BMC Genomics | |
The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity | |
Methodology Article | |
Jonathan Schug1  Klaus H. Kaestner1  Kate Mackiewicz2  Peter M. Clark2  Dimitri Monos3  Johannes Dapprich4  Deborah Ferriola5  Eric Rappaport6  Xiaowu Gai7  Monica D’Arcy7  Ariella Sasson7  | |
[1] Department of Genetics, University of Pennsylvania, 19104, Philadelphia, PA, USA;Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, 19104, Philadelphia, PA, USA;Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, 19104, Philadelphia, PA, USA;The Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, 19104, Philadelphia, PA, USA;Generation Biotech, 08648, Lawrenceville, NJ, USA;Generation Biotech, 08648, Lawrenceville, NJ, USA;Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, 19104, Philadelphia, PA, USA;Nucleic Acids & Protein Core Facility, The Children’s Hospital of Philadelphia, 19104, Philadelphia, PA, USA;The Center for Biomedical Informatics, The Children’s Hospital of Philadelphia, 19104, Philadelphia, PA, USA; | |
关键词: DNA target capture; Targeted enrichment; Genomic resequencing; DNA sequencing; MHC haplotype; | |
DOI : 10.1186/s12864-016-2836-6 | |
received in 2015-08-04, accepted in 2016-06-15, 发布年份 2016 | |
来源: Springer | |
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【 摘 要 】
BackgroundThe 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.ResultsRSE 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 %.ConclusionsRSE 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.
【 授权许可】
CC BY
© The Author(s). 2016
【 预 览 】
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