Next‐Generation Sequencing for Identification of EMS‐Induced Mutations in Caenorhabditis elegans

Nicolas J. Lehrbach1, Fei Ji1, Ruslan Sadreyev2

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.29
DOI:  10.1002/cpmb.27
Online Posting Date:  January, 2017
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Forward genetic analysis using chemical mutagenesis in model organisms is a powerful tool for investigation of molecular mechanisms in biological systems. In the nematode, Caenorhabditis elegans, mutagenesis screens using ethyl methanesulfonate (EMS) have led to important insights into genetic control of animal development and physiology. A major bottleneck to this approach is identification of the causative mutation underlying a phenotype of interest. In the past, this has required time‐consuming genetic mapping experiments. More recently, next‐generation sequencing technologies have allowed development of new methods for rapid mapping and identification of EMS‐induced lesions. In this unit we describe a protocol to map and identify EMS‐induced mutations in C. elegans. © 2017 by John Wiley & Sons, Inc.

Keywords: C. elegans; EMS mutagenesis; next‐generation sequencing

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Table of Contents

  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1:

  • C. elegans parental strain
  • Mutant of interest
  • 5‐cm nematode growth medium (NGM) agar plates seeded with E. coli strain OP50 (see Stiernagle )
  • Distilled water
  • Ice
  • Puregene Cell and Tissue Kit (Qiagen) containing:
    • Cell lysis solution
    • Proteinase K
    • RNase A solution
    • Protein precipitation solution
    • DNA hydration solution
  • Isopropanol
  • 70% (v/v) Ethanol
  • Deionized water
  • TE buffer
  • NEBNext DNA library prep kit for Illumina (NEB) containing:
    • End repair reaction buffer
    • End prep enzyme mix
    • NEBNext High fidelity 2 × PCR master mix
    • dA‐tailing reaction buffer
    • Klenow fragment
    • Quick ligation reaction buffer (5×)
    • Quick T4 DNA ligase
    • USER enzyme
    • Universal PCR primer (25 μM)
    • Index PCR primer
  • NEBNext Multiplex oligos for Illumina (NEB)
  • Zymo DNA Clean and Concentrator Kit (Zymo Research)
  • AMPure XP Beads (Agencourt)
  • 2.5% agarose gel
  • Qubit dsDNA HS Assay kit (Life Technologies)
  • 15‐ml Falcon tubes
  • Benchtop centrifuge
  • 55°C water bath
  • 37°C incubator or water bath
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • NanoDrop microvolume spectrophotometer
  • Covaris S2 Sonicator
  • 6 × 16–mm microTUBE AFA Fiber Crimp‐Cap (Covaris)
  • Covaris microTUBE holder S 5001144
  • Heat block
  • Magnetic stand
  • 200 μl PCR tubes
  • Thermal cycler
  • Qbit Fluorometer (Life Technologies)
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Literature Cited

Literature Cited
  Afgan, E., Baker, D., van den Beek, M., Blankenberg, D., Bouvier, D., Čech, M., Chilton, J., Clements, D., Coraor, N., Eberhard, C., Grüning, B., Guerler, A., Hillman‐Jackson, J., Kuster, G., Rasche, E., Soranzo, N., Turaga, N., Taylor, J., Nekrutenko, A., and Goecks, J. 2016. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 44:W3‐W10 gkw343.
  Chu, J.S.‐C., Chua, S.‐Y., Wong, K., Davison, A., Johnsen, R., Baillie, D.L., and Rose, A.M. 2014. High‐throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans. BMC Genomics 15:361. doi: 10.1186/1471‐2164‐15‐361.
  Davis, M., Hammarlund, M., Harrach, T., Hullett, P., Olsen, S., and Jorgensen, E. 2005. Rapid single nucleotide polymorphism mapping in C. elegans. BMC Genomics 6:118. doi: 10.1186/1471‐2164‐6‐118.
  Desjardins, P. R. and Conklin, D. S. 2011. Microvolume Quantitation of Nucleic Acids. Curr. Protoc. Molec. Biol. 93:3J:A.3J.1‐A.3J.16.
  Doitsidou, M., Poole, R.J., Sarin, S., Bigelow, H., and Hobert, O. 2010. C. elegans mutant identification with a one‐step whole‐genome‐sequencing and SNP mapping strategy. PLoS One 5:e15435. doi: 10.1371/journal.pone.0015435.
  Fay, D. (2006). Genetic mapping and manipulation: Chapter 1‐Introduction and basics. WormBook. doi: 10.1895/wormbook.1.90.1.
  Frøkjaer‐Jensen, C., Davis, M.W., Sarov, M., Taylor, J., Flibotte, S., LaBella, M., Pozniakovsky, A., Moerman, D.G., and Jorgensen, E.M. 2014. Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon. Nat. Methods 11:529‐534. doi: 10.1038/nmeth.2889.
  Hu, P.J. 2014. Whole genome sequencing and the transformation of C. elegans forward genetics. Methods 68:437‐440. doi: 10.1016/j.ymeth.2014.05.008.
  Jaramillo‐Lambert, A., Fuchsman, A.S., Fabritius, A.S., Smith, H.E., and Golden, A. 2015. Rapid and Efficient Identification of Caenorhabditis elegans legacy mutations using Hawaiian SNP‐based mapping and whole‐genome sequencing. G3 (Bethesda) 5:1007‐1019. doi: full_text.
  Minevich, G., Park, D.S., Blankenberg, D., Poole, R.J., and Hobert, O. 2012. CloudMap: A cloud‐based pipeline for analysis of mutant genome sequences. Genetics 192:1249‐1269. doi: 10.1534/genetics.112.144204.
  Sarin, S., Prabhu, S., O'Meara, M.M., Pe'er, I., and Hobert, O. 2008. Caenorhabditis elegans mutant allele identification by whole‐genome sequencing. Nat. Methods 5:865‐867. doi: 10.1038/nmeth.1249.
  Schneeberger, K. 2014. Using next‐generation sequencing to isolate mutant genes from forward genetic screens. Nat. Rev. Genet. 15:662‐676. doi: 10.1038/nrg3745.
  Stiernagle, T. 2006. Maintenance of C. elegans. WormBook.
  Thompson, O., Edgley, M., Strasbourger, P., Flibotte, S., Ewing, B., Adair, R., Au, V., Chaudhry, I., Fernando, L., Hutter, H., Kieffer, A., Lau, J., Lee, N., Miller, A., Raymant, G., Shen, B., Shendure, J., Taylor, J., Turner, E.H., Hillier, L.W., Moerman, D.G., and Waterston, R.H. 2013. The million mutation project: A new approach to genetics in Caenorhabditis elegans. Genome Res. 23:1749‐1762. doi: 10.1101/gr.157651.113.
  Zuryn, S. and Jarriault, S. 2014. Deep sequencing strategies for mapping and identifying mutations from genetic screens. Worm 2:e25081. doi: 10.4161/worm.25081.
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