Multigene amplification and massively parallel sequencing for cancer mutation discovery
Multigene amplification and massively parallel sequencing for cancer mutation discovery
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DOI：
10.1073/pnas.0702165104
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发表时间：
2007-05-29
影响因子：
11.1
通讯作者：
Ji, Hanlee
中科院分区：
文献类型：
--
作者：
Dahl, Fredrik;Stenberg, Johan;Ji, Hanlee
我们开发了一种将高度多路复用和目标特异性扩增过程与高通量平行测序技术相结合的多种人类基因大规模平行重测序方法。扩增过程是基于寡核苷酸结构，称为选择器，它引导特定DNA目标区域的循环。随后，循环的目标序列被多重放大，并使用高度并行的合成测序技术进行分析。作为一项概念验证性研究，我们展示了覆盖177个外显子的10个癌症基因的平行重测序，每个样本的平均序列覆盖率为93%。研究了7个癌细胞系和1个正常基因组DNA样本，在10个基因中发现了多个突变和多态性。TP53基因的突变和多态性通过传统测序得到证实。
We have developed a procedure for massively parallel resequencing of multiple human genes by combining a highly multiplexed and target-specific amplification process with a high-throughput parallel sequencing technology. The amplification process is based on oligonucleotide constructs, called selectors, that guide the circularization of specific DNA target regions. Subsequently, the circularized target sequences are amplified in multiplex and analyzed by using a highly parallel sequencing-by-synthesis technology. As a proof-of-concept study, we demonstrate parallel resequencing of 10 cancer genes covering 177 exons with average sequence coverage per sample of 93%. Seven cancer cell lines and one normal genomic DNA sample were studied with multiple mutations and polymorphisms identified among the 10 genes. Mutations and polymorphisms in the TP53 gene were confirmed by traditional sequencing.