We  report  long-range  sequencing  of nine  rice-infecting Magnaporthe oryzae isolates from  different  rice-growing  regions of Italy using Oxford  Nanopore Technology.  We aquired chromosome-level genome assemblies, polished with Illumina short reads, and removed  mitochondrial  sequences  to  improve the quality  of  the  assemblies.We provide the genome assemblies to the public with open access

Adeline Harant

Angus Malmgren

Anna Maria Picco

Hernán A. Burbano

Joe Win

Nicholas J. Talbot

Ram-Krishna Shrestha

Sergio M. Latorre

Sophien Kamoun

Thorsten Langner

Vincent Were

English

We report long-range sequencing of nine rice-infecting Magnaporthe oryzae isolates from different rice-growing regions of Italy using Oxford Nanopore Technology. We aquired chromosome-level genome assemblies, polished with Illumina short reads, and removed mitochondrial sequences to improve the quality of the assemblies. We provide the genome assemblies to the public with open access

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Large scale genome assemblies of Magnaporthe oryzae rice isolates from Italy

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Hernán A. Burbano

Archivio Istituzionale della Ricerca - Università degli Studi di Pavia

 1 Large scale genome assemblies of Magnaporthe oryzae rice isolates from Italy   Joe Win1, Adeline Harant1, Angus Malmgren1, Thorsten Langner1, Ram-Krishna Shrestha1, Sergio M. Latorre2, Vincent Were1, Nicholas J. Talbot1, Hernán A. Burbano2,3, Anna Maria Picco4, Sophien Kamoun1  1The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich, United Kingdom 2Research Group for Ancient Genomics and Evolution, Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany 3Centre for Life’s Origins and Evolution, Department of Genetics, Evolution and Environment, University College London, London, United Kingdom 4Department of Earth and Environmental Sciences, University of Pavia, Pavia, Italy  We report long-range sequencing of nine rice-infecting Magnaporthe oryzae isolates from different rice-growing regions of Italy using Oxford Nanopore Technology. We aquired chromosome-level genome assemblies, polished with Illumina short reads, and removed mitochondrial sequences to improve the quality of the assemblies. We provide the genome assemblies to the public with open access.    Introduction  Magnaporthe oryzae (Syn. Pyricularia oryzae) is the number one fungal plant pathogen that poses a clear threat to global food security (Dean et al., 2012; Fisher et al., 2012). M. oryzae causes blast disease on rice, wheat and a range of other cereals including oat, finger millet and foxtail millet as well as wild grasses. M. oryzae is found all over the world wherever warm temperature and high humidity are common. This includes Italy which is the largest rice producing country in Europe. In Italy, M. oryzae is thought to be co-evolving with rice since its introduction to the country sometimes after the 15th century. We aim to test a hypothesis that M. oryzae has adapted to the rice cultivars and growing conditions employed in Italy, and in doing so, its genome has accumulated signatures of adaptation including genome-level structural variations. To generate the genomic data that would enable such analyses, we sequenced nine isolates of M. oryzae using a long-range sequencing strategy based on Oxford Nanopore Technology. We deposited these chromosome-level genome assemblies in public databases for open access.    Results  We sequenced nine M. oryzae isolates collected from different regions of Italy using PromethION flow cells (Table 1). We obtained N50 read lengths ranging from 23.4 to 32.3 kbp and total base counts of ~4.3 to 7.5 Gbp (~100x to 180x genome coverage). The sequence reads were assembled using Canu software (Koren et al., 2017). The number of contigs varied from 17 to 38 with the largest contigs in the ~10-11 Mbp range (Table 2).   To improve the quality of the assemblies, we acquired short sequence reads (Table 3) from the same fungal isolates using Illumina technology and polished the genomes using Pilon (Walker et al., 2014) and Racon (Vaser et al., 2017) programs. We then removed the contigs in the assemblies that had high similarities to mitochondrial sequences (Table 4). We re-ordered the contigs in our assemblies to follow the chromosome structure of M. oryzae rice isolate 70-15 (Dean et al., 2005; Okagaki et al., 2015) by aligning our contigs to the 70-15 chromosomes using Mauve (Darling et al., 2004).  To assess the quality of the assemblies, we compared the assemblies of Italian isolates to the chromosome quality assembly of M. oryzae rice isolate 70- 15 by whole-genome sequence alignment using MUMmer 3 package (Kurtz et al., 2004). We noted significant co-linearities  2 between our Italian assemblies and that of 70-15 indicating that our nanopore assemblies are of acceptable quality (Figure 1).       Figure 1. Genome assemblies of Magnaporthe oryzae Italian isolates are highly contiguous and colinear to the refererence genome of isolate 70-15. Colinearity plot of whole genome alignments between Italian rice blast isolates to the 70-15 reference genome. Contig IDs of Italian isolates are given on the Y-axis. Supercontig IDs of the reference genome are given on the X-axis. The color of the alignments shows the sequence similarity to 70-15.  3 To evaluate the completeness of the assemblies and any improvement in polishing process, we run Benchmarking sets of Universal Single-Copy Orthologs (BUSCO) (Seppey et al., 2019) using fungi_obd10 database for each genome assembly. We found that the polishing step improved the completeness of the assemblies from 72.2-83.4% before polishing to 97.7-98.8% after polishing, and gene fragmentation from 3.4-7.4% to 0-0.3% (Tables 5 and 6).   The final polished genome assemblies of M. oryzae we built in this project will give us useful insights into how this fungus evolves and adapts to Italian environmental conditions at the genome structure level. This project is part of the OpenRiceBlast community on Zenodo: https://zenodo.org/communities/openriceblast. The data can also be accessed via the OpenRiceBlast portal http://openriceblast.org.    Conclusions  The total lengths of the polished genome assemblies (44.6 to 48.5 Mbp) indicate near-complete sequencing of the M. oryzae isolates shown in Table 4. This is complemented by near complete BUSCO score (97.7-98.8%) in polished assemblies (Table 6) for all the isolates indicating that the fungal orthologous gene space is highly represented. The individual contigs were also long enough to enable structural variation studies and appear to be of acceptable quality judging from the level of collinearity observed between our assemblies and the reference 70-15 genome. We ensured open access to these genome data to inspire community involvement in analyzing these data and tackling the blast disease of rice, wheat and other crops using cutting-edge genomic tools.   Materials and methods  Single-spore cultures of M. oryzae isolates (Table 1) from different regions of Italy were used for genomic DNA extraction and sequencing.  High molecular weight genomic DNA from M. oryzae was extracted from mycelia of 7-day old cultures by following the method described by (Schwessinger and Rathjen, 2017). Genomic DNA was quantified on a TapeStation (Agilent) and treated with DNAse-free RNAse. RNAse- treated DNA was sheared using either a gTUBE or a 22 Gauge needle. Sheared DNA was captured using AMPure beads (Beckman Coulter, Indianapolis, US) and eluted in 45 μl water and used for library construction following the 1D protocol from Oxford Nanopore. Sequencing runs were performed using PromethION platform (Oxford Nanopore Technologies, Oxford, UK). Sequence reads were assembled into contigs using Canu software (v1.8) (Koren et al., 2017). Simultaneously, Illumina sequencing libraries were prepared from the same genomic DNA samples by following a NextTera protocol (Caruccio, 2011) modified by Karasov et al., (2018), and sequence reads were obtained using the Illumina HiSeq 3000 platform located at the Genome Center facility at the Max Planck Institute for Developmental Biology.  Canu assemblies were subjected to two rounds of polishing with Illumina short sequence reads generated from the same isolates using Pilon version 1.22 (Walker et al., 2014) and Racon version 1.3.2 (Vaser et al., 2017). To remove mitochondrial sequences, we performed blastn (Altschul et al., 1990) search against our assemblies using the mitochondrial sequences from the reference genome 70-15 and removed any contigs that had high similarity to the mitochondrial sequences. We ran BUSCO v4.0.6 (Seppey et al., 2019) using fungi_obd10 database with default parameters.  Whole genome alignments between each Italian isolate genome assembly and the reference genome of isolate 70-15 were performed using the nucmer function of the MUMmer 3 package (Kurtz et al., 2004). The resulting alignments were filtered for the alignments that  4 span over >10 kb using the “delta-filter” function with the option “-l 10000”and colinearity plots were generated using mummerplot with the “--color” option.   Acknowledgments  This research was funded by The Gatsby Charitable Foundation, The Max Planck Society, and the European Research Council BLASTOFF project. Nanopore sequencing and assembly services were sourced from Future Genomics Technologies (Leiden, The Netherlands). Illumina sequencing was provided by the Sequencing Centre of the Max Planck Institute for Developmental Biology (Tübingen, Germany). M. oryzae isolates were collected within the “Risinnova Project”, AGER Foundation.   References  Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403-410. Caruccio N. 2011. Preparation of next-generation sequencing libraries using Nextera™ technology: Simultaneous DNA fragmentation and adaptor tagging by in vitro transposition. In: Kwon Y., Ricke S. (eds) High-Throughput Next Generation Sequencing. Methods in Molecular Biology 733:241-255. Humana Press, New York, NY. Darling AC, Mau B, Blattner FR, Perna NT. 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res 14:1394-403. Dean RA, Van Kan JAL, Pretorius ZA, Hammond-Kosack K.E., Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Khamann R, Ellis J, Foster GD. 2012. The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414-430. Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. 2005. The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986. Fisher M, Henk D, Briggs C, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ. 2012. Emerging fungal threats to animal, plant and ecosystem health. Nature 484:186–194. Karasov TL, Almario J, Friedemann C, Ding W, Giolai M, Heavens D, Kersten S, Lundberg DS, Neumann M, Regalado J, Neher RA, Kemen E, Weigel D. 2018. Arabidopsis thaliana and Pseudomonas pathogens exhibit stable associations over evolutionary timescales. Cell Host Microbe. 24:168-179.e4. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722-736. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. 2004. Versatile and open software for comparing large genomes. Genome Biol 5:R12. Okagaki LH, Nunes CC, Sailsbery J, et al. 2015. Genome sequences of three phytopathogenic species of the Magnaporthaceae family of fungi. G3. 5:2539-2545. Schwessinger B, Rathjen JP. 2017. Extraction of high molecular weight DNA from fungal rust spores for long read sequencing. Methods Mol Biol 1659:49-57. Seppey M, Manni M, Zdobnov EM. 2019. BUSCO: Assessing genome assembly and annotation completeness. In: Kollmar M. eds. Gene Prediction. Methods in Molecular Biology. 1962:227-245. Humana Press, New York, NY. Vaser R, Sović I, Nagarajan N, Šikić M. 2017. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res 27:737-746. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963.   Table 1. M. oryzae isolates used in this study and summary statistics for their nanopore sequencing runs  M. oryzae isolate Host Source Year collected Flowcell ID Sequencing platform1 Basecaller (version) No. Reads  Base count (bp) Read N50 ENA accession AG002 Oryza sativa Sozzago, Italy 2010 PAD04813 PromethION, ONT Guppy (2.1.3) 469,224 7,522,559,914 24,130 ERR4704598 AG006 Oryza sativa Vigevano, Italy 2011 PAD04813 PromethION, ONT Guppy (2.1.3) 321,713 5,198,920,186 29,066 ERR4704596 AG032 Oryza sativa Ferrara, Italy 2011 PAD04813 PromethION, ONT Guppy (2.1.3) 333,608 5,596,762,033 28,813 ERR4704597 AG038 Oryza sativa Olcenengo, Italy 2011 PAD04813 PromethION, ONT Guppy (2.1.3) 284,715 5,438,810,944 32,347 ERR4704599 AG039 Oryza sativa Vercelli, Italy 2011 PAD04813 PromethION, ONT Guppy (2.1.3) 305,056 5,380,738,368 30,721 ERR4704592 AG059 Oryza sativa Siziano, Italy 2011 PAD04813 PromethION, ONT Guppy (2.1.3) 417,916 6,602,278,461 23,457 ERR4704594 AG098 Oryza sativa Oristano, Italy N/D PAD04813 PromethION, ONT Guppy (2.1.3) 314,015 4,665,230,724 28,118 ERR4704600 PR003 Oryza sativa Dossena, Italy 2003 PAD04813 PromethION, ONT Guppy (2.1.3) 245,744 4,274,949,588 30,333 ERR4704595 San andrea Oryza sativa N/D 2001 PAD04813 PromethION, ONT Guppy (2.1.3) 360,461 5,562,064,787 26,468 ERR4704593 1Sequencing was performed by Future Genomics Technologies, Leiden; ONT = Oxford Nanopore Technology N/D = No data; bp = base pair; ENA = European Nucleotide Archive   Table 2. Summary statistics for M. oryzae genomes assembled from nanopore reads  M. oryzae isolate Assembly Software (version) No. contigs Assembly length (bp) N50 (bp) Max length (bp) Min length (bp) GenBank Accession AG002 Canu (1.8) 38 45,963,345 4,529,404 6,354,330 1,902 GCA_905067045.1 AG006 Canu (1.8) 26 46,895,299 6,579,242 10,942,893 51,636 GCA_905067025.1 AG032 Canu (1.8) 25 45,799,422 5,926,258 8,797,420 1,932 GCA_905067055.1 AG038 Canu (1.8) 20 46,144,830 5,640,447 9,106,624 45,956 GCA_905067005.1 AG039 Canu (1.8) 29 47,340,277 6,212,358 10,940,842 1,858 GCA_905067035.1 AG059 Canu (1.8) 38 47,498,635 5,859,208 7,099,302 42,191 GCA_905066965.1 AG098 Canu (1.8) 34 47,650,771 6,089,662 7,827,513 1,894 GCA_905067015.1 PR003 Canu (1.8) 17 44,525,684 6,029,236 8,528,881 40,948 GCA_905067075.1 San andrea Canu (1.8) 37 48,327,069 4,099,541 6,266,075 13,134 GCA_905067085.1 Table 3. Illumina short sequence reads used to polish the genome assemblies  M. oryzae Isolate Sequencing Platform1 Library Layout Base count (bp) No. reads ENA accession AG002 HiSeq 3000, Illumina Paired 1,818,725,400 3,031,209 ERR4757123 AG006 HiSeq 3000, Illumina Paired 962,429,400 1,604,049 ERR4757124 AG032 HiSeq 3000, Illumina Paired 1,493,714,400 2,489,524 ERR4757125 AG038 HiSeq 3000, Illumina Paired 1,252,386,000 2,087,310 ERR4757126 AG039 HiSeq 3000, Illumina Paired 1,256,038,200 2,093,397 ERR4757127 AG059 HiSeq 3000, Illumina Paired 1,258,650,600 2,097,751 ERR4757128 AG098 HiSeq 3000, Illumina Paired 1,345,979,400 2,243,299 ERR4757129 PR003 HiSeq 3000, Illumina Paired 994,322,400 1,657,204 ERR4757130 San andrea HiSeq 3000, Illumina Paired 1,111,573,200 1,852,622 ERR4757131 1Sequencing was performed by Max Planck Institute, Tübingen   Table 4. Polished genome assemblies of Italian Magnaporthe oryzae isolates without mitochondrial contigs  M. oryzae isolate Software (version) No. contigs Assembly length (bp) N50 (bp) Max length (bp) Min length (bp) GenBank accession AG002 Pilon (1.22), Racon (1.3.2) 36 46,086,469 4,555,161 6,389,779 1,896 GCA_905067045.2 AG006 Pilon (1.22), Racon (1.3.2) 24 47,005,811 6,618,557 11,008,917 51,574 GCA_905067025.2 AG032 Pilon (1.22), Racon (1.3.2) 24 45,916,910 5,961,411 8,843,348 1,925 GCA_905067055.2 AG038 Pilon (1.22), Racon (1.3.2) 19 46,291,169 5,673,907 9,163,141 46,127 GCA_905067005.2 AG039 Pilon (1.22), Racon (1.3.2) 26 47,495,958 6,249,570 11,002,933 1,852 GCA_905067035.2 AG059 Pilon (1.22), Racon (1.3.2) 37 47,743,121 5,900,770 7,152,446 42,369 GCA_905066965.2 AG098 Pilon (1.22), Racon (1.3.2) 33 47,810,826 6,094,221 7,877,299 500 GCA_905067015.2 PR003 Pilon (1.22), Racon (1.3.2) 16 44,615,198 6,063,740 8,582,813 41,659 GCA_905067075.2 San andrea Pilon (1.22), Racon (1.3.2) 35 48,509,714 4,122,582 6,303,665 53,723 GCA_905067085.2       Table 5. BUSCO1 scores of the genome assemblies before polishing  Assembly name DB2 Mode Completed SingleCopy Duplicated Fragmented Missing Total AG002 fungi_odb10 genome 632 (83.4%) 630 (83.1%) 2 (0.3%) 26 (3.4%) 100 (13.2%) 758 AG006 fungi_odb10 genome 599 (79.0%) 599 (79.0%) 0 (0.0%) 42 (5.5%) 117 (15.5%) 758 AG032 fungi_odb10 genome 613 (80.9%) 613 (80.9%) 0 (0.0%) 28 (3.7%) 117 (15.4%) 758 AG038 fungi_odb10 genome 616 (81.3%) 616 (81.3%) 0 (0.0%) 29 (3.8%) 113 (14.9%) 758 AG039 fungi_odb10 genome 610 (80.5%) 603 (79.6%) 7 (0.9%) 32 (4.2%) 116 (15.3%) 758 AG059 fungi_odb10 genome 547 (72.2%) 545 (71.9%) 2 (0.3%) 56 (7.4%) 155 (20.4%) 758 AG098 fungi_odb10 genome 610 (80.4%) 609 (80.3%) 1 (0.1%) 30 (4.0%) 118 (15.6%) 758 San andrea fungi_odb10 genome 627 (82.7%) 621 (81.9%) 6 (0.8%) 29 (3.8%) 102 (13.5%) 758 1BUSCO = Benchmarking Universal Single-Copy Orthologs; Seppey et. al., 2019. Methods in Molecular Biology. 1962: 227-245 2DB = Database used in BUSCO  Table 6. BUSCO1 scores of the genome assemblies after polishing  Assembly name DB2 Mode Completed SingleCopy Duplicated Fragmented Missing Total AG002 fungi_odb10 genome 747 (98.6%) 744 (98.2%) 3 (0.4%) 0 (0.0%) 11 (1.4%) 758 AG006 fungi_odb10 genome 745 (98.3%) 744 (98.2%) 1 (0.1%) 2 (0.3%) 11 (1.4%) 758 AG032 fungi_odb10 genome 745 (98.3%) 743 (98.0%) 2 (0.3%) 0 (0.0%) 13 (1.7%) 758 AG038 fungi_odb10 genome 745 (98.3%) 744 (98.2%) 1 (0.1%) 1 (0.1%) 12 (1.6%) 758 AG039 fungi_odb10 genome 741 (97.8%) 736 (97.1%) 5 (0.7%) 0 (0.0%) 17 (2.2%) 758 AG059 fungi_odb10 genome 745 (98.3%) 744 (98.2%) 1 (0.1%) 0 (0.0%) 13 (1.7%) 758 AG098 fungi_odb10 genome 749 (98.8%) 747 (98.5%) 2 (0.3%) 1 (0.1%) 8 (1.1%) 758 PR003 fungi_odb10 genome 742 (97.9%) 740 (97.6%) 2 (0.3%) 0 (0.0%) 16 (2.1%) 758 San andrea fungi_odb10 genome 741 (97.7%) 737 (97.2%) 4 (0.5%) 0 (0.0%) 17 (2.3%) 758 1BUSCO = Benchmarking Universal Single-Copy Orthologs; Seppey et. al., 2019. Methods in Molecular Biology. 1962: 227-245 2DB = Database used in BUSCO  

Large scale genome assemblies of Magnaporthe oryzae rice isolates from Italy

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Archivio Istituzionale della Ricerca - Università degli Studi di Pavia