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

Content Provider	Springer Nature : BioMed Central
Author	Dapprich, Johannes Ferriola, Deborah Mackiewicz, Kate Clark, Peter M. Rappaport, Eric D’Arcy, Monica Sasson, Ariella Gai, Xiaowu Schug, Jonathan Kaestner, Klaus H. Monos, Dimitri
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.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-016-2836-6.pdf
Ending Page	14
Page Count	14
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/s12864-016-2836-6
Journal	BMC Genomics
Issue Number	1
Volume Number	17
Language	English
Publisher	BioMed Central
Publisher Date	2016-07-09
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics DNA target capture Targeted enrichment Genomic resequencing DNA sequencing MHC haplotype
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
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9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

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