title: Serial sequencing of isolength RAD tags for cost-efficient genome-wide profiling of genetic and epigenetic variations.
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.
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.|Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
authors.forename: Shi
authors.identifier: http://orcid.org/0000-0002-9571-9864
authors.initials: S
authors.lastname: Wang
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Liu
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Lv
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
authors.forename: Yangping
authors.initials: Y
authors.lastname: Li
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
authors.forename: Taoran
authors.initials: T
authors.lastname: Cheng
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.|Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
authors.forename: Hongzhen
authors.initials: H
authors.lastname: Sun
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.|Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Hu
authors.affiliation: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.|Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
authors.forename: Zhenmin
authors.initials: Z
authors.lastname: Bao
mesh_terms: D003362:Cost-Benefit Analysis; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics*; D023281:Genomics; D017422:Sequence Analysis, DNA / Q000191:economics* / Q000379:methods*
chemical_list: D004247:DNA