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  • Review Article
  • Published:

Using next-generation sequencing to isolate mutant genes from forward genetic screens

Key Points

  • Next-generation sequencing enables simultaneous mapping and identification of a causal mutation though sequencing bulk populations of mutant recombinant organisms in an approach known as mapping-by-sequencing.

  • The initial mapping interval can be found in several ways. The ratio of homozygous and heterozygous markers, as well as the difference in the allele counts between pools of mutant and wild-type organisms, allows the analysis of linkage without prior knowledge of any genetic marker. Allele frequency analyses are more accurate but require a genome-wide list of genetic markers.

  • The analysis of large genomes can be simplified using various methods. High-throughput RNA sequencing (RNA-seq), targeted enrichment sequencing or restriction-site-associated DNA sequencing (RAD-seq) are powerful alternatives to whole-genome sequencing that can also allow the mapping of causal regions.

  • Mutation identification by direct sequencing of mutant genomes is challenged by the large amounts of background mutations in a mutant genome. Additional information, such as prior mapping intervals, can be used narrow down the list of possible candidate mutations.

  • Mutation identification can be as simple as comparing the genomes of independently generated allelic mutant lines and searching for genes that are affected in all genomes.

  • Mapping-by-sequencing methods can be used to decipher the genetic architecture of complex traits.

Abstract

The long-lasting success of forward genetic screens relies on the simple molecular basis of the characterized phenotypes, which are typically caused by mutations in single genes. Mapping the location of causal mutations using genetic crosses has traditionally been a complex, multistep procedure, but next-generation sequencing now allows the rapid identification of causal mutations at single-nucleotide resolution even in complex genetic backgrounds. Recent advances of this mapping-by-sequencing approach include methods that are independent of reference genome sequences, genetic crosses and any kind of linkage information, which make forward genetics amenable for species that have not been considered for forward genetic screens so far.

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Figure 1: Mapping-by-sequencing reveals the genetic basis of an inner ear mutant of zebrafish.
Figure 2: Analysis strategies for mapping-by-sequencing.
Figure 3: Pros and cons of different sequence data types for mapping-by-sequencing.
Figure 4: Mutation identification without linkage information, reference sequences and recombination.

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References

  1. Patton, E. E. & Zon, L. I. The art and design of genetic screens: zebrafish. Nature Rev. Genet. 2, 956–966 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Forsburg, S. L. The art and design of genetic screens: yeast. Nature Rev. Genet. 2, 659–668 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Casselton, L. & Zolan, M. The art and design of genetic screens: filamentous fungi. Nature Rev. Genet. 3, 683–697 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Jorgensen, E. M. & Mango, S. E. The art and design of genetic screens: Caenorhabditis elegans. Nature Rev. Genet. 3, 356–369 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. St Johnston, D. The art and design of genetic screens: Drosophila melanogaster. Nature Rev. Genet. 3, 176–188 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Page, D. R. & Grossniklaus, U. The art and design of genetic screens: Arabidopsis thaliana. Nature Rev. Genet. 3, 124–136 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Shuman, H. A. & Silhavy, T. J. The art and design of genetic screens: Escherichia coli. Nature Rev. Genet. 4, 419–431 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Kile, B. T. & Hilton, D. J. The art and design of genetic screens: mouse. Nature Rev. Genet. 6, 557–567 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Candela, H. & Hake, S. The art and design of genetic screens: maize. Nature Rev. Genet. 9, 192–203 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Michelmore, R. W., Paran, I. & Kesseli, R. V. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl Acad. Sci. USA 88, 9828–9832 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Darvasi, A. & Soller, M. Selective DNA pooling for determination of linkage between a molecular marker and a quantitative trait locus. Genetics 138, 1365–1373 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Winzeler, E. A. et al. Direct allelic variation scanning of the yeast genome. Science 281, 1194–1197 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Lindblad-Toh, K. et al. Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nature Genet. 24, 381–386 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H. & Plasterk, R. H. Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nature Genet. 28, 160–164 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Stickney, H. L. et al. Rapid mapping of zebrafish mutations with SNPs and oligonucleotide microarrays. Genome Res. 12, 1929–1934 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Borevitz, J. O. et al. Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res. 13, 513–523 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hazen, S. P. et al. Rapid array mapping of circadian clock and developmental mutations in Arabidopsis. Plant Physiol. 138, 990–997 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Brauer, M. J., Christianson, C. M., Pai, D. A. & Dunham, M. J. Mapping novel traits by array-assisted bulk segregant analysis in Saccharomyces cerevisiae. Genetics 173, 1813–1816 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lai, C. Q. et al. Speed-mapping quantitative trait loci using microarrays. Nature Methods 4, 839–841 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Liu, S. et al. High-throughput genetic mapping of mutants via quantitative single nucleotide polymorphism typing. Genetics 184, 19–26 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wolyn, D. J. et al. Light-response quantitative trait loci identified with composite interval and eXtreme array mapping in Arabidopsis thaliana. Genetics 167, 907–917 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schneeberger, K. et al. SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nature Methods 6, 550–551 (2009). This is the first report on mapping-by-sequencing. It introduces two different analysis principles based on MAFs and density of heterozygous markers.

    Article  CAS  PubMed  Google Scholar 

  23. Davey, J. W. et al. Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nature Rev. Genet. 12, 499–510 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Lister, R., Gregory, B. D. & Ecker, J. R. Next is now: new technologies for sequencing of genomes, transcriptomes, and beyond. Curr. Opin. Plant Biol. 12, 107–118 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cuperus, J. T. et al. Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proc. Natl Acad. Sci. USA 107, 466–471 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Austin, R. S. et al. Next-generation mapping of Arabidopsis genes. Plant J. 67, 715–725 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Manavella, P. A. et al. Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151, 859–870 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Uchida, N., Sakamoto, T., Kurata, T. & Tasaka, M. Identification of EMS-induced causal mutations in a non-reference Arabidopsis thaliana accession by whole genome sequencing. Plant Cell Physiol. 52, 716–722 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Wenger, J. W., Schwartz, K. & Sherlock, G. Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae. PLoS Genet. 6, e1000942 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Doitsidou, M., Poole, R. J., Sarin, S., Bigelow, H. & Hobert, O. C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy. PLoS ONE 5, e15435 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Pomraning, K. R., Smith, K. M. & Freitag, M. Bulk segregant analysis followed by high-throughput sequencing reveals the Neurospora cell cycle gene, ndc-1, to be allelic with the gene for ornithine decarboxylase, spe-1. Eukaryot. Cell 10, 724–733 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Leshchiner, I. et al. Mutation mapping and identification by whole genome sequencing. Genome Res. 22, 1541–1548 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Voz, M. L. et al. Fast homozygosity mapping and identification of a zebrafish ENU-induced mutation by whole-genome sequencing. PLoS ONE 7, e34671 (2012). This study introduces mapping-by-sequencing based on the principle of homozygosity mapping in zebrafish using low-coverage sequencing.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bowen, M. E., Henke, K., Siegfried, K. R., Warman, M. L. & Harris, M. P. Efficient mapping and cloning of mutations in zebrafish by low-coverage whole-genome sequencing. Genetics 190, 1017–1024 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Obholzer, N. et al. Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing. Development 139, 4280–4290 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lindner, H. et al. SNP-Ratio Mapping (SRM): identifying lethal alleles and mutations in complex genetic backgrounds by next-generation sequencing. Genetics 191, 1381–1386 (2012). This study introduces a crossing scheme for mapping-by-sequencing of lethal alleles. Within these mapping populations, the causal allele segregates at a slightly different allele frequency from those of background mutations. It also discusses the problems in identifying subtle allele frequency differences in mapping-by-sequencing data.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Galvão, V. C. et al. Synteny-based mapping-by-mequencing enabled by targeted enrichment. Plant J. 71, 517–526 (2012).

    PubMed  Google Scholar 

  38. Greenberg, M. V. et al. Identification of genes required for de novo DNA methylation in Arabidopsis. Epigenetics 6, 344–354 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Abe, A. et al. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotech. 30, 174–178 (2012). This paper introduces mapping-by-sequencing within isogenic mapping populations using mutagen-induced markers only. This showcase analysis was carried out in non-reference rice lines and showed the general applicability of this method.

    Article  CAS  Google Scholar 

  40. Hartwig, B., James, G. V., Konrad, K., Schneeberger, K. & Turck, F. Fast isogenic mapping-by-sequencing of ethyl methanesulfonate-induced mutant bulks. Plant Physiol. 160, 591–600 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Birkeland, S. R. et al. Discovery of mutations in Saccharomyces cerevisiae by pooled linkage analysis and whole-genome sequencing. Genetics 186, 1127–1137 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nowrousian, M., Teichert, I., Masloff, S. & Kück, U. Whole-genome sequencing of Sordaria macrospora mutants identifies developmental genes. G3 (Bethesda) 2, 261–270 (2012).

    Article  CAS  Google Scholar 

  43. Zhu, Y. et al. Gene discovery using mutagen-induced polymorphisms and deep sequencing: application to plant disease resistance. Genetics 192, 139–146 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Nordström, K. J. et al. Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers. Nature Biotech. 31, 325–330 (2013). This paper introduces mutation identification in the absence of reference sequences, genetic maps and segregating populations by directly sequencing two alleles of the same mutant.

    Article  CAS  Google Scholar 

  45. Allen, R. S., Nakasugi, K., Doran, R. L., Millar, A. A. & Waterhouse, P. M. Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines. Front. Plant Sci. 4, 362 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Zuryn, S., Le Gras, S., Jamet, K. & Jarriault, S. A. Strategy for direct mapping and identification of mutations by whole genome sequencing. Genetics 186, 427–430 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Velikkakam James, G. et al. User guide for mapping-by-sequencing in Arabidopsis. Genome Biol. 14, R61 (2013). This paper introduces the best practice of mapping-by-sequencing in plants assessed through a simulation study that highlights the most influential experimental parameters.

    Article  CAS  Google Scholar 

  48. Fekih, R. et al. MutMap+: genetic mapping and mutant identification without crossing in rice. PLoS ONE 8, e68529 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lander, E. S. & Botstein, D. Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children. Science 236, 1567–1570 (1987).

    Article  CAS  PubMed  Google Scholar 

  50. Hildebrandt, F. et al. A systematic approach to mapping recessive disease genes in individuals from outbred populations. PLoS Genet. 5, e1000353 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Singh, R. et al. The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK. Nature 500, 340–344 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. del Viso, F., Bhattacharya, D., Kong, Y., Gilchrist, M. J. & Khokha, M. K. Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing. BMC Genomics 13, 649 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Smith, D. R. et al. Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res. 18, 1638–1642 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Srivatsan, A. et al. High-precision, whole-genome sequencing of laboratory strains facilitates genetic studies. PLoS Genet. 4, e1000139 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Irvine, D. V. et al. Mapping epigenetic mutations in fission yeast using whole-genome next-generation sequencing. Genome Res. 19, 1077–1083 (2009). This paper introduces direct sequencing of genomes of mutant recombinants that were generated by backcrossing to wild-type isolates in fission yeast. Filtering for background mutations efficiently removes natural variation.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ashelford, K. et al. Full genome re-sequencing reveals a novel circadian clock mutation in Arabidopsis. Genome Biol. 12, R28 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Sarin, S., Prabhu, S., O'Meara, M. M., Pe'er, I. & Hobert, O. Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nature Methods 5, 865–867 (2008). This study is one of the very first NGS experiments that show the potential of WGS for mutation identification while analysing complete mutant genomes combined with mapping information.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Blumenstiel, J. P. et al. Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing. Genetics 182, 25–32 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Sobreira, N. L. et al. Whole-genome sequencing of a single proband together with linkage analysis identifies a Mendelian disease gene. PLoS Genet. 6, e1000991 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. McCluskey, K. et al. Rediscovery by whole genome sequencing: classical mutations and genome polymorphisms in Neurospora crassa. G3 (Bethesda) 1, 303–316 (2011).

    Article  CAS  Google Scholar 

  61. Arnold, C. N. et al. Rapid identification of a disease allele in mouse through whole genome sequencing and bulk segregation analysis. Genetics 187, 633–641 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Dutcher, S. K. et al. Whole-genome sequencing to identify mutants and polymorphisms in Chlamydomonas reinhardtii. G3 (Bethesda) 2, 15–22 (2012).

    Article  CAS  Google Scholar 

  63. Krothapalli, K. et al. Forward genetics by genome sequencing reveals that rapid cyanide release deters insect herbivory of Sorghum bicolor. Genetics 195, 309–318 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Lin, H., Miller, M. L., Granas, D. M. & Dutcher, S. K. Whole genome sequencing identifies a deletion in protein phosphatase 2A that affects its stability and localization in Chlamydomonas reinhardtii. PLoS Genet. 9, e1003841 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Schneeberger, K. & Weigel, D. Fast-forward genetics enabled by new sequencing technologies. Trends Plant Sci. 16, 282–288 (2011).

    Article  CAS  PubMed  Google Scholar 

  66. Gerhold, A. R., Richter, D. J., Yu, A. S. & Hariharan, I. K. Identification and characterization of genes required for compensatory growth in Drosophila. Genetics 189, 1309–1326 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Belfield, E. J. et al. Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana. Genome Res. 22, 1306–1315 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Hill, J. T. et al. MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq. Genome Res. 23, 687–697 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Minevich, G., Park, D. S., Blankenberg, D., Poole, R. J. & Hobert, O. CloudMap: a cloud-based pipeline for analysis of mutant genome sequences. Genetics 192, 1249–1269 (2012). This paper describes CloudMap, which is a comprehensive analysis tool for mapping-by-sequencing by implementing different analysis principles and background variation filtering steps.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Edwards, M. D. & Gifford, D. K. High-resolution genetic mapping with pooled sequencing. BMC Bioinformatics 13 (Suppl. 6), S8 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Magwene, P. M., Willis, J. H. & Kelly, J. K. The statistics of bulk segregant analysis using next generation sequencing. PLoS Comput. Biol. 7, e1002255 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Henke, K., Bowen, M. E. & Harris, M. P. Perspectives for identification of mutations in the zebrafish: making use of next-generation sequencing technologies for forward genetic approaches. Methods 62, 185–196 (2013).

    Article  CAS  PubMed  Google Scholar 

  73. Zuryn, S. & Jarriault, S. Deep sequencing strategies for mapping and identifying mutations from genetic screens. Worm 2, e25081 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  74. Wijnker, E. et al. The genomic landscape of meiotic crossovers and gene conversions in Arabidopsis thaliana. Elife 2, e01426 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. Long, Q. et al. Massive genomic variation and strong selection in Arabidopsis thaliana lines from Sweden. Nature Genet 45, 884–890 (2013).

    Article  CAS  PubMed  Google Scholar 

  76. Fairfield, H. et al. Mutation discovery in mice by whole exome sequencing. Genome Biol. 12, R86 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Andrews, T. D. et al. Massively parallel sequencing of the mouse exome to accurately identify rare, induced mutations: an immediate source for thousands of new mouse models. Open Biol. 2, 120061 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Gupta, T. et al. Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte. PLoS Genet. 6, e1001073 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Wang, H. et al. Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing. Genome Res. 20, 981–988 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Bontems, F. et al. Efficient mutation identification in zebrafish by microarray capturing and next generation sequencing. Biochem. Biophys. Res. Commun. 405, 373–376 (2011).

    Article  CAS  PubMed  Google Scholar 

  81. O'Rourke, S. M. et al. Rapid mapping and identification of mutations in Caenorhabditis elegans by RAD mapping and genomic interval pull-down sequencing. Genetics 189, 767–778 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Sun, M. et al. Multiplex chromosomal exome sequencing accelerates identification of ENU-induced mutations in the mouse. G3 (Bethesda) 2, 143–150 (2012).

    Article  CAS  Google Scholar 

  83. Mascher, M. et al. Mapping-by-sequencing accelerates forward genetics in barley. Genome Biol. 15, R78 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  84. Pankin, A. et al. Mapping-by-sequencing identifies HvPHYTOCHROME C as a candidate gene for the early maturity 5 locus modulating the circadian clock and photoperiodic flowering in barley. Genetics http://dx.doi.org/10.1534/genetics.114.165613 (2014).

  85. Ryan, S. et al. Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish. Development 140, 4445–4451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Mokry, M. et al. Identification of factors required for meristem function in Arabidopsis using a novel next generation sequencing fast forward genetics approach. BMC Genomics 12, 256 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  87. Lewis, Z. A. et al. High-density detection of restriction-site-associated DNA markers for rapid mapping of mutated loci in Neurospora. Genetics 177, 1163–1171 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Miller, M. R., Dunham, J. P., Amores, A., Cresko, W. A. & Johnson, E. A. Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers. Genome Res. 17, 240–248 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Baird, N. A. et al. Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS ONE 3, e3376 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  90. Miller, A. C., Obholzer, N. D., Shah, A. N., Megason, S. G. & Moens, C. B. RNA-seq based mapping and candidate identification of mutations from forward genetic screens. Genome Res. 23, 679–686 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Liu, S., Yeh, C. T., Tang, H. M., Nettleton, D. & Schnable, P. S. Gene mapping via bulked segregant RNA-seq (BSR-seq). PLoS ONE 7, e36406 (2012). This study introduces mapping-by-sequencing using transcriptomic data. Besides showing the advantages of RNA-based mapping in complex genomes such as the one of maize, it also describes the difficulties in such an analysis.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Wurtzel, O., Dori-Bachash, M., Pietrokovski, S., Jurkevitch, E. & Sorek, R. Mutation detection with next-generation resequencing through a mediator genome. PLoS ONE 5, e15628 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Livaja, M. et al. BSTA: a targeted approach combines bulked segregant analysis with next-generation sequencing and de novo transcriptome assembly for SNP discovery in sunflower. BMC Genomics 14, 628 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Ratan, A., Zhang, Y., Hayes, V. M., Schuster, S. C. & Miller, W. Calling SNPs without a reference sequence. BMC Bioinformatics 11, 130 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  95. Peterlongo, P., Schnel, N., Pisanti, N., Sagot, M. F. & Lacroix, V. in String Processing and Information Retrieval 147–158 (Springer, 2010).

    Book  Google Scholar 

  96. Iqbal, Z., Caccamo, M., Turner, I., Flicek, P. & McVean, G. De novo assembly and genotyping of variants using colored de Bruijn graphs. Nature Genet. 44, 226–232 (2012).

    Article  CAS  PubMed  Google Scholar 

  97. Huang, X. et al. High-throughput genotyping by whole-genome resequencing. Genome Res. 19, 1068–1076 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Sturtevant, A. H. The linear arrangement of six sex-linked factors in Drosophila, as shown by their mode of association. J. Exp. Zool. 14, 43–59 (1913).

    Article  Google Scholar 

  99. Harper, M. A. et al. Phenotype sequencing: identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants. PLoS ONE 6, e16517 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Jiménez-Gómez, J. M. Next generation quantitative genetics in plants. Front. Plant Sci. 2, 77 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  101. Lebowitz, R. J., Soller, M. & Beckmann, J. S. Trait-based analyses for the detection of linkage between marker loci and quantitative trait loci in crosses between inbred lines. Theor. Appl. Genet. 73, 556–562 (1987).

    Article  CAS  PubMed  Google Scholar 

  102. Takagi, H. et al. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J. 74, 174–183 (2013).

    Article  CAS  PubMed  Google Scholar 

  103. Swinnen, S. et al. Identification of novel causative genes determining the complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis. Genome Res 22, 975–984 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Trick, M. et al. Combining SNP discovery from next-generation sequencing data with bulked segregant analysis (BSA) to fine-map genes in polyploid wheat. BMC Plant Biol. 12, 14 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Carter, R., Hunt, P. & Cheesman, S. Linkage Group Selection — a fast approach to the genetic analysis of malaria parasites. Int. J. Parasitol. 37, 285–293 (2007).

    Article  CAS  PubMed  Google Scholar 

  106. Parts, L. et al. Revealing the genetic structure of a trait by sequencing a population under selection. Genome Res. 21, 1131–1138 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Van Leeuwen, T. et al. Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods. Proc. Natl Acad. Sci. USA 109, 4407–4412 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Kinga Modrzynska, K. et al. Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria. BMC Genomics 13, 106 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  109. Ehrenreich, I. M. et al. Dissection of genetically complex traits with extremely large pools of yeast segregants. Nature 464, 1039–1042 (2010). This study highlights the power of BSA for deciphering the genetic basis of complex traits by sequencing large populations from both ends of a phenotypic distribution.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Yang, Z. et al. Mapping of quantitative trait loci underlying cold tolerance in rice seedlings via high-throughput sequencing of pooled extremes. PLoS ONE 8, e68433 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Nolte, V., Pandey, R. V., Kofler, R. & Schlötterer, C. Genome-wide patterns of natural variation reveal strong selective sweeps and ongoing genomic conflict in Drosophila mauritiana. Genome Res. 23, 99–110 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Turner, T. L., Bourne, E. C., Von Wettberg, E. J., Hu, T. T. & Nuzhdin, S. V. Population resequencing reveals local adaptation of Arabidopsis lyrata to serpentine soils. Nature Genet 42, 260–263 (2010).

    Article  CAS  PubMed  Google Scholar 

  113. Takagi, H. et al. MutMap-Gap: whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii. New Phytol. 200, 276–283 (2013).

    Article  CAS  PubMed  Google Scholar 

  114. Tobler, R. et al. Massive habitat-specific genomic response in D. melanogaster populations during experimental evolution in hot and cold environments. Mol Biol Evol. 31, 364–375 (2014).

    Article  CAS  PubMed  Google Scholar 

  115. Keightley, P. D. & Bulfield, G. Detection of quantitative trait loci from frequency changes of marker alleles under selection. Genet. Res. 62, 195–203 (1993).

    Article  CAS  PubMed  Google Scholar 

  116. Burke, M. K. et al. Genome-wide analysis of a long-term evolution experiment with Drosophila. Nature 467, 587–590 (2010).

    Article  CAS  PubMed  Google Scholar 

  117. Turner, T. L., Stewart, A. D., Fields, A. T., Rice, W. R. & Tarone, A. M. Population-based resequencing of experimentally evolved populations reveals the genetic basis of body size variation in Drosophila melanogaster. PLoS Genet. 7, e1001336 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Sham, P., Bader, J. S., Craig, I., O'Donovan, M. & Owen, M. DNA pooling: a tool for large-scale association studies. Nature Rev. Genet. 3, 862–871 (2002).

    Article  CAS  PubMed  Google Scholar 

  119. Goecks, J., Nekrutenko, A., Taylor, J. & Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  120. Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The author thanks J. M. Jiménez-Gómez, E.-M. Willing and H. Sun for comments on the manuscript, and N. Obholzer for comments on figure 1. The development of methods for mapping-by-sequencing in K.S.'s group has been generously supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG SPP-1530).

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Glossary

Forward genetic screens

Genetic screens in which mutants are isolated on the basis of their phenotypes. The mutations responsible are identified by positional cloning or by a candidate gene approach.

Genetic markers

Genetic differences that are used to distinguish between different alleles of the respective DNA loci.

Next-generation sequencing

(NGS). In the context of this Review, sequencing methods that have emerged since 2005 and that produce millions of typically short sequence reads (50–400 bases) from amplified DNA clones.

Dominance

A genetic interaction between the two alleles at a locus, such that the phenotype of heterozygotes deviates from the average of the two homozygotes.

Penetrance

The proportion of individuals with a specific genotype who manifest the genotype at the phenotypic level. If the penetrance of an allele is 100%, then all individuals carrying that allele will express the associated phenotype, and the genotype is said to be completely penetrant.

Fixed

Pertaining to the point at which an allele (such as a mutation) has completely displaced other alleles; that is, the allele is present in a homozygous state in every individual in the population.

Non-synonymous nucleotide substitution

A change in nucleotide sequence that alters the encoded amino acid.

Second-site mutations

Mutations introduced in organisms that are already genetically compromised for a given pathway or process in order to isolate mutations that either suppress or enhance the effect of the first mutation.

RAD sequencing

(RAD-seq). A DNA sequencing method in which DNA is cut with restriction enzymes prior to sequencing, which exclusively targets the DNA associated with restriction sites.

Syntenic

Pertaining to the presence of collinear homologous DNA sequences in related chromosomal regions.

Quantitative trait loci

(QTLs). Genetic loci that control quantitative traits. They are identified on the basis of statistical association between genetic markers and phenotypes that can be measured.

Structural sequence variation

A genomic alteration that changes the number of copies or the arrangement of regions of the genome.

Epistatic interactions

Non-additive interactions between two or more mutations at different loci, such that their combined effect on a phenotype deviates from the sum of their individual effects.

Saturated screens

Genetic screens that have reached the point at which no new gene mutations can be found.

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Schneeberger, K. Using next-generation sequencing to isolate mutant genes from forward genetic screens. Nat Rev Genet 15, 662–676 (2014). https://doi.org/10.1038/nrg3745

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