Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2836-6) contains supplementary material, which is available to authorized users.

Highlights

  • The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions

  • The optimal spacing of capture primers depends in part on the particular DNA extraction method that was used to prepare the DNA from blood, tissue or cells: Fig. 2 Effects of Region-Specific Extraction (RSE) capture primer spacing on target enrichment. a Schematic representation of the distribution of captured genomic DNA copy number obtained around the primer hybridization site, indicated with a red triangle, as measured by qPCRs, placed at increasing distances from the primer hybridization site and shown with black inverted triangles

  • To study the dependence of capture efficiency at variable distances from the point of primer hybridization, we designed qPCR assays at varying distances from single RSE primers at multiple genomic regions: CLEC16A, EGFR, BRCA2, KCNE1, NOS3, KCNJ2 and CETP (Fig. 2b)

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Summary

Introduction

The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. The DNA enrichment methodology described 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. As a result, existing methods for genomic partitioning remain a severely limiting factor for comprehensively characterizing complex genomic loci because they cannot provide the larger size fragments that are required to successfully span confounding sequence elements, such as extended repeats, or resolve sections of unknown or unexpected sequence that have been inserted or rearranged within the targeted region [18, 19]

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