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The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity
Journal article   Open access   Peer reviewed

The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity

Johannes Dapprich, Deborah Ferriola, Kate Mackiewicz, Peter M. Clark, Eric Rappaport, Monica D'Arcy, Ariella Sasson, Xiaowu Gai, Jonathan Schug, Klaus H. Kaestner, …
BMC genomics, v 17(1), pp 486-486
09 Jul 2016
DOI: https://doi.org/10.1186/s12864-016-2836-6
PMID: 27393338
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1186/s12864-016-2836-6View
Published, Version of Record (VoR) Open

Abstract

Biotechnology & Applied Microbiology Genetics & Heredity Life Sciences & Biomedicine Science & Technology
Background: The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. Results: RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3'-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. Conclusions: RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.

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57 citations in Web of Science
61 citations in Scopus

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Web of Science research areas
Biotechnology & Applied Microbiology
Genetics & Heredity
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