Strains and media
Strains used are listed in Supplementary Table 2 and plasmids in Supplementary Table 3. Strains and plasmids are available on request. Hanseniaspora strains were grown in standard rich medium (YPD; yeast extract, peptone, dextrose) or in synthetic complete dropout medium at 30 °C.
DNA extraction and Nanopore sequencing
DNA for nanopore sequencing was prepared as previously described in ref. 66. In brief, overnight cultures of Hanseniaspora spp. (roughly 5 ml of YPD) were pelleted by centrifugation, washed with 1× PBS and resuspended in 5 ml of spheroplast buffer (1 M sorbitol, 50 mM potassium phosphate, 5 mM EDTA, pH 7.5) supplemented with 5 mM dithiothreitol and 50 mg ml−1 zymolyase. Cultures were incubated at 30 °C with shaking at 210 rpm for 1 h. The resulting spheroplasts were collected by centrifugation at 2,500g (4 °C), gently washed with 1 M sorbitol and treated with a proteinase K solution (final concentration 25 mM EDTA, 0.5% SDS, 0.5 mg ml−1 proteinase K) for 2 h at 65 °C, with gentle inversion every 30 min. Genomic DNA was extracted twice using a 1:1:1 ratio of phenol:chloroform:isoamyl alcohol. The aqueous layer was treated with roughly 10 µg of RNase A at 37 °C for 30 min, followed by a final 1:1 extraction with chloroform:isoamyl alcohol. DNA precipitation was carried out using 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold 100% ethanol, with inversion until visible DNA strands formed. High-molecular-weight DNA was spooled onto a pipette tip, washed in 70% ethanol, air-dried and dissolved overnight in Tris-EDTA buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). DNA quantification was performed using the Qubit 1× dsDNA HS Assay reagent (Thermo, Q33231) on the Qubit Flex Fluorometer. For sequencing, genomic DNA was simultaneously tagmented and barcoded with the Oxford Nanopore Rapid Barcoding Kit (SQK-RBK004) following the manufacturer’s protocol. Barcoded libraries were pooled, purified and concentrated using Sera-Mag beads (Cytiva, 29343052). The prepared library was immediately loaded onto a MinION R9.4.1 flow cell (SKU, FLO-MIN106.001) and sequenced on a GridION Mk1 device for 46 h.
Hi-C library generation
Cultures (YPD) were inoculated with the desired strain and grown overnight at 30 °C. The following morning, each was diluted into 150 ml of YPD at a starting optical density (OD) with absorbance at 600 nm (A 600 ) of 0.25 and each was grown until an OD of A 600 = 0.8–1.0. Cells were crosslinked in formaldehyde [3% (v/v)] for 20 min at room temperature and the reaction was quenched with glycine (300 mM). Cells were then collected by centrifugation and washed twice in fresh YPD. Last, the cell pellet was frozen in liquid nitrogen and kept at −80 °C until further processing. Hi-C experiments and library generation were then performed as previously described in refs. 67,68,69.
Genome sequencing and assembly
Raw fastq reads were first processed with porechop (v.0.2.4) to remove barcodes and adaptor sequences. We then generated de novo genome assemblies using Canu (v.2.2; genomeSize = 10 maxInputCoverage = 100). Contigs were polished in the following manner: first, raw contigs were corrected using the ultrafast consensus module Racon (v.1.4.17), followed by two sequential rounds of contig polishing with Medaka (v.1.7). Finally, we performed three rounds of contig polishing with Pilon (v.1.23) using publicly available Illumina sequencing datasets from each species (Sequence Read Archive (SRA) accession numbers SRX5619117, SRX5619118 and SRX5619119). Next, the polished contigs were scaffolded using chromatin conformation sequencing data (Hi-C) with the 3D-DNA pipeline (v.180922)70. Scaffolded assemblies were manually corrected using the Hi-C map visualization and editor Juicebox (https://github.com/aidenlab/Juicebox). The final assembly statistics are reported in Supplementary Table 1.
We generated a reference-assisted assembly of the Sa. ludwigii strain PC99/R1 using publicly available shotgun sequencing data (SRA SRR12082187). First, we generated a de novo genome assembly using the SPAdes (v.4.1.0) assembler. This resulted in an assembly of 408 contigs larger than 500 bp, with a contig N50 of 87,147 bp. We scaffolded the contigs using ragtag (v.2.1.0) with the assembly UHD_SCDLUD_16 (strain NBRC 1722) as the reference. The PC99/R1 assembly was used to extract extra centromere regions as they were divergent from strain NBRC 1722. In total, the assembly of PC99/R1 contained five out of seven centromeric regions.
We generated de novo genome assemblies of three strains of Sa. pseudoludwigii using the Plasmidsaurus hybrid yeast genome service. Cells were grown in YPD at 30 °C until saturation, collected by centrifugation, washed with 1× PBS and 75 mg of wet cells were resuspended in 500 µl of Zymo 1× DNA/RNA Shield (catalogue no. R1100-50). The preserved specimens were then shipped to Plasmidsaurus. Genome sequencing and assembly were completed using Oxford Nanopore Technologies and Illumina sequencing with Plasmidsaurus’s custom analysis and annotation pipeline.
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