Chapter 3
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, and Ya-Ping Zhang
Abstract
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.
KeywordsMitochondrial genome, Capture hybridization, Long-range PCR, NGSList of Abbreviations
12s rRNA 12s ribosomal RNA
16s rRNA 16s ribosomal RNA
apt6 ATP synthase subunit 6
apt8 ATP synthase subunit 8
bp Base pair
CO1 Barcoding gene from COX1
COX1 Cytochrome c oxidase subunit I
COX2 Cytochrome c oxidase subunit II
COX3 Cytochrome c oxidase subunit III
cytb Cytochrome b
Tao Huang (ed.),Computational Systems Biology: Methods and Protocols, Methods in Molecular Biology, vol. 1754,
https://doi.org/10.1007/978-1-4939-7717-8_3,©Springer Science+Business Media, LLC, part of Springer Nature 2018
Authors Xing Chen, Gang Ni, Kai He, and Zhao-Li Ding contributed equally to this chapter.
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