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Biotechniques. 2009 Mar;46(3):229-31. doi: 10.2144/000113082.

Method for improving sequence coverage uniformity of targeted genomic intervals amplified by LR-PCR using Illumina GA sequencing-by-synthesis technology.

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Scripps Genomic Medicine, Scripps Translational Science Institute, Scripps Research Institute, La Jolla, CA, USA.


One approach for high-throughput population-based sequencing of targeted intervals in the human genome is to amplify the regions using long-range PCR (LR-PCR) followed by sequencing with next-generation sequencing (NGS) technologies. Utilizing this method, we have observed that the 50 bp located at the amplicon ends account for more than 50% of the sequenced bases and that the sequence coverage depth of base pairs within an amplicon is highly variable. Here we propose an explanation for the overrepresentation of the amplicon ends and show that the use of 5'-blocked primers for the LR-PCR reaction reduces their overrepresentation. Furthermore, we demonstrate that using a 600-bp library insert size rather than the standard 200-bp insert size results in more uniform sequence coverage depth. The capability to increase sequence coverage uniformity greatly improves the effective throughput of NGS platforms.

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