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 all ThruPLEX DNA-Seq, ThruPLEX Plasma-Seq, and ThruPLEX Tag-seq kits.
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)
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 ThruPLEX DNA-seq, Plasma-seq, or Tag-seq 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.
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:
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).
Related Products
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.
Questions? Protocols of your own that you would like to share?