Reference Genome Assembly and Alignment

We generated a long read-based de novo genome assembly using Oxford Nanopore and Hi-C approaches, below.

High Molecular Weight DNA isolation and Oxford Nanopore sequencing. A total of 0.4 g Cochlearia excelsa leaf material from one individual plant was ground using liquid nitrogen before the addition of 10 ml of CTAB DNA extraction buffer (100 mM Tris-HCl, 2% CTAB, 1.4 M NaCl, 20 mM EDTA, and 0.004 mg/ml Proteinase K). The mixture was incubated at 55°C for 1 hour then cooled on ice before the addition of 5 ml Chloroform. This was then centrifuged at 3000 rpm for 30 minutes and the upper phase taken, this was added to 1X volume of phenol:chloroform:isoamyl- alcohol and spun for 30 minutes at 3000 rpm. Again, the upper phase was taken and mixed with a 10% volume of 3M NaOAc and 2.5X volume of 100% ethanol at 4 °C. This was incubated on ice for 30 minutes before being centrifuged for 30 minutes at 3000 rpm and 4 °C. Three times the pellet was washed in 4ml 70% ethanol at 4 °C before being centrifuged again for 10 minutes at 3000 rpm and 4°C. The pellet was then air-dried and resuspended in 300 μl nuclease-free water containing 0.0036 mg/ml RNase A. The quantity and quality of high molecular weight DNA was checked on a Qubit Fluorometer 2.0 (Invitrogen) using the Qubit dsDNA HS Assay kit. Fragment sizes were assessed using a Q-card (OpGen Argus) and the Genomic DNA Tapestation assay (Agilent). Removal of short DNA fragments and final purification to HMW DNA was performed with the Circulomics Short Read Eliminator XS kit.

Long-read libraries were prepared using the Genomic DNA by Ligation kit (SQK-LSK109; Oxford Nanopore Technologies) following the manufacturer’s procedure. Libraries were then loaded onto a R9.4.1 PromethION Flow Cell (Oxford Nanopore Technologies) and run on a PromethION Beta sequencer. Due to the rapid accumulation of blocked flow cell pores or due to apparent read length anomalies on some Cochlearia runs, flow cells used in runs were treated with a nuclease flush to digest blocking DNA fragments before loading with fresh library according to the Oxford Nanopore Technologies Nuclease Flush protocol, version NFL_9076_v109_revD_08Oct2018.

Genome size estimation and computational ploidy inference. We used KMC to create a k- mer frequency spectrum (Kmer length=21) of trimmed Illumina reads. We then used GenomeScope 2.0 (parameters: -k 21 -m 61) and Smudgeplot to estimate genome size and heterozygosity from k-mer spectra.

Data processing and assembly. Fast5 sequences produced by PromethION sequencing were base called using the Guppy 6 high accuracy base calling model (dna_r9.4.1_450bps_hac.cfg) and the resulting fastq files were quality filtered by the base caller. A total of 17.2 GB base called data were generated for the primary assembly, resulting in 60x expected coverage. Primary assembly was performed in Flye and Necat. The contigs were polished to improve the single-base accuracy in a single round of polishing in Medaka and Pilon.

Pseudomolecule construction by Hi-C, assembly cleanup, and polishing. To scaffold the assembled contigs into pseudomolecules, we performed chromosome conformation capture using HiC. Leaves from a single plant were snap-frozen in liquid N and ground to a fine powder using mortar and pestle. The sample was then homogenised, cross-linked and shipped to Phase Genomics (Seattle, USA), who prepared and sequenced an in vivo Hi-C library. After filtering low-quality reads with Trimmomatic, we aligned the Hi-C reads against the contig-level assembly using bwa-mem (settings -5 -S -P) and removed PCR duplicated using Picard Tools (https://broadinstitute.github.io/picard/). We used 3D-DNA to conduct the initial scaffolding, followed by a manual curation in Juicebox. After manually assigning chromosome boundaries, we searched for centromeric and telomeric repeats to orient the chromosome arms and to assess the completeness of the assembled pseudomolecules. To identify the centromeric repeat motif in C. excelsa, we used the RepeatExplorer pipeline to search for repetitive elements from short-read sequence data originating from the reference individual. RepeatExplorer discovered a highly abundant 102 nucleotide repeat element (comprising 21% of the short-read sequence), which we confirmed as the centromeric repeat motif by fluorescence in situ hybridisation. Using BLAST, we localised the centromeric and telomeric (TTTAGGG) repeats and used them to orient the chromosome arms. We performed a final assembly cleanup in Blobtools (Fig.S1). Gene space completeness was assessed using BUSCO version 3.0.2).