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Serial sequencing of isolength RAD tags for cost-efficient genome-wide profiling of genetic and epigenetic variations

Nature Protocols volume 11, pages 2189–2200 (2016)Cite this article

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Abstract

Isolength restriction site–associated DNA (isoRAD) sequencing is a very simple but powerful approach that was originally developed for genome-wide genotyping at minimal labor and cost, and it has recently extended its applicability to allow quantification of DNA methylation levels. The isoRAD method is distinct from other genotyping-by-sequencing (GBS) methods because of its use of special restriction enzymes to produce isolength tags (32–36 bp), and sequencing of these uniform tags can bring many benefits. However, the relatively short tags produced by the original protocol are mostly suited to single-end (SE) sequencing (36–50 bp), and therefore they cannot efficiently match the gradually increased sequencing capacity of next-generation sequencing (NGS) platforms. To address this issue, we describe an advanced protocol that allows the preparation of five concatenated isoRAD tags for Illumina paired-end (PE) sequencing (100–150 bp). The configuration of the five concatenated tags is highly flexible, and can be defined by users to work with a desired combination of samples and/or restriction enzymes to suit specific research purposes. In comparison with the original protocol, the advanced protocol has an additional digestion and ligation step, and library preparation can be completed in ∼8 h.

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Figure 1: Schematic overview of the Multi-isoRAD and isoRAD procedures.
Figure 2: Representative gel images from the library preparation procedure.
Figure 3: Performance demonstration of the Multi-isoRAD protocol.

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Acknowledgements

The isoRAD methodology and the advanced version described here were developed under grants from the National Natural Science Foundation of China (grant 31322055), the National High Technology Research and Development Program of China (863 Program; grant 2012AA10A405), the Natural Science Foundation for Distinguished Young Scholars of Shandong Province of China (grant JQ201308), and the AoShan Talents Program, supported by Qingdao National Laboratory for Marine Science and Technology (no. 2015ASTP-ES02), to S.W. and from the National Natural Science Foundation of China (grant 31630081) to Z.B.

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Author notes

  1. Shi Wang and Pingping Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China

    Shi Wang, Pingping Liu, Jia Lv, Yangping Li, Taoran Cheng, Lingling Zhang, Yu Xia, Hongzhen Sun, Xiaoli Hu & Zhenmin Bao

  2. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China

    Shi Wang

  3. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China

    Lingling Zhang, Xiaoli Hu & Zhenmin Bao

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  1. Shi Wang
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Contributions

S.W. and Z.B. conceived and designed the protocol; S.W., P.L., J.L., T.C., Y.X. and H.S. participated in testing and optimizing the protocol; Y.L. developed bioinformatic scripts; and S.W., P.L., L.Z. and X.H. participated in data analysis; S.W., P.L., L.Z., X.H. and Z.B. wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Shi Wang or Zhenmin Bao.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Tables

Supplementary Tables 1–5, References (PDF 251 kb)

Supplementary Data

Bioinformatic scripts for preprocessing of Multi-isoRAD sequencing data. (ZIP 148 kb)

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Wang, S., Liu, P., Lv, J. et al. Serial sequencing of isolength RAD tags for cost-efficient genome-wide profiling of genetic and epigenetic variations. Nat Protoc 11, 2189–2200 (2016). https://doi.org/10.1038/nprot.2016.133

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