Skip to main content

The first genome assembly of fungal pathogen Pyrenophora tritici-repentis race 1 isolate using Oxford Nanopore MinION sequencing

Abstract

Objectives

The assembly of fungal genomes using short-reads is challenged by long repetitive and low GC regions. However, long-read sequencing technologies, such as PacBio and Oxford Nanopore, are able to overcome many problematic regions, thereby providing an opportunity to improve fragmented genome assemblies derived from short reads only. Here, a necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) isolate 134 (Ptr134), which causes tan spot disease on wheat, was sequenced on a MinION using Oxford Nanopore Technologies (ONT), to improve on a previous Illumina short-read genome assembly and provide a more complete genome resource for pan-genomic analyses of Ptr.

Results

The genome of Ptr134 sequenced on a MinION using ONT was assembled into 28 contiguous sequences with a total length of 40.79 Mb and GC content of 50.81%. The long-read assembly provided 6.79 Mb of new sequence and 2846 extra annotated protein coding genes as compared to the previous short-read assembly. This improved genome sequence represents near complete chromosomes, an important resource for large scale and pan genomic comparative analyses.

Introduction

The necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) is the causal agent of tan (or yellow) spot a major disease of wheat (Triticum aestivum) [1]. A number of genomic sequencing projects have been undertaken for Ptr [2,3,4,5,6], the majority derived solely from Illumina sequence. Many of these short-read assemblies are incomplete as many genomic regions in Ptr contain long repetitive regions and identical gene copies that are not resolved by short reads [5]. We therefore undertook the currently more affordable Oxford Nanopore Technologies (ONT) long-read sequencing of an Australian Ptr isolate 134 (Ptr134) that was previously sequenced by short read (150 bp paired end) Illumina technology [3].

Main text

Methods

Isolate collection and sequencing

The pathogenic isolate Ptr134 was isolated from tan spot infected leaves collected from Queensland, Australia in 2001. Ptr134 was cultured in vitro from a single spore [7]. Ptr134 genomic DNA was extracted from 3-day old mycelia grown in vitro in Fries 3 liquid medium, using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). DNA was further treated with phenol/chloroform extraction, followed by precipitation with sodium acetate and ethanol, and finally resuspension in TE buffer [3]. The Ptr134 genomic DNA was sequenced using a MinION (MIN-101B) Oxford Nanopore StarterPack, R9 (FLO-MINSP6) flow cell, flow cell priming kit (XP-FLP001) and Rapid Sequencing Kit SQK-RAD004, following manufacturers (Oxford Nanopore Technologies, Oxford, UK) protocol. ONT sequencing after 24 h yielded 4,37,865 passed long reads with a total length of 2.6 Gb (65 × genome coverage), base called in real time using MinKNOW version 127.0.0.1 software on a MacBook Pro (version 10.13.6, 2.6 GHz Intel Core i7 processor and 16 GB 2400 MHz DDR4 memory) to a 1 TB Seagate Backup Plus Slim portable storage device (model SRC0VN2), at the Centre for Crop Disease and Management, Perth, Western Australia. ONT sequence data was based called in real time using the MinKNOW Fast basecalling model from Fast5 into FastQ file format. Raw reads were classed as passed by MinKNOW based on the average read quality score > 7. The Ptr134 genome was also previously sequenced via Illumina HiSeq stranded (150 bp paired end reads) by Novogene Co., Ltd (Hong Kong) to yield 3.2 Gb at 80× coverage [3]. The median and maximum read lengths obtained from the MinION were 4253 bp and 91,723 bp, respectively.

Genome assembly of Ptr134

The passed FastQ data was error-corrected and assembled using linux-amd64 Canu 1.8 software [8] guided by a genome size of 40 Mb and option for raw nanopore data. Illumina PE reads were quality trimmed for random hexamer primers on the 5′ read end using Trimmomatic v0.22 [9]. The high quality trimmed Illumina reads were aligned to the Canu genome assembly using BWA 0.7.14-r1138 [10] and filtered for concordant PE read alignments using samtools 0.1.19-96b5f2294a [11]. The genome assembly was then corrected with the high quality Illumina alignments using Pilon 1.23 [12] to generate a final polished Ptr134 sequence assembly with 2407 SNPs, 1,64,237 small insertions (totalling 208,176 bases) and 123 small deletions (totalling 151 bases) corrected. Post Canu and Pilon error corrections, the average weighted Phred score base qualities for Ptr134 ONT sequence and a previously PacBio RSII sequenced M4 isolate [3] were 36 and 37, respectively.

Ptr134 was then aligned to M4 [3] scaffolded chromosomes using NUCmer [13] v3.1 (-maxmatch -coords).

Gene prediction and functional annotation

Ptr134 Illumina RNA-seq data [3] was aligned to the Ptr134 nanopore assembled genome using TopHat v2.0.12 [14] (-N 2 -i 10 -I 5000 -p 16 –no-discord- ant –no-mixed –report-secondary-alignments –micro- exon-search –library-type fr-firststrand) for supporting ab initio gene predictions by CodingQuarry v1.2 [15] in pathogen mode (PM). Ab initio gene predictions were also made with GeneMark-ES v4.33 [16].

Pt-1C-BFP [2] and M4 reference proteins [3] were aligned to Ptr134 using Exonerate v2.2.0 [17] (–showvulgar no –showalignment no –minintron 10 –maxintron 3000) in mode protein2genome. The ab initio gene predictions and exonerate alignments were then combined using EvidenceModeller v1.1.1 [18] with a minimum intron length of 10 bp and weightings of CodingQuarry:1, GeneMark.hmm:1, protein exonerate:2.

Gene annotations were assigned by BLASTX [19, 20] v2.3.0 + searches across NCBI RefSeq and NR (taxon = Ascomycota) (February 2020) databases and RPSTBLASTN v2.7.1 + of COG, Pfam, Smart and CDD domain databases (February 2020). Final gene annotations were summarised by AutoFACT v3.4 [21]. BUSCO [22] v5.1.2 analysis was conducted on predicted protein sequences using the lineage for pleosporales_odb10.

The ONT Ptr134 annotated genome has been deposited with DDBJ/ENA/GenBank under the updated accession MVBF02000000.

Results and discussion

Genome assembly and annotation of Ptr134

The Ptr134 genome assembled into 28 contiguous sequences with of total length 40.79 Mb and GC content of 50.81% (Table 1). Ptr134 ONT (Version 2) contig length statistics showed marked improvements in comparison to the short-read assembly (Version 1) [3]. In comparison to the previous short read assembly, the long-read assembly provided 6.79 Mb of new sequence. A total of 13,918 protein coding genes were also predicted for the Ptr134 ONT assembly, 2,846 more than the previous short read assembly (Table 1). Although there was no improvement in the BUSCO scores for predicted protein coding genes the new predictions are possible pathogen specific genes found in the more complex regions which are harder to assemble with short reads. The ONT Ptr134 annotated genome has been deposited with DDBJ/ENA/GenBank under the updated accession MVBF02000000 (Table 1).

Table 1 Pyrenophora tritici-repentis race 1 isolate Ptr134 Oxford Nanopore genome information and assembly statistics compared to race 1 isolate M4 and version 1 short read assembly of Ptr134

The improved Ptr134 genome assembly contains many near complete chromosomes (chromosomes 2, 4, 5, 6, 8, and 9) (Fig. 1). Whole genome alignment of Ptr134 version 2 (Fig. 1A) and Ptr134 version 1 [3] (Fig. 1B) to M4 [3] (PacBio RSII) showed few large-scale rearrangements. However, distinct smaller rearrangements were more clearly observed in the ONT assembly, as compared to the Illumina assembly, in particular a small central sequence inversion in chromosome 5 (Fig. 1A). Furthermore, sequence breaks in Ptr134 relative to M4 chromosomes 1, 3, 7 and 10 reflect sequence variations between the two isolates. In particular, the Ptr134 sequence break relative to M4 chromosome 10 coincides with the chromosome 10 and 11 fusion site revealed previously by optical mapping of M4 [3].

Fig. 1
figure1

A Ptr134 Oxford Nanopore Technology contiguous genome sequences (vertical axis) aligned in a dot matrix plot to M4 assembled chromosomes (horizontal axis). B Ptr134 Illumina contiguous genome sequences (vertical axis) aligned in a dot matrix plot against M4 assembled chromosomes (horizontal axis)

This is the first ONT sequenced, assembled and annotated genome for a Ptr race 1 isolate. The improved ONT genome assembly of Ptr134, over the former Illumina assembly, will enable the better characterization of important genes involved in pathogenicity that are often contained in highly complex genomic regions [5], and contribute to improved pan genomic analyses of this important fungal pathogen.

We demonstrate that ONT is a viable option for sequencing less fragmented and near complete genome assemblies for fungal species. Using these methods researchers can sequence and assemble ‘in house’ isolates of interest to create quality reference genomes.

Limitations

All methods have been made as consistent as possible for comparative analyses, this analysis has used databases, software and PacBio sequencing versions currently available, which may be updated in the future. The comparison of the two Australian long-read assemblies is only an indication of potential genome stability in Australia.

Availability of data and materials

The assembled and annotated genome for isolate Ptr134 described in this Data Note can be freely and openly accessed at DDBJ/ENA/GenBank repository under Accession Number- https://www.ncbi.nlm.nih.gov/nuccore/MVBF00000000 (whole genome project) [23].

Abbreviations

BUSCO:

Benchmarking Universal Single-Copy Orthologs

CDD:

Conserved Domain Database

COG:

Clusters of Orthologous Groups

DDBJ:

DNA Data Bank of Japan

ENA:

European Nucleotide Archive

Kb:

Kilo bases

Mb:

Mega bases

NCBI:

National Centre for Biotechnology Information

NR:

Non-redundant

Pfam:

Protein families

ONT:

Oxford Nanopore Technologies

SMART:

Simple Modular Architecture Research Tool

SNP:

Single nucleotide polymorphism

References

  1. 1.

    Moffat CS, Santana MF. Diseases affecting wheat: tan spot. In: Oliver R, editor. Integrated disease management of wheat and barley. Cambridge: Burleigh dodds Science Publishing; 2018.

    Google Scholar 

  2. 2.

    Manning VA, Pandelova I, Dhillon B, Wilhelm LJ, Goodwin SB, Berlin AM, et al. Comparative genomics of a plant-pathogenic fungus, Pyrenophora tritici-repentis, reveals transduplication and the impact of repeat elements on pathogenicity and population divergence. G3. 2013;3(1):41–63.

    CAS  Article  Google Scholar 

  3. 3.

    Moolhuijzen P, See PT, Hane JK, Shi G, Liu Z, Oliver RP, et al. Comparative genomics of the wheat fungal pathogen Pyrenophora tritici-repentis reveals chromosomal variations and genome plasticity. BMC Genomics. 2018;19(1):279.

    Article  Google Scholar 

  4. 4.

    Moolhuijzen P, See PT, Moffat CS. A new PacBio genome sequence of an Australian Pyrenophora tritici-repentis race 1 isolate. BMC Res Notes. 2019;12(1):642.

    Article  Google Scholar 

  5. 5.

    Moolhuijzen P, See PT, Moffat CS. PacBio genome sequencing reveals new insights into the genomic organisation of the multi-copy ToxB gene of the wheat fungal pathogen Pyrenophora tritici-repentis. BMC Genomics. 2020;21(1):645.

    CAS  Article  Google Scholar 

  6. 6.

    Moolhuijzen PM, See PT, Oliver RP, Moffat CS. Genomic distribution of a novel Pyrenophora tritici-repentis ToxA insertion element. PLoS ONE. 2018;13(10):e0206586.

    Article  Google Scholar 

  7. 7.

    Moffat CS, See PT, Oliver RP. Leaf yellowing of the wheat cultivar Mace in the absence of yellow spot disease. Australas Plant Pathol. 2015;44(2):161–6.

    Article  Google Scholar 

  8. 8.

    Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722–36.

    CAS  Article  Google Scholar 

  9. 9.

    Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20.

    CAS  Article  Google Scholar 

  10. 10.

    Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754–60.

    CAS  Article  Google Scholar 

  11. 11.

    Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–9.

    Article  Google Scholar 

  12. 12.

    Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE. 2014;9(11):e112963.

    Article  Google Scholar 

  13. 13.

    Delcher AL, Salzberg SL, Phillippy AM. Using MUMmer to identify similar regions in large sequence sets. Curr Protoc Bioinformatics. 2003. https://doi.org/10.1002/0471250953.bi1003s00 (Chapter 10:Unit 10.3).

    Article  PubMed  Google Scholar 

  14. 14.

    Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25(9):1105–11.

    CAS  Article  Google Scholar 

  15. 15.

    Testa AC, Hane JK, Ellwood SR, Oliver RP. CodingQuarry: highly accurate hidden Markov model gene prediction in fungal genomes using RNA-seq transcripts. BMC Genomics. 2015;16:170.

    Article  Google Scholar 

  16. 16.

    Borodovsky M, Lomsadze A. Eukaryotic gene prediction using GeneMark.hmm-E and GeneMark-ES. Curr Protoc Bioinformatics. 2011. https://doi.org/10.1002/0471250953.bi0406s35 (Chapter 4:Unit 4.6.1–10).

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Slater GS, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005;6:31.

    Article  Google Scholar 

  18. 18.

    Haas BJ, Salzberg SL, Zhu W, Pertea M, Allen JE, Orvis J, et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the program to assemble spliced alignments. Genome Biol. 2008;9(1):R7.

    Article  Google Scholar 

  19. 19.

    Kent WJ. BLAT—the BLAST-like alignment tool. Genome Res. 2002;12(4):656–64.

    CAS  Article  Google Scholar 

  20. 20.

    Shiryev SA, Papadopoulos JS, Schaffer AA, Agarwala R. Improved BLAST searches using longer words for protein seeding. Bioinformatics. 2007;23(21):2949–51.

    CAS  Article  Google Scholar 

  21. 21.

    Koski LB, Gray MW, Lang BF, Burger G. AutoFACT: an automatic functional annotation and classification tool. BMC Bioinformatics. 2005;6:151.

    Article  Google Scholar 

  22. 22.

    Seppey M, Manni M, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness. Methods Mol Biol. 2019;1962:227–45.

    CAS  Article  Google Scholar 

  23. 23.

    Moolhuijzen P, See PT, Moffat C. The improved genome of an Australian Pyrenophora tritici-repentis race 1 isolate using Oxford Nanopore MinION sequencing 2021. https://www.ncbi.nlm.nih.gov/nuccore/MVBF00000000.

Download references

Acknowledgements

We thank the Australian grain growers for their continued support of research through the Grains Research and Development Corporation (GRDC) and the Australian Government National Collaborative Research Infrastructure Strategy (NCRIS) for providing access to Pawsey Supercomputing under a National Computational Merit Allocation Scheme (NCMAS), Nectar Research and Pawsey Nimbus Cloud resources.

Funding

This work was generously supported through co-investment by Grains Research and Development Corporation (GRDC) and Curtin University (Project code CUR00023) as well as Australian Government National Collaborative Research Infrastructure Strategy and Education Investment Fund Super Science Initiative. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Author information

Affiliations

Authors

Contributions

PM conducted the bioinformatics analysis and wrote the manuscript. PTS conducted the molecular analysis. PTS and PM conducted the Oxford Nanopore sequencing. CM and PM led the project conceptualization. All authors contributed to reviewing and editing this manuscript. All authors agree to the publication policies of BMC Genomic Data Note. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Paula Moolhuijzen.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Moolhuijzen, P., See, P.T. & Moffat, C.S. The first genome assembly of fungal pathogen Pyrenophora tritici-repentis race 1 isolate using Oxford Nanopore MinION sequencing. BMC Res Notes 14, 334 (2021). https://doi.org/10.1186/s13104-021-05751-0

Download citation

Keywords

  • Pyrenophora tritici-repentis
  • Genome assembly
  • Oxford Nanopore
  • Comparative analysis
  • ToxA
  • Tan spot
  • Yellow spot
  • MinION