Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Simon, Fabrice; Banér, Johan; Dahl, Fredrik A.; Chu, Angela M.; Ji, Hanlee P.; Welch, Kimo M.; Davis, Ronald W.

doi:10.1093/nar/gkm078

Cited by 54 publications

(39 citation statements)

References 21 publications

(26 reference statements)

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“…In this proof-of-concept study, a large number of individual PCR assays were done before pooling for sequencing. Alternately, one could incorporate the Gene-Collector method, which generates uniformly distributed multiplexed amplification products with less bias (23), to increase the efficiency of amplicon generation. Furthermore, careful optimization of the amplification protocols, designing PCR amplicons of consistent length, and addition of spiked controls should be considered in future studies.…”

Section: Discussionmentioning

confidence: 99%

Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Taylor¹,

et al. 2007

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We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (<20) typically analyzed by traditional bisulfite sequencing. Comprehensive analysis of CpG methylation patterns at a single DNA molecule level using clustering algorithms revealed differential methylation patterns between diseases. A significant increase in methylation was detected in ALL and FL samples compared with CLL and MCL. Furthermore, a progressive spreading of methylation was detected from the periphery toward the center of select CpG islands in the ALL and FL samples. The ultradeep sequencing also allowed simultaneous analysis of genetic and epigenetic data and revealed an association between a single nucleotide polymorphism and the methylation present in the LRP1B promoter. This new generation of methylome sequencing will provide digital profiles of aberrant DNA methylation for individual human cancers and offers a robust method for the epigenetic classification of tumor subtypes. [Cancer Res 2007;67(18):8511-8]

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Section: Discussionmentioning

confidence: 99%

Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Taylor¹,

et al. 2007

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“…Another potential approach to normalizing the distribution of amplified targets is to separate the pool of probes in one high-abundant and one low-abundant reaction, before PCR amplification. Furthermore, we recently developed an alternative sample preparation strategy, called ''gene-collector,'' which potentially generates a more uniformly distributed multiplexed amplification product compared with selector technology (24).…”

Section: Discussionmentioning

confidence: 99%

Multigene amplification and massively parallel sequencing for cancer mutation discovery

Dahl

Stenberg

Simon

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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155

105

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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.cancer analysis ͉ high-throughput sequencing ͉ multiplex amplification

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“…Recently, several new methods have been developed for the multiplexed selection, amplification, and sequencing of genomics subsets (Bashiardes et al 2005;Dahl et al 2005Dahl et al , 2007Albert et al 2007;Fredriksson et al 2007;Hodges et al 2007;Meuzelaar et al 2007;Okou et al 2007;Porreca et al 2007). Several of these methods achieve higher levels of multiplexing Dahl et al 2007;Hodges et al 2007;Okou et al 2007;Porreca et al 2007), but they do not perform as well in other areas, such as the precise definition of target boundaries Dahl et al 2007;Hodges et al 2007;Okou et al 2007), the reproducible capture of some target regions (Porreca et al 2007), or the fraction of reads matching target sequences Hodges et al 2007;Okou et al 2007).…”

Section: Discussionmentioning

confidence: 99%

Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes

Varley¹,

Mitra²

2008

Genome Res.

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Medical resequencing of candidate genes in individual patient samples is becoming increasingly important in the clinic and in clinical research. Medical resequencing requires the amplification and sequencing of many candidate genes in many patient samples. Here we introduce Nested Patch PCR, a novel method for highly multiplexed PCR that is very specific, can sensitively detect SNPs and mutations, and is easy to implement. This is the first method that couples multiplex PCR with sample-specific DNA barcodes and next-generation sequencing to enable highly multiplex mutation discovery in candidate genes for multiple samples in parallel. In our pilot study, we amplified exons from colon cancer and matched normal human genomic DNA. From each sample, we successfully amplified 96% (90 of 94) targeted exons from across the genome, totaling 21.6 kbp of sequence. Ninety percent of all sequencing reads were from targeted exons, demonstrating that Nested Patch PCR is highly specific. We found that the abundance of reads per exon was reproducible across samples. We reliably detected germline SNPs and discovered a colon tumor specific nonsense mutation in APC, a gene causally implicated in colorectal cancer. With Nested Patch PCR, candidate gene mutation discovery across multiple individual patient samples can now utilize the power of second-generation sequencing.

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Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Cited by 54 publications

References 21 publications

Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Multigene amplification and massively parallel sequencing for cancer mutation discovery

Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes

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