Telomere-to-Telomere Genome Assembly of Bursaphelenchus okinawaensis Strain SH1

Bursaphelenchus okinawaensis is a self-fertilizing, hermaphroditic, fungal-feeding nematode used as a laboratory model for the genus Bursaphelenchus, which includes the important pathogen Bursaphelenchus xylophilus. Here, we report the nearly complete genome sequence of B. okinawaensis. The 70-Mbp assembly contained six scaffolds (>11 Mbp each) with telomere repeats on their ends, indicating complete chromosomes.

ABSTRACT

Bursaphelenchus okinawaensis is a self-fertilizing, hermaphroditic, fungus-feeding nematode used as a laboratory model for the genus Bursaphelenchus, which includes the important pathogen Bursaphelenchus xylophilus. Here, we report the nearly complete genome sequence of B. okinawaensis. The 70-Mbp assembly contained six scaffolds (>11 Mbp each) with telomere repeats on their ends, indicating complete chromosomes.

ANNOUNCEMENT

Bursaphelenchus okinawaensis is a fungus-feeding nematode associated with longhorn beetles (Monochamus maruokai) and the beetles’ host trees (1). Because these nematodes self-fertilize, B. okinawaensis has recently emerged as a laboratory model for the genus Bursaphelenchus (2), which includes the important plant pathogen Bursaphelenchus xylophilus (3). Here, we generated a nearly complete genome sequence of B. okinawaensis.

Botrytis cinerea grown on autoclaved barley grains was fed to Bursaphelenchus okinawaensis strain SH1 (maintained at Meiji University) for 13 days. Mixed-stage worms were collected using a modified Baermann funnel technique (4). Briefly, worm culture was suspended in distilled water (dH₂O) complemented with streptomycin, amphotericin B, and penicillin (antibiotic/antimycotic [anti/anti]; Gibco), and live worms were passed through a sieve lined with Kimwipes (Crecia) followed by discontinuous sucrose gradient centrifugation to remove culture debris (5). Cleaned worms were incubated in a worm lysis solution (buffer G2 with 0.8 mg/ml proteinase K [Qiagen], 50 mM dithiothreitol [Wako], and 0.5 mg/ml RNase A [Invitrogen]; 55°C; 4 h) following two freeze-thaw treatments. High-molecular-weight genomic DNA was extracted via phenol-chloroform extraction and ethanol precipitation. A Nanopore library was prepared from 1 μg of genomic DNA using a ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies) according to the manufacturer’s protocol. A single 24-h sequencing run was performed with an R9.4.1 MinION flow cell; thereafter, 2.8 Gbp of sequence data (173,396 reads; N₅₀, 33.6 kbp) was obtained. The Nanopore reads were base called to generate FASTQ files using the Guppy v4.0.15 basecaller (Oxford Nanopore Technologies) with the supplied dna_r9.4.1_450bps_hac configuration and were quality checked using NanoPlot v1.31.0 (6). An Illumina sequencing library was separately prepared from 1 μg of extracted DNA using a TruSeq DNA sample preparation kit (Illumina) according to the manufacturer’s protocol. The library was sequenced on an Illumina HiSeq 2000 instrument according to the manufacturer’s protocol, producing 101-bp paired-end reads (4.2 Gbp). Raw Illumina sequence data were subjected to the Real-Time Analysis (RTA) v1.12.4.2 analysis pipeline (Illumina). A Hi-C library was prepared from ∼10,000 fresh worms using an Arima-HiC kit (Arima Genomics) followed by a Collibri ES DNA library prep kit (Thermo Fisher Scientific) according to the manufacturers’ protocols and was sequenced using a MiSeq system with the MiSeq reagent kit v3 (101 cycles × 2), and the 3.6 million short reads were quality checked using the Hi-C quality control pipeline (https://phasegenomics.github.io/2019/09/19/hic-alignment-and-qc.html).

Nanopore long reads were assembled using Flye v2.7.1 (7) with the following parameters: --genome size 70 M and -iteration 4. The Flye assembly was highly contiguous, comprising eight long (1- to 12-Mb) contigs and one short (6-kb) contig. Base correction was then performed with the Illumina paired-end reads with two rounds of Pilon v1.23 (8). To scaffold contigs and confirm assembly fidelity, we performed Hi-C analysis on the Flye assembly using the 3D-DNA pipeline v180114 with the following parameters: -g 50 -r 2 --editor-coarse-resolution 2500000 --editor-coarse-region 10000000 --editor-fine-resolution 1000000 --polisher-input-size 20000000 --splitter-input-size 20000000 (9). Juicebox v1.11.08 (10) was used for visualization of the Hi-C results.

The resulting 70.0-Mbp assembly had a GC content of 36.2%. It comprised six scaffolds (>11 Mbp each) with four gaps and one ∼6-kbp contig. BUSCO v3.1 completeness analyses (11) of the assembled genome revealed that 92.1% of core eukaryote genes are present in this assembly. Notably, we identified a telomere repeat signature (TTAGGC)n at both ends of five scaffolds, indicating complete chromosomal sequences at one end of the sixth scaffold (Fig. 1).

FIG 1.

Six chromosomes of the Bursaphelenchus okinawaensis genome assembly. (a) Scaffold map of the 70-Mbp B. okinawaensis genome. Blue boxes represent telomere repeat signatures, and green boxes indicate remaining gaps in the assembly. (b) Hi-C contact map representing 6 chromosomes (blue boxes) of the B. okinawaensis genome. (c) Nonrandom distribution of repetitive DNA sequences in the 6 chromosomes. A species-specific repeat library was generated using RepeatModeler v1.0.8 (https://github.com/Dfam-consortium/RepeatModeler) with the default options, and repeats were identified using RepeatMasker v4.0.9 (http://www.repeatmasker.org/) with the default options.

Data availability.

The B. okinawaensis v2 assembly has been deposited in DDBJ/EMBL/GenBank under BioProject number PRJEB40023. The raw Illumina, Nanopore, and Hi-C read data are available in the Sequence Read Archive with accession numbers DRR243691, DRR243689, and DRR243690, respectively.