Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions |
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Authors: | YeYang Su Lin Lin Geng Tian Chen Chen Tao Liu Xingya Xu XinPeng Qi XiuQing Zhang HuanMing Yang |
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Institution: | (1) Graduate School of Chinese Academy of Sciences, Beijing, 100049, China;(2) Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100029, China;(3) Beijing Genomics Institute, Beijing, 101300, China |
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Abstract: | To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies
provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory
methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have
significant usage in association studies like cancer research, haven’t benefited much from those next-generation sequencing
technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer
may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies.
Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based
technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast,
we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal
Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and
sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences
of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully
amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both
a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation ‘synthesis by sequencing technology’ Illumina/Solexa
Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each
target sequence and the SNPs detection effectiveness) of using the LBA protocol in facilitating PCR-based re-sequencing and
genetic-variant-detection studies on the next-generation sequencing machine, raising the prospect of various PCR-based genomic/genetic
studies using this strategy.
National High-Tech Research & Development Program of China (Grant No.2006 AA02A301) |
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Keywords: | health genomics the next-generation sequencing techonology sequencing technology large-scale genomics PCR |
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