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.
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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
- HTS:
-
High-throughput sequencing
- K2P:
-
Kimura 2-parameter
- kb:
-
Kilobase
- LR-LCH:
-
Long-range library capture hybridization
- LR-PCR:
-
Long-range polymerase chain reaction
- mtDNA:
-
Mitochondrial DNA
- Mitogenome:
-
Mitochondrial genome
- ND1:
-
NADH dehydrogenase subunit 1
- ND2:
-
NADH dehydrogenase subunit 2
- ND3:
-
NADH dehydrogenase subunit 3
- ND4:
-
NADH dehydrogenase subunit 4
- ND5:
-
NADH dehydrogenase subunit 5
- ND6:
-
NADH dehydrogenase subunit 6
- PGM:
-
Ion Torrent Personal Genome Machine
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Acknowledgments
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 (http://www.ncbi.nlm.nih.gov/sra) under accession number SRP090718.
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Chen, X. et al. (2018). Capture Hybridization of Long-Range DNA Fragments for High-Throughput Sequencing. In: Huang, T. (eds) Computational Systems Biology. Methods in Molecular Biology, vol 1754. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7717-8_3
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DOI: https://doi.org/10.1007/978-1-4939-7717-8_3
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