DOI QR코드

DOI QR Code

Mutation Analysis of Synthetic DNA Barcodes in a Fission Yeast Gene Deletion Library by Sanger Sequencing

  • Lee, Minho (Catholic Precision Medicine Research Center, College of Medicine, The Catholic University of Korea) ;
  • Choi, Shin-Jung (Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB)) ;
  • Han, Sangjo (Data Analytics CoE, SK Telecom) ;
  • Nam, Miyoung (Department of New Drug Development, Chungnam National University) ;
  • Kim, Dongsup (Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST)) ;
  • Kim, Dong-Uk (Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB)) ;
  • Hoe, Kwang-Lae (Department of New Drug Development, Chungnam National University)
  • Received : 2018.05.11
  • Accepted : 2018.05.16
  • Published : 2018.06.30

Abstract

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (~28%) and well above the predicted error of Sanger sequencing (~2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

Acknowledgement

Supported by : Chungnam National University

References

  1. Nurse P, Thuriaux P, Nasmyth K. Genetic control of the cell di- vision cycle in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet 1976;146:167-178. https://doi.org/10.1007/BF00268085
  2. Hedges SB. The origin and evolution of model organisms. Nat Rev Genet 2002;3:838-849. https://doi.org/10.1038/nrg929
  3. Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, Stewart A, et al. The genome sequence of Schizosaccharomyces pombe. Nature 2002;415:871-880. https://doi.org/10.1038/nature724
  4. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 1999;285:901-906. https://doi.org/10.1126/science.285.5429.901
  5. Kim DU, Hayles J, Kim D, Wood V, Park HO, Won M, et al. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 2010;28: 617-623. https://doi.org/10.1038/nbt.1628
  6. Hayles J, Wood V, Jeffery L, Hoe KL, Kim DU, Park HO, et al. A genome-wide resource of cell cycle and cell shape genes of fission yeast. Open Biol 2013;3:130053. https://doi.org/10.1098/rsob.130053
  7. Shoemaker DD, Lashkari DA, Morris D, Mittmann M, Davis RW. Quantitative phenotypic analysis of yeast deletion mu- tants using a highly parallel molecular bar-coding strategy. Nat Genet 1996;14:450-456. https://doi.org/10.1038/ng1296-450
  8. Han S, Lee M, Chang H, Nam M, Park HO, Kwak YS, et al. Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach. Biochem Biophys Res Commun 2013;436:613-618. https://doi.org/10.1016/j.bbrc.2013.05.138
  9. Eason RG, Pourmand N, Tongprasit W, Herman ZS, Anthony K, Jejelowo O, et al. Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains. Proc Natl Acad Sci U S A 2004;101:11046-11051. https://doi.org/10.1073/pnas.0403672101
  10. Smith AM, Heisler LE, Mellor J, Kaper F, Thompson MJ, Chee M, et al. Quantitative phenotyping via deep barcode sequencing. Genome Res 2009;19:1836-1842. https://doi.org/10.1101/gr.093955.109
  11. Han TX, Xu XY, Zhang MJ, Peng X, Du LL. Global fitness profiling of fission yeast deletion strains by barcode sequencing. Genome Biol 2010;11:R60. https://doi.org/10.1186/gb-2010-11-6-r60
  12. Forsburg SL, Rhind N. Basic methods for fission yeast. Yeast 2006;23:173-183. https://doi.org/10.1002/yea.1347
  13. Rice P, Longden I, Bleasby A. EMBOSS: the European molec- ular biology open software suite. Trends Genet 2000;16:276-277. https://doi.org/10.1016/S0168-9525(00)02024-2
  14. Needleman SB, Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 1970;48:443-453. https://doi.org/10.1016/0022-2836(70)90057-4
  15. Nam M, Lee SJ, Han S, Kim D, Lee M, Kang EJ, et al. Systematic targeted gene deletion using the gene-synthesis method in fission yeast. J Microbiol Methods 2014;106:72-77. https://doi.org/10.1016/j.mimet.2014.08.005
  16. Richterich P. Estimation of errors in “raw” DNA sequences: a validation study. Genome Res 1998;8:251-259. https://doi.org/10.1101/gr.8.3.251