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. 2007 May 29;104(22):9387-92.
doi: 10.1073/pnas.0702165104. Epub 2007 May 17.

Multigene amplification and massively parallel sequencing for cancer mutation discovery

Affiliations

Multigene amplification and massively parallel sequencing for cancer mutation discovery

Fredrik Dahl et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

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.

Figures

Fig. 1.
Fig. 1.
The selector and sequencing assay. (A) A DNA sample is digested to defined fragments by using restriction enzyme(s). The color bars represent the targets of interest. (B) Targeted circularization is performed by using selectors. (I) Selectors contain two oligonucleotides: a selector probe and a general vector oligonucleotide. The selector probe has two single-stranded target-complementary end sequences (orange) that are linked by a general sequence motif (gray) and the vector oligonucleotide that is complementary to the general sequence motif in the selector probes (gray). (II) The circularization reaction can be carried out by using two different approaches. Either both ends of the selected fragment connect to the vector oligonucleotide by hybridizing and ligation or the vector oligonucleotide forms a branched structure in an optional position at the 5′ end of the fragment. This latter structure is recognized and processed by the added endonucleolytic enzyme, forming ends suitable for ligation. (C) The circles are amplified in a multiplex PCR by using a primer pair complementary to the general vector sequence introduced in every circle. (D) The first step in the 454-sequencing procedure is to attach general, 454-optimized, adaptor sequences to each end of each PCR product. (E) The PCR products are separated into single strands and bound to beads in limiting dilutions, resulting in one unique fragment per bead. (F) The beads are clonally amplified in droplets of an oil-emulsion-based PCR, resulting in beads carrying millions of target sequences. (G) The beads are finally deposited into picoliter-sized wells, one bead per well, where solid-phase pyrosequencing is performed and monitored.
Fig. 2.
Fig. 2.
Sequencing depth. A normal sample, circularized and amplified in triplicate reactions, and a cancer cell line sample, PC/JW, were sequenced in one 454-experiment. The x axis shows number of reads (n), and the y axis shows the fraction of the target region with a sequencing depth of n or more.

References

    1. Bodmer WF. J Hum Genet. 2006;51:391–396. - PMC - PubMed
    1. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, et al. Science. 2004;304:1497–1500. - PubMed
    1. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, et al. N Engl J Med. 2004;350:2129–2139. - PubMed
    1. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, et al. Science. 2006;314:268–274. - PubMed
    1. Patil N, Berno AJ, Hinds DA, Barrett WA, Doshi JM, Hacker CR, Kautzer CR, Lee DH, Marjoribanks C, McDonough DP, et al. Science. 2001;294:1719–1723. - PubMed

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