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Target-Enrichment Through Amplification of Hairpin-Ligated Universal Targets for Next-Generation Sequencing Analysis.
Methods Mol Biol. 2011;733:267-278
Authors: Singh P, Nayak R, Kwon YM
With rapid development of next-generation sequencing (NGS) technologies, it is becoming increasingly feasible to sequence entire genomes of various organisms from virus to human. However, in many occasions, it is still more practical to sequence and analyze only small regions of the entire genome that are informative for the purpose of the experiment. Although many target-enrichment or target capture methods exist, each method has its own strength and weakness in terms of the number of enriched targets, specificity, drop-off rate, and uniformity in capturing target DNA sequences. Many applications require a consistently low drop-off rate and high uniformity of enriched targets for routine collection of meaningful data. Here, we describe a simple and robust PCR-based protocol that can allow simultaneous amplification of numerous target regions. This method employs target-specific hairpin selectors to create DNA templates that contain target regions flanked by common universal priming sequences. We demonstrated the utility of this method by applying it for simultaneous amplification of 21 targets in the range of 191-604 bp from 41 different Salmonella strains using bar-coded universal primers. Analysis of 454 FLX pyrosequencing data demonstrated the promising performance of this method in terms of specificity and uniformity. This method, with great potential for robust amplification of hundreds of targets, should find broad applications for efficient analysis of multiple genomic targets for various experimental goals.
PMID: 21431777 [PubMed - as supplied by publisher]
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Target-Enrichment Through Amplification of Hairpin-Ligated Universal Targets for Next-Generation Sequencing Analysis.
Methods Mol Biol. 2011;733:267-278
Authors: Singh P, Nayak R, Kwon YM
With rapid development of next-generation sequencing (NGS) technologies, it is becoming increasingly feasible to sequence entire genomes of various organisms from virus to human. However, in many occasions, it is still more practical to sequence and analyze only small regions of the entire genome that are informative for the purpose of the experiment. Although many target-enrichment or target capture methods exist, each method has its own strength and weakness in terms of the number of enriched targets, specificity, drop-off rate, and uniformity in capturing target DNA sequences. Many applications require a consistently low drop-off rate and high uniformity of enriched targets for routine collection of meaningful data. Here, we describe a simple and robust PCR-based protocol that can allow simultaneous amplification of numerous target regions. This method employs target-specific hairpin selectors to create DNA templates that contain target regions flanked by common universal priming sequences. We demonstrated the utility of this method by applying it for simultaneous amplification of 21 targets in the range of 191-604 bp from 41 different Salmonella strains using bar-coded universal primers. Analysis of 454 FLX pyrosequencing data demonstrated the promising performance of this method in terms of specificity and uniformity. This method, with great potential for robust amplification of hundreds of targets, should find broad applications for efficient analysis of multiple genomic targets for various experimental goals.
PMID: 21431777 [PubMed - as supplied by publisher]
More...