Abstract
Random mutagenesis followed by screening for phenotypes of interest is a widely used strategy for genetic dissection of biological pathways; however, identifying the causal gene traditionally required time-consuming mapping approaches based on iterative linkage analysis. Mapping-by-sequencing accelerates this process, efficiently linking the phenotype of a mutant to a narrow candidate genomic region, using next-generation sequencing (NGS) data from a mapping population segregating for the mutant phenotype. To enable researchers at any bioinformatics skill level to conduct mapping-by-sequencing, we developed the Easymap mapping software. In this protocol we break down the steps involved in mapping-by-sequencing. First, we describe different ways of obtaining a mapping population and the steps used to generate NGS data. Next, we show how to analyze the NGS data using Easymap and how to obtain a list of candidate mutations, along with comprehensive information for assessing the potential causality of each candidate. Thus, this protocol enables the user to conduct mapping-by-sequencing using Easymap, facilitating the identification of causal loci for a mutant phenotype of interest.
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Abbreviations
- NGS:
-
next-generation sequencing
- EMS:
-
ethyl methanesulfonate
- SNP:
-
single-nucleotide polymorphism
- AF:
-
allele frequency
- RD:
-
read depth
- Gbp:
-
giga base pairs
References
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Acknowledgments
This work was supported by grants from the Ministerio de Ciencia e Innovación of Spain (PGC2018-093445-B-I00 [MCI/AEI/FEDER, UE]) and the Generalitat Valenciana (PROMETEO/2019/117) to JLM. SDL held a predoctoral fellowship (ACIF/2018/005) from the Generalitat Valenciana.
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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Lup, S.D., Wilson-Sánchez, D., Micol, J.L. (2022). Mapping-by-Sequencing of Point and Insertional Mutations with Easymap. In: Lambing, C. (eds) Plant Gametogenesis. Methods in Molecular Biology, vol 2484. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2253-7_23
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DOI: https://doi.org/10.1007/978-1-0716-2253-7_23
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2252-0
Online ISBN: 978-1-0716-2253-7
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