Targeted DNA enrichment enables one to sequence genomic regions of interest efficiently and cost-effectively. Current targeted sequencing methods are time-consuming, labor-intensive, and often require large amount of input genomic DNA. We have developed a simple method for preparing libraries for targeted DNA sequencing, which takes less than eight hours. The method works by hybridizing single primers to DNA targets on adapter ligated gDNA fragments, extending the primers over the regions of interest and adapter, and amplifying the regions of interest by common PCR primers. The amount of starting genomic DNA can be as little as 10 ng and the assay can be easily customized for any genomic region. We show the sequencing results of a 1 Mb targeted region of cancer related genes with >90% alignment, >80% on target rate, high uniformity, and low drop-out rate. We also show the method is suitable for use with FFPE DNA.
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