User-generated DNA target enrichment protocols for ThruPLEX kits
Remarkably, the sequencing data from the PicoPLEX DNA-seq libraries of PGD embryos clearly showed two small, unbalanced segments consistent with the predicted patterns from high resolution fish re-testing of a maternal blood sample that was initially scored as normal. This is a significant example of the sequencing data from embryos exposing a cryptic translocation missed by microarrays."
—Brian Mariani, Ph.D., Chief Scientist, Scientific Director, GENETICS & IVF INSTITUTE
Target enrichment is a sample preparation strategy used to isolate and sequence only genes of interest, reducing costs and improving informatics efficiency. For example, in exome enrichment the genes of interest are the entire exome of the human genome, which accounts for about 1% of the human genome and about 30 Mb of sequence. While excellent enrichment tools exist, many available library preparation products struggle to perform effectively with low-input samples. ThruPLEX kits are able to create libraries with greatly reduced input DNA levels as compared to other kits while providing at least equivalent data.
ThruPLEX target enrichment workflow
In a typical targeted sequencing workflow, libraries are constructed from purified DNA, enriched for specific targets or genes through solution-based hybridization using oligonucleotide probes, and then sequenced. Tools for enriching specific targets are now offered by several companies, who have developed target enrichment kits and panels for use with libraries prepared from a variety of DNA sources, including plasma, fresh tissue, and FFPE samples. These kits contain oligonucleotides designed to capture the genes of interest (for example, the entire exome) by hybridization. Libraries prepared from ThruPLEX kits are compatible with these target enrichment kits and provide superior performance, particularly with low-input samples.
Libraries are prepared following the standard ThruPLEX protocol. If multiplexing, unique indexes must be used to create the libraries. The libraries are pooled, and xGen Universal Blockers are added to inhibit daisy-chaining (binding of the library fragments to each other). This sample is then used in the target enrichment protocol following the manufacturer's directions.
Purify DNA
Shear DNA (if necessary)
Prepare ThruPLEX Libraries: repair, add adapters, amplify
Cleanup with AMPure XP
Quantify and pool (if necessary)
Combine with blocking oligos
Hybridize, capture, and wash
Amplify
Pool (if necessary) and cleanup
Quantify enriched libraries
Sequence
Protocols for integrating ThruPLEX kits with target enrichment systems:
Illumina Nextera protocol
Protocol for exome enrichment of ThruPLEX libraries using the Illumina Nextera Rapid Capture Exome Enrichment Kit.
Roche NimbleGen SeqCap EZ protocol
Protocol for target enrichment of ThruPLEX libraries using the Roche NimbleGen SeqCap EZ System.
xGen lockdown probe protocol
Protocol for target enrichment of ThruPLEX libraries using the IDT xGen target enrichment panels
Agilent SureSelectXT protocol
Protocol for target enrichment of ThruPLEX libraries using the Agilent SureSelectXT Target Enrichment System.
Agilent SureSelectXT2 protocol
Protocol for target enrichment of ThruPLEX libraries using the Agilent SureSelectXT2 Target Enrichment System.
Agilent SureSelectQXT protocol
Protocol for target enrichment of ThruPLEX libraries using the Agilent SureSelectQXT Target Enrichment System.
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?
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