Enrichment is a sample preparation strategy used to isolate and sequence only those genes of interest, reducing the cost and improving the informatics efficiency. 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. In a typical exome sequencing workflow, libraries are constructed from purified DNA, enriched for the exon through solution-based hybridization with oligonucleotide probes and then sequenced.
Tools to analyze the exome are now offered by several companies who have developed and now sell exome enrichment kits for use with libraries prepared from a variety of human sources including plasma, fresh tissue, and FFPE samples. These kits contain oligonucleotides designed to capture the genes of interest (in this case the entire exome) by hybridization. The kits are provided by Agilent and Roche; other companies such as IDT prepare custom panels that are use to capture a “panel” of genes.
While the vendors have excellent enrichment tools, the library preparation products offered to complement these often fail to perform effectively with low sample amounts. ThruPLEX kits are able to create libraries with input DNA amounts at levels much reduced from other kits while providing at least equivalent data at this lower amount. In our application note (166-001), we demonstrate that libraries prepared from ThruPLEX Kits are compatible with Agilent’s SureSelect technology and provide superior performance when compared to the Agilent SureSelect Library Prep Kit.
Libraries are prepared following ThruPLEX standard protocol. If multiplexing, unique barcodes must be created on each library. The libraries are pooled and xGen® Universal Blocking Oligos (Integrated DNA Technologies) are added to inhibit daisy-chaining (binding of the library fragments to each other). ThruPLEX libraries prepared with dual barcodes require a different set of xGen® Universal Blocking Oligos than those prepared with single barcodes (see information below). This sample is then used in the enrichment protocol following the manufacturers directions.
xGen® Universal Blocking Oligos Information
All blocking oligos are available from IDT (Integrated DNA Technologies). When using ThruPLEX DNA-seq with 8 nt single or dual indexes, order xGen® Universal Blocking Oligo – TS HT-i5 and xGen® Universal Blocking Oligo – TS HT-i7
Protocols specific for each technology are available:
Exome Capture of ThruPLEX® Libraries with Roche NimbleGen® SeqCap® Library
Exome Capture of ThruPLEX® Libraries with Agilent SureSelect® Capture Library