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

Autor: Taoran Cheng, Yu Xia, Jia Lv, Yangping Li, Zhenmin Bao, Lingling Zhang, Shi Wang, Xiaoli Hu, Pingping Liu, Hongzhen Sun
Rok vydání: 2016
Předmět:
Zdroj: Nature Protocols. 11:2189-2200
ISSN: 1750-2799
1754-2189
DOI: 10.1038/nprot.2016.133
Popis: 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.
Databáze: OpenAIRE
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