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- DNA-seq protocols
DNA target enrichment protocols for SMARTer ThruPLEX kits
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. SMARTer ThruPLEX kits are able to create libraries with greatly reduced input DNA levels as compared to other kits while providing at least equivalent data.
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 SMARTer ThruPLEX kits are compatible with these target enrichment kits and provide superior performance, particularly with low-input samples.
Libraries are prepared following the standard SMARTer 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.
Protocols for integrating SMARTer ThruPLEX kits with target enrichment systems:
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