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High-throughput tetrad analysis

Nature Methods volume 10, pages 671–675 (2013)Cite this article

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Abstract

Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious.

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Figure 1: BEST method.
Figure 2: FACS plot of yeast spores transformed with a fluorescent reporter.
Figure 3: Sequence-based tetrad reconstruction and genotyping.

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Acknowledgements

We thank B. Cohen, A. Sherman and members of the Dudley lab for helpful discussions, and we thank B. Cohen (Washington University in St. Louis) for providing S. cerevisiae strain BC257. We thank D. Nickerson and B. Paeper for assistance with Illumina HiSeq 2000 sequencing and S. Bloom for assistance with Illumina GA IIx sequencing. This work was supported by a US National Institutes of Health–National Human Genome Research Institute (NIH/NHGRI) Genome Scholar/Faculty Transition Award (K22 HG002908) to A.M.D. and a strategic partnership between the Institute for Systems Biology and the University of Luxembourg. We note with sadness the passing of our friend and coauthor T.L. Gilbert.

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Author notes

  1. Teresa L Gilbert: Deceased.

  2. Catherine L Ludlow and Adrian C Scott: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Systems Biology, Seattle, Washington, USA

    Catherine L Ludlow, Adrian C Scott, Gareth A Cromie, Eric W Jeffery, Amy Sirr, Patrick May, Jake Lin, Michelle Hays & Aimée M Dudley

  2. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg

    Patrick May

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  1. Catherine L Ludlow
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Contributions

C.L.L., A.C.S., A.S., E.W.J., T.L.G. and A.M.D. contributed to experimental design; A.C.S. and C.L.L. contributed strains and plasmids; A.C.S., A.S., M.H., C.L.L., E.W.J. and T.L.G. contributed to experiments; G.A.C., A.C.S., P.M. and J.L. contributed to data analysis and scripts; and A.C.S., C.L.L., G.A.C. and A.M.D. wrote the manuscript.

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Correspondence to Aimée M Dudley.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 9–11 and Supplementary Note (PDF 351 kb)

Supplementary Table 1

Polymorphism table for ∑1278B (TXT 1146 kb)

Supplementary Table 2

Polymorphism table for YPS163 (TXT 2133 kb)

Supplementary Table 3

RAD-seq genotypes of FY4 × ∑1278b (pilot) progeny strains (no imputation) (TXT 331 kb)

Supplementary Table 4

RAD-seq genotypes of FY4 × ∑1278b (pilot) progeny strains in three- or four-spore tetrads (including imputed markers and inferred genotypes of missing spores) (TXT 290 kb)

Supplementary Table 5

RAD-seq genotypes of S288c × YPS163 (pilot) progeny strains (no imputation) (TXT 592 kb)

Supplementary Table 6

RAD-seq genotypes of S288c × YPS163 (pilot) progeny strains in three- or four-spore tetrads (including imputed markers and inferred genotypes of missing spores) (TXT 505 kb)

Supplementary Table 7

RAD-seq genotypes of FY4 × ∑1278b (full scale) progeny strains (no imputation) (TXT 4355 kb)

Supplementary Table 8

RAD-seq genotypes of FY4 × ∑1278b (full scale) progeny strains in three- or four-spore tetrads (including imputed markers and inferred genotypes of missing spores) (TXT 3325 kb)

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Ludlow, C., Scott, A., Cromie, G. et al. High-throughput tetrad analysis. Nat Methods 10, 671–675 (2013). https://doi.org/10.1038/nmeth.2479

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