High-Quality Genome Assembly of Fusarium oxysporum f. sp. lini

Abstract

In the present work, a highly pathogenic isolate of Fusarium oxysporum f. sp. lini, which is the most harmful pathogen affecting flax (Linum usitatissimum L.), was sequenced for the first time. To achieve a high-quality genome assembly, we used the combination of two sequencing platforms-Oxford Nanopore Technologies (MinION system) with long noisy reads and Illumina (HiSeq 2500 instrument) with short accurate reads. Given the quality of DNA is crucial for Nanopore sequencing, we developed the protocol for extraction of pure high-molecular-weight DNA from fungi. Sequencing of DNA extracted using this protocol allowed us to obtain about 85x genome coverage with long (N50 = 29 kb) MinION reads and 30x coverage with 2 × 250 bp HiSeq reads. Several tools were developed for genome assembly; however, they provide different results depending on genome complexity, sequencing data volume, read length and quality. We benchmarked the most requested assemblers (Canu, Flye, Shasta, wtdbg2, and MaSuRCA), Nanopore polishers (Medaka and Racon), and Illumina polishers (Pilon and POLCA) on our sequencing data. The assembly performed with Canu and polished with Medaka and POLCA was considered the most full and accurate. After further elimination of redundant contigs using Purge Haplotigs, we achieved a high-quality genome of F. oxysporum f. sp. lini with a total length of 59 Mb, N50 of 3.3 Mb, and 99.5% completeness according to BUSCO. We also obtained a complete circular mitochondrial genome with a length of 38.7 kb. The achieved assembly expands studies on F. oxysporum and plant-pathogen interaction in flax.

Nanopore and Illumina Genome Sequencing of Fusarium oxysporum f. sp. lini Strains of Different Virulence

“…Since F. oxysporum f. sp. lini genome size is about 60-70 Mb (Kanapin et al, 2020;Krasnov et al, 2020), the obtained data corresponded to 10x genome coverage with ONT reads and 50x coverage with Illumina reads on average. The Nanopore and Illumina sequencing data for these five strains and isolate #39, which was sequenced by us earlier, were deposited in NCBI under the BioProject accession number PRJNA721899.…”

“…Following the previously developed protocol, pure highmolecular-weight DNA of the fungal samples was obtained (Krasnov et al, 2020). In brief, the DNA was extracted using the CTAB method followed by its isolation with Blood and Cell Culture DNA Mini Kit (Qiagen, USA).…”

“…Reads with an average quality below 6 (Q < 6) were discarded with Trimmomatic 0.39 (Bolger et al, 2014). To perform the initial genome assemblies, we chose the Canu tool (version 2.1) developed for long-read datasets (Koren et al, 2017), as it provided us with the best results when assembling the genome of isolate #39 in our previous study (Krasnov et al, 2020). The approximate size of the genomes was set as 60 Mb, and the other Canu parameters were kept default.…”

“…These assemblies were not good enough for further analysis that can be explained by the low coverage of genomes with ONT reads (about 6-13x). So, we decided to perform hybrid assemblies of the genomes of the five strains with MaSuRCA (version 3.4.2, CA algorithm) (Zimin et al, 2013) using both ONT and Illumina data, as the obtained coverage with Illumina reads was high (30-80x) and MaSuRCA showed decent results for isolate #39 earlier (Krasnov et al, 2020). The N50 parameter of the hybrid assemblies of The obtained assemblies are a useful resource for comparative genomic studies of the flax pathogen strains.…”

“…Besides, the genome of F. oxysporum f. sp. lini isolate #39 was reassembled with Canu, using the earlier obtained data (Krasnov et al, 2020) and according to the same strategy as described above (ONT read processing with Guppy, Porechop, Trimmomatic, assembling with Canu), and with MaSuRCA (basecalling with Guppy, assembling from ONT and Illumina reads with MaSuRCA) (Table 1). Polishing of the Canu assembly was conducted using ONT reads according to the previously optimized scheme-two iterations with the Racon polisher and one with Medaka (Dmitriev et al, 2020).…”