Multigene amplification and massively parallel sequencing for cancer mutation discovery.
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.
Dahl
Fredrik
F
Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA. fdahl@stanford.edu
Stenberg
Johan
J
Fredriksson
Simon
S
Welch
Katrina
K
Zhang
Michael
M
Nilsson
Mats
M
Bicknell
David
D
Bodmer
Walter F
WF
Davis
Ronald W
RW
Ji
Hanlee
H
D001483:Base Sequence; D045744:Cell Line, Tumor; D052138:Genes, Neoplasm / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D016159:Tumor Suppressor Protein p53