Multigene amplification and massively parallel sequencing for cancer mutation discovery

*Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304;
^‡Department of Medicine, Division of Oncology, Stanford University School of Medicine, Clark Center W300, 318 Campus Drive, Stanford, CA 94305-5440;
^§Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden; and
^¶Cancer Research UK Cancer and Immunogenetics Laboratory, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom

Contributed by Ronald W. Davis, March 8, 2007 (received for review February 22, 2007)

Abstract

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.

Footnotes

^†To whom correspondence may be addressed. E-mail: dbowe{at}stanford.edu, fdahl{at}stanford.edu, or genomics_ji{at}stanford.edu
Author contributions: F.D. and J.S. contributed equally to this work; F.D., J.S., S.F., M.N., W.F.B., R.W.D., and H.J. designed research; F.D., J.S., S.F., K.W., and M.Z. performed research; F.D., J.S., D.B., and W.F.B. contributed new reagents/analytic tools; F.D., J.S., and H.J. analyzed data; and F.D., J.S., and H.J. wrote the paper.
Conflict of interest statement: M.N. and S.F. are cofounders of Olink AB and hold commercial rights to selector technology, and F.D and J.S. have business agreements with Olink AB.
This article contains supporting information online at www.pnas.org/cgi/content/full/0702165104/DC1.
Freely available online through the PNAS open access option.