Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments
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TLDR
The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences.Abstract:
We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.read more
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Solution Hybrid Selection with Ultra-long Oligonucleotides for Massively Parallel Targeted Sequencing
Andreas Gnirke,Alexandre Melnikov,Jared Maguire,Peter Rogov,Emily M LeProust,William Brockman,William Brockman,Timothy Fennell,Georgia Giannoukos,Sheila Fisher,Carsten Russ,Stacey Gabriel,David B. Jaffe,Eric S. Lander,Eric S. Lander,Eric S. Lander,Chad Nusbaum +16 more
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TL;DR: The experiences with the leading target-enrichment technologies, the optimizations that are performed, and typical results that can be obtained using each are described and detailed protocols for each are provided so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.
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Genome Structure of the Legume, Lotus japonicus
Shusei Sato,Yasukazu Nakamura,Takakazu Kaneko,Erika Asamizu,Tomohiko Kato,Mitsuteru Nakao,Shigemi Sasamoto,Akiko Watanabe,Akiko Ono,Kumiko Kawashima,Tsunakazu Fujishiro,Midori Katoh,Mitsuyo Kohara,Yoshie Kishida,Chiharu Minami,Shinobu Nakayama,Naomi Nakazaki,Yoshimi Shimizu,Sayaka Shinpo,Chika Takahashi,Tsuyuko Wada,Manabu Yamada,Nobuko Ohmido,Makoto Hayashi,Kiichi Fukui,Tomoya Baba,Tomoko Nakamichi,Hirotada Mori,Satoshi Tabata +28 more
TL;DR: Structural features of the L. japonicus genome are reported, providing the first opportunity to look into the complex and unique genetic system of legumes.
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Multiplex amplification of large sets of human exons
Gregory J. Porreca,Kun Zhang,Kun Zhang,Jin Billy Li,Bin Xie,Derek Austin,Sara L Vassallo,Emily M LeProust,Bill J. Peck,Christopher J. Emig,Fredrik A. Dahl,Fredrik A. Dahl,Yuan Gao,George M. Church,Jay Shendure,Jay Shendure +15 more
TL;DR: It is shown that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify ∼10,000 human exons in a single multiplex reaction, and it is anticipated that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing ofhuman exons at a fraction of the cost of whole-genome resequenced.
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Massively Parallel Sequencing: The Next Big Thing in Genetic Medicine
TL;DR: Routine clinical use of massively parallel sequencing will require higher accuracy, better ways to select genomic subsets of interest, and improvements in the functionality, speed, and ease of use of data analysis software, which will increase the responsibility of geneticists to ensure that the information obtained is used in a medically and socially responsible manner.
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