Capture Hybridization of Long-Range DNA Fragments for High-Throughput Sequencing

  • Xing Chen
  • Gang Ni
  • Kai He
  • Zhao-Li Ding
  • Gui-Mei Li
  • Adeniyi C. Adeola
  • Robert W. Murphy
  • Wen-Zhi Wang
  • Ya-Ping Zhang
Part of the Methods in Molecular Biology book series (MIMB, volume 1754)


Capture hybridization coupled with high-throughput sequencing (HTS) has become one of the most popular approaches to address some scientific problems not only for fundamental evolution but also for ecology and human disease in recent years. However, the technical problem of limited probe capture ability affects its widespread application. Here, we propose to capture hybridize long-range DNA fragments for HTS (termed LR-LCH). We provide a case of three amphibian samples to examine LR-LCH with 2 kb libraries and comparison of standard capture hybridization with 480 bp libraries. Capture sensitivity increased from an average 13.57% of standard capture hybridization to an average 19.80% of LR-LCH; capture efficiency also increased from an average 72.56% of standard capture hybridization to an average 97.71% of LR-LCH. These indicate that longer fragments in the library generally contain both relatively variable regions and relatively conservative regions. The divergent parts of target DNA are enriched along with conservative parts of DNA sequence that effectively captured during hybridization. We present a protocol that allows users to overcome the low capture sensitivity problem for high divergent regions.

Key words

Mitochondrial genome Capture hybridization Long-range PCR NGS 

List of Abbreviations

12s rRNA

12s ribosomal RNA

16s rRNA

16s ribosomal RNA


ATP synthase subunit 6


ATP synthase subunit 8


Base pair


Barcoding gene from COX1


Cytochrome c oxidase subunit I


Cytochrome c oxidase subunit II


Cytochrome c oxidase subunit III


Cytochrome b


High-throughput sequencing


Kimura 2-parameter




Long-range library capture hybridization


Long-range polymerase chain reaction


Mitochondrial DNA


Mitochondrial genome


NADH dehydrogenase subunit 1


NADH dehydrogenase subunit 2


NADH dehydrogenase subunit 3


NADH dehydrogenase subunit 4


NADH dehydrogenase subunit 5


NADH dehydrogenase subunit 6


Ion Torrent Personal Genome Machine



We thank Jing Che’s research group for specimen collection and identification, and especially Hong-man Chen who examined species information and its CO1 sequence. We thank Dong Wang, Chun-Yan Yang, Kong-Wah Sing, and Elizabeth Georgian for reviewing the manuscript. This work was supported by the Ministry of Science and Technology of China (MOST no. 2012FY110800 to W.W.) and the National Natural Science Foundation of China (NSFC no. 31090251 to Y.Z.). Raw data from next-generation sequencing is available at SRA ( under accession number SRP090718.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Xing Chen
    • 1
  • Gang Ni
    • 1
    • 2
  • Kai He
    • 1
  • Zhao-Li Ding
    • 3
    • 4
  • Gui-Mei Li
    • 3
    • 4
  • Adeniyi C. Adeola
    • 1
    • 5
    • 6
  • Robert W. Murphy
    • 1
    • 7
  • Wen-Zhi Wang
    • 11
    • 12
    • 8
    • 9
  • Ya-Ping Zhang
    • 1
    • 2
    • 6
    • 10
  1. 1.State Key Laboratory of Genetic Resources and EvolutionKunmingChina
  2. 2.Yunnan Laboratory of Molecular Biology of Domestic AnimalsKunmingChina
  3. 3.Kunming Biological Diversity Regional Centre of Large Apparatus and EquipmentsKunmingChina
  4. 4.Public Technology Service CentreKunmingChina
  5. 5.China-Africa Centre for Research and Education & Yunnan Laboratory of Molecular Biology of Domestic AnimalsKunmingChina
  6. 6.Animal Branch of the Germplasm Bank of Wild Species, Kunming Institute of ZoologyChinese Academy of SciencesKunmingChina
  7. 7.Centre for Biodiversity and Conservation BiologyRoyal Ontario MuseumTorontoCanada
  8. 8.Wildlife Forensics Science ServicesKunmingChina
  9. 9.Guizhou Academy of Testing and AnalysisGuiyangChina
  10. 10.Laboratory for Conservation and Utilization of Bio-resource and Key Laboratory for Microbial Resources of the Ministry of EducationYunnan UniversityKunmingChina
  11. 11.State Key Laboratory of Genetic Resources and EvolutionKunming Institute of Zoology, Chinese Academy of SciencesKunmingChina
  12. 12.Animal Branch of the Germplasm Bank of Wild SpeciesKunming Institute of Zoology, Chinese Academy of SciencesKunmingChina

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