Advertisement

Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing

  • Benjamin SchwessingerEmail author
  • John P. Rathjen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1659)

Abstract

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

Key words

Rust fungi Puccinia striiformis f. sp. tritici Puccinia graminis f. sp. tritici Long-read sequencing PacBio Nanopore 10× Genomics HMW DNA 

Notes

Acknowledgments

We thank Drs Megan McDonald, Melania Figueroa, Claire Anderson, Andrii Gryganskyi, and David Hayward for useful input while developing this protocol. We would like to thank several users for their positive feedback on successful application of this protocol to various fungal and oomycete species. This was enabled by early access to previous versions of this protocol on the platform “protocols.io,” which provides the opportunity to share and update protocols online. B.S. is supported by an Australian Research Council Discovery Early Career Research Award (DE150101897).

References

  1. 1.
    Singh RP, Hodson DP, Jin Y, Lagudah ES, Ayliffe MA, Bhavani S, Rouse MN, Pretorius ZA, Szabo LJ, Huerta-Espino J, Basnet BR, Lan C, Hovmøller MS (2015) Emergence and spread of new races of wheat stem rust fungus: continued threat to food security and prospects of genetic control. Phytopathol 105:872–884CrossRefGoogle Scholar
  2. 2.
    Schwessinger B (2017) Fundamental wheat stripe rust research in the 21st century. New Phytol 213:1625–1631CrossRefPubMedGoogle Scholar
  3. 3.
    Kolmer J (2013) Leaf rust of wheat: pathogen biology, variation and host resistance. Forests. 4:70–84CrossRefGoogle Scholar
  4. 4.
    Cantu D, Segovia V, MacLean D, Bayles R, Chen X, Kamoun S, Dubcovsky J, Saunders DG, Uauy C (2013) Genome analyses of the wheat yellow (stripe) rust pathogen Puccinia striiformis f. sp. tritici reveal polymorphic and haustorial expressed secreted proteins as candidate effectors. BMC Genomics 14:270CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kiran K, Rawal HC, Dubey H, Jaswal R, Devanna BN, Gupta DK, Bhardwaj SC, Prasad P, Pal D, Chhuneja P, Balasubramanian P, Kumar J, Swami M, Solanke AU, Gaikwad K, Singh NK, Sharma TR (2016) Draft genome of the wheat rust pathogen (Puccinia triticina) unravels genome-wide structural variations during evolution. Genome Biol Evol 8:2702–2721CrossRefPubMedGoogle Scholar
  6. 6.
    Cuomo CA, Bakkeren G, Khalil HB, Panwar V, Joly D, Linning R, Sakthikumar S, Song X, Adiconis X, Fan L, Goldberg JM, Levin JZ, Young S, Zeng Q, Anikster Y, Bruce M, Wang M, Yin C, McCallum B, Szabo LJ, Hulbert S, Chen X, Fellers JP (2017) Comparative analysis highlights variable genome content of wheat rusts and divergence of the mating loci. Genes Genomes Genet 7:361–376Google Scholar
  7. 7.
    Upadhyaya NM, Garnica DP, Karaoglu H, Sperschneider J, Nemri A, Xu B, Mago R, Cuomo CA, Rathjen JP, Park RF, Ellis JG, Dodds PN (2015) Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes. Front Plant Sci 5:759CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Duplessis S, Cuomo CA, Lin YC, Aerts A, Tisserant E, Veneault-Fourrey C, Joly DL, Hacquard S, Amselem J, Cantarel BL, Chiu R, Coutinho PM, Feau N, Field M, Frey P, Gelhaye E, Goldberg J, Grabherr MG, Kodira CD, Kohler A, Kues U, Lindquist EA, Lucas SM, Mago R, Mauceli E, Morin E, Murat C, Pangilinan JL, Park R, Pearson M, Quesneville H, Rouhier N, Sakthikumar S, Salamov AA, Schmutz J, Selles B, Shapiro H, Tanguay P, Tuskan GA, Henrissat B, Van de Peer Y, Rouze P, Ellis JG, Dodds PN, Schein JE, Zhong S, Hamelin RC, Grigoriev IV, Szabo LJ, Martin F (2011) Obligate biotrophy features unraveled by the genomic analysis of rust fungi. Proc Natl Acad Sci USA 108(22):9166–9171CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Zheng W, Huang L, Huang J, Wang X, Chen X, Zhao J, Guo J, Zhuang H, Qiu C, Liu J, Liu H, Huang X, Pei G, Zhan G, Tang C, Cheng Y, Liu M, Zhang J, Zhao Z, Zhang S, Han Q, Han D, Zhang H, Zhao J, Gao X, Wang J, Ni P, Dong W, Yang L, Yang H, Xu J, Zhang G, Kang Z (2013) High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus. Nat Commun 4:2673PubMedPubMedCentralGoogle Scholar
  10. 10.
    Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B, Christians F, Cicero R, Clark S, Dalal R, deWinter A, Dixon J, Foquet M, Gaertner A, Hardenbol P, Heiner C, Hester K, Holden D, Kearns G, Kong X, Kuse R, Lacroix Y, Lin S, Lundquist P, Ma C, Marks P, Maxham M, Murphy D, Park I, Pham T, Phillips M, Roy J, Sebra R, Shen G, Sorenson J, Tomaney A, Travers K, Trulson M, Vieceli J, Wegener J, Wu D, Yang A, Zaccarin D, Zhao P, Zhong F, Korlach J, Turner S (2009) Real-time DNA sequencing from single polymerase molecules. Science 323:133–138CrossRefPubMedGoogle Scholar
  11. 11.
    Wang M, Beck CR, English AC, Meng Q, Buhay C, Han Y, Doddapaneni HV, Yu F, Boerwinkle E, Lupski JR, Muzny DM, Gibbs RA (2015) PacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations. BMC Genomics 16:214CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Jain M, Olsen HE, Paten B, Akeson M (2016) The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol 17:239CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zheng GXY, Lau BT, Schnall-Levin M, Jarosz M, Bell JM, Hindson CM, Kyriazopoulou-Panagiotopoulou S, Masquelier DA, Merrill L, Terry JM, Mudivarti PA, Wyatt PW, Bharadwaj R, Makarewicz AJ, Li Y, Belgrader P, Price AD, Lowe AJ, Marks P, Vurens GM, Hardenbol P, Montesclaros L, Luo M, Greenfield L, Wong A, Birch DE, Short SW, Bjornson KP, Patel P, Hopmans ES, Wood C, Kaur S, Lockwood GK, Stafford D, Delaney JP, Wu I, Ordonez HS, Grimes SM, Greer S, Lee JY, Belhocine K, Giorda KM, Heaton WH, McDermott GP, Bent ZW, Meschi F, Kondov NO, Wilson R, Bernate JA, Gauby S, Kindwall A, Bermejo C, Fehr AN, Chan A, Saxonov S, Ness KD, Hindson BJ, Ji HP (2016) 0 Haplotyping germline and cancer genomes with high-throughput linked-read sequencing. Nat Biotechnol 34:303–311CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Spies N, Weng Z, Bishara A, McDaniel J, Catoe D, Zook JM, Salit M, West RB, Batzoglou S, Sidow A (2016) Genome-wide reconstruction of complex structural variants using read clouds. bioRxiv:74518. doi: 10.1101/074518
  15. 15.
    Mostovoy Y, Levy-Sakin M, Lam J, Lam ET, Hastie AR, Marks P, Lee J, Chu C, Lin C, Džakula Z, Cao H, Schlebusch SA, Giorda K, Schnall-Levin M, Wall JD, Kwok PY (2016) A hybrid approach for de novo human genome sequence assembly and phasing. Nat Methods 13:587–590CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chin CS, Peluso P, Sedlazeck FJ, Nattestad M, Concepcion GT, Clum A, Dunn C, O’Malley R, Figueroa-Balderas R, Morales-Cruz A, Cramer GR, Delledonne M, Luo C, Ecker JR, Cantu D, Rank DR, Schatz MC (2016) Phased diploid genome assembly with single-molecule real-time sequencing. Nat Methods 13:1050–1054CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Fulton TM, Chunwongse J, Tanksley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Report 13:207–209CrossRefGoogle Scholar
  18. 18.
    High quality DNA from fungi for long read sequencing e.g. PacBio (2016) https://www.protocols.io/view/High-quality-DNA-from-fungi-for-long-read-sequencing-ewtbfen. Accessed on February 2, 2017Google Scholar
  19. 19.
    Extraction of high quality DNA for genome sequencing, 1000 fungal genomes project (2012). http://1000.fungalgenomes.org/home/protocols/high-quality-genomic-dna-extraction/. Accessed on February 2, 2017

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Research School of BiologyAustralian National UniversityCanberraAustralia

Personalised recommendations