 1 μl xGen Universal Blocking Oligo - TS HT-i7
- 50 μl buffer EHB (from Nextera kit)
- 10 μl capture oligos CEX, EEX, or RCO (from Nextera kit)
- Continue with Procedure Step 2, page 22 of the probe hybridization per the Nextera Rapid Capture Enrichment Reference Guide, Catalog # FC-140-9001DOC, Part # 15037436 Rev. J, "Shake at 1200 rpm…"
NOTE: This protocol was developed using the Nextera Rapid Capture Exome Enrichment Kit (FC-140-1083).
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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.
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