The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity
Public DepositedAdd to collection
You do not have access to any existing collections. You may create a new collection.
Downloadable Content
Download PDFCitation
MLA
Dapprich, Johannes, et al. The Next Generation of Target Capture Technologies - Large Dna Fragment Enrichment and Sequencing Determines Regional Genomic Variation of High Complexity. BioMed Central, 2016. https://doi.org/10.17615/fhf7-wb38APA
Dapprich, J., Ferriola, D., Mackiewicz, K., Clark, P., Rappaport, E., D’arcy, M., Sasson, A., Gai, X., Schug, J., Kaestner, K., & Monos, D. (2016). The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity. BioMed Central. https://doi.org/10.17615/fhf7-wb38Chicago
Dapprich, Johannes, Deborah Ferriola, Kate Mackiewicz, Peter M Clark, Eric Rappaport, Monica D’arcy, Ariella Sasson et al. 2016. The Next Generation of Target Capture Technologies - Large Dna Fragment Enrichment and Sequencing Determines Regional Genomic Variation of High Complexity. BioMed Central. https://doi.org/10.17615/fhf7-wb38- Creator
-
Dapprich, Johannes
- Other Affiliation: Generation Biotech
-
Ferriola, Deborah
- Other Affiliation: The Children’s Hospital of Philadelphia
-
Mackiewicz, Kate
- Other Affiliation: The Children’s Hospital of Philadelphia
-
Clark, Peter M
- Other Affiliation: The Children’s Hospital of Philadelphia
-
Rappaport, Eric
- Other Affiliation: The Children’s Hospital of Philadelphia
-
D’Arcy, Monica
- Other Affiliation: The Children’s Hospital of Philadelphia
-
Sasson, Ariella
- Other Affiliation: The Children’s Hospital of Philadelphia
-
Gai, Xiaowu
- Other Affiliation: The Children’s Hospital of Philadelphia
-
Schug, Jonathan
- Other Affiliation: University of Pennsylvania
-
Kaestner, Klaus H
- Other Affiliation: University of Pennsylvania
-
Monos, Dimitri
- Other Affiliation: The Children’s Hospital of Philadelphia
- Abstract
- 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.
- Date of publication
- July 9, 2016
- DOI
- Identifier
- Resource type
- Article
- Rights statement
- In Copyright
- Rights holder
- The Author(s).
- Journal title
- BMC Genomics
- Journal volume
- 17
- Journal issue
- 1
- Page start
- 486
- Language
- English
- Bibliographic citation
- BMC Genomics. 2016 Jul 09;17(1):486
- Publisher
- BioMed Central
Relations
- Parents:
This work has no parents.
Items
Thumbnail | Title | Date Uploaded | Visibility | Actions |
---|---|---|---|---|
12864_2016_article_2836.pdf | 2019-05-07 | Public | Download | |
12864_2016_2836_moesm1_esm.pdf | 2019-05-07 | Public | Download |