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Home › Learning centers › Next-generation sequencing › DNA-seq protocols › Targeted capture with Roche NimbleGen SeqCap EZ

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User-generated protocol

Targeted capture of ThruPLEX libraries with Roche NimbleGen SeqCap EZ

Introduction  

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 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, or ThruPLEX Tag-seq kits listed in the Related Products section at the bottom of this page)
  • Two blocking oligos (both required)
    • xGen Universal Blocking Oligo - TS HT-i5 (Integrated DNA Technologies; IDT)
    • xGen Universal Blocking Oligo - TS HT-i7 (IDT)
  • 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)
    • SeqCap EZ Library
  • Other consumables as specified in the the SeqCap EZ Library SR User's Guide (version 5.0) under "Consumables Purchased from Other Vendors"

Equipment

As specified in the "Laboratory Equipment" section of the SeqCap EZ Library SR User's Guide (version 5.0)

Protocol  

ThruPLEX library preparation

  1. Prepare ThruPLEX libraries according to the ThruPLEX DNA-Seq, Plasma-Seq, or Tag-seq kit user manual.
  2. 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

  1. Resuspend xGen Universal Blocking Oligos to 1 µl per reaction (or 1 nmol/µl) in nuclease-free water.
  2. 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.
  3. 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
  4. 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 to generate captured ThruPLEX libraries that are ready to be quantified and sequenced using Illumina NGS platforms.

NOTE: This protocol was developed using the SeqCap EZ Human Exome v3.0 Library.

Related Products

Cat. # Product Size Price License Quantity Details
R400584 ThruPLEX® Tag-seq 6S (12) Kit 12 Rxns USD $850.00

The ThruPLEX Tag-seq Kit includes all necessary reagents for generating and multiplexing DNA-seq libraries with the incorporation of Unique Molecular Indexes (UMIs), and includes 6 unique single index PCR primer sets. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols. Only 50 pg to 50 ng of fragmented double-stranded DNA is required for library preparation. The entire three-step workflow takes place in a single tube or well in about two hours. No intermediate purification steps or sample transfers are necessary, preventing handling errors and loss of valuable samples. This kit includes reagents sufficient for 12 reactions with 6 single-index primer sets.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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R400586 ThruPLEX® Tag-seq 96D Kit 96 Rxns USD $4987.00

The ThruPLEX Tag-seq Kit includes all necessary reagents for generating and multiplexing DNA-seq libraries with the incorporation of Unique Molecular Indexes (UMIs), and includes 96 dual index PCR primer sets. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols. Only 50 pg to 50 ng of fragmented double-stranded DNA is required for library preparation. The entire three-step workflow takes place in a single tube or well in about two hours. No intermediate purification steps or sample transfers are necessary, preventing handling errors and loss of valuable samples. This kit includes reagents sufficient for 96 reactions with 96 dual-index primer sets.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The ThruPLEX Tag-seq Kit includes all necessary reagents for generating and multiplexing DNA-seq libraries with the incorporation of Unique Molecular Indexes (UMIs), and includes 48 unique single index PCR primer sets. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols. Only 50 pg to 50 ng of fragmented double-stranded DNA is required for library preparation. The entire three-step workflow takes place in a single tube or well in about two hours. No intermediate purification steps or sample transfers are necessary, preventing handling errors and loss of valuable samples. This kit includes reagents sufficient for 48 reactions with 48 single-index primer sets.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The ThruPLEX DNA-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from as little as 50 pg of DNA. Single index and unique dual index kits are available and must be purchased separately. This product contains reagents for 24 reactions.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The ThruPLEX DNA-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from as little as 50 pg of DNA. Single index and unique dual index kits are available and must be purchased separately. This product contains reagents for 48 reactions.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The ThruPLEX DNA-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from as little as 50 pg of DNA. Single index and unique dual index kits are available and must be purchased separately. This product contains reagents for 96 reactions.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data
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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The ThruPLEX Plasma-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from cell-free DNA isolated from plasma. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 96 reactions.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data
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The ThruPLEX Plasma-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from cell-free DNA isolated from plasma. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product is composed of five 96-reaction kits (Cat. # R400681), 480 reactions total, of ThruPLEX Plasma-Seq reagents.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components

User-generated protocols

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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