title: The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity.
abstract: 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.
  
  
  
  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 %.
  
  
  
  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.
authors.affiliation: Generation Biotech, Lawrenceville, NJ, 08648, USA. jdapprich@generationbiotech.com.
authors.forename: Johannes
authors.initials: J
authors.lastname: Dapprich
authors.affiliation: Generation Biotech, Lawrenceville, NJ, 08648, USA.|Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Ferriola
authors.affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
authors.forename: Kate
authors.initials: K
authors.lastname: Mackiewicz
authors.affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Clark
authors.affiliation: Nucleic Acids & Protein Core Facility, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Rappaport
authors.affiliation: The Center for Biomedical Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: D'Arcy
authors.affiliation: The Center for Biomedical Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
authors.forename: Ariella
authors.initials: A
authors.lastname: Sasson
authors.affiliation: The Center for Biomedical Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
authors.forename: Xiaowu
authors.initials: X
authors.lastname: Gai
authors.affiliation: Department of Genetics, University of Pennsylvania, Philadelphia, PA, 19104, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Schug
authors.affiliation: Department of Genetics, University of Pennsylvania, Philadelphia, PA, 19104, USA.
authors.forename: Klaus H
authors.initials: KH
authors.lastname: Kaestner
authors.affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA. monosd@email.chop.edu.|The Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. monosd@email.chop.edu.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Monos
mesh_terms: D055028:Comparative Genomic Hybridization / Q000379:methods; D017931:DNA Primers; D030541:Databases, Genetic; D014644:Genetic Variation; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008285:Major Histocompatibility Complex / Q000235:genetics; D009693:Nucleic Acid Hybridization; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers
keywords: DNA sequencing; DNA target capture; Genomic resequencing; MHC haplotype; Targeted enrichment