Identification of Mutations in Zebrafish Using Next‐Generation Sequencing

Katrin Henke1, Margot E. Bowen1, Matthew P. Harris1

1 Department of Genetics, Harvard Medical School, and Department of Orthopedics, Boston Children's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.13
DOI:  10.1002/0471142727.mb0713s104
Online Posting Date:  October, 2013
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Whole‐genome sequencing (WGS) has been used in many invertebrate model organisms as an efficient tool for mapping and identification of mutations affecting particular morphological or physiological processes. However, the application of WGS in highly polymorphic, larger genomes of vertebrates has required new experimental and analytical approaches. As a consequence, a wealth of different analytical tools has been developed. As the generation and analysis of data stemming from WGS can be unwieldy and daunting to researchers not accustomed to many common bioinformatic analyses and Unix‐based computational tools, we focus on how to manage and analyze next‐generation sequencing datasets without an extensive computational infrastructure and in‐depth bioinformatic knowledge. Here we describe methods for the analysis of WGS for use in mapping and identification of mutations in the zebrafish. We stress key elements of the experimental design and the analytical approach that allow the use of this method across different sequencing platforms and in different model organisms with annotated genomes. Curr. Protoc. Mol. Biol. 104:7.13.1‐7.13.33. © 2013 by John Wiley & Sons, Inc.

Keywords: whole‐genome sequencing; WGS; mutation mapping; zebrafish; next‐generation sequencing

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Table of Contents

  • Introduction
  • Strategic Planning of Mapping Experiments
  • Basic Protocol 1: Preparation of the DNA Library for Next‐Generation Sequencing
  • Basic Protocol 2: Sequence Data Alignment and Variant Identification
  • Support Protocol 1: Software and Datasets Used for Data Analysis
  • Basic Protocol 3: Linkage Mapping Based on Homozygosity‐by‐Descent
  • Support Protocol 2: Verification of Linkage
  • Basic Protocol 4: Identification of Candidate Mutations
  • Support Protocol 3: Identifying Candidate Causative Mutations in Regions Covered by Only One Read
  • Basic Protocol 5: Identification of Small Insertions or Deletions within a Linked Interval as Candidate Mutations
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of the DNA Library for Next‐Generation Sequencing

  • F2 generation of a genetic cross, sorted by phenotype into mutants and siblings (Fig. A)
  • Reagents for DNA extraction: e.g., DNeasy Blood & Tissue Kit (Qiagen, cat. no. 69504)
  • Optional: Kit for library preparation for next‐generation sequencing, e.g., TruSeq DNA Sample Preparation kit (Illumina, cat. no. CES FC‐121‐2001)
  • Spectrophotometer, e.g., NanoDrop (see APPENDICES & )
  • Additional reagents and equipment for DNA extraction (unit 2.1), quantitation of nucleic acids (APPENDICES & ), and library preparation for Illumina sequencing (Son and Taylor, )
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Key Reference
  Bowen, et al., 2012. See above.
  The protocol described here is based on the technique developed by the authors of this paper. More detailed information about the limitations of the technique, for example minimal number of reads needed for mapping, as well as how many potential candidate mutations can be expected to be identified, can be found in this paper.
Internet Resource
  See for useful Internet links and resources. An extensive list of algorithms used in next‐generation sequence analysis software can be found at the URL above.
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