The genome sequence of the Velvet Shank, Flammulina velutipes (Curtis) Singer, 1951 [version 1; peer review: awaiting peer review]

We present a genome assembly from a specimen of Flammulina velutipes (the Velvet Shank; Basidiomycota; Agaricomycetes; Agaricales; Physalacriaceae). The genome sequence is 37.4 megabases in span. Most of the assembly is scaffolded into 12 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 79.6 kilobases in length


Background
Flammulina velutipes (Velvet Shank) is a lamellate agaric that can grow individually, but more commonly forms densely fasciculate clusters of sporocarps, with viscid to very slimy, orange to yellow pileus, often with a yellow/red brown centre which may be umbonate, each measuring 7-60(-90) mm across. Lamellae are medium spaced to crowded, cream to pale yellow, adnate to emarginate. The stipe is cylindrical, often compressed, tough, velutinous, cream yellow at the top where it joins the cap, and dark red brown throughout the lower areas, and measures 10-80 × 2-10 mm.
In culture, the mycelium is concentrically zonate, clamped and branched, with considerable aerial hyphae surrounding the inoculum, often with creamy white to yellow brown globular hyphal clusters. Spherical to oval chlamydospores are produced in aging cultures (Balaeş & Tănase, 2012;Ingold, 1980). This species grows rapidly and aggressively in culture and is capable of producing sporocarps in short time periods on a wide range of cellulose and lignin-based substrates. It is considered a good edible fungus but is not commonly cultivated. There is a long-standing taxonomic confusion between this fungus and F. filiformis (Wang et al., 2018), also known as Enokitake, which is widely cultivated in Asia and produces fine, greatly elongated sporocarps, which can be white to translucent gold in colour depending on the strain when grown in dark conditions (Sakamoto et al., 2004).
F. velutipes is known from Europe and North America. Though its range may extend to other areas, it is unclear due to recent taxonomic changes that have shifted the species concept (Wang et al., 2018). Throughout Europe it is known to cause a white-rot in the dead wood of a broad range of angiosperm trees, particularly Ulmus and Fagus. In the UK, F. velutipes is widespread and common wherever host trees can be found.
Fungi from this genus are known to contain a number of bioactive compounds that have potential therapeutic and industrial applications (Sharma et al., 2021), but most of this type of research would have focused on F. filiformis instead of F. velutipes due to the above-mentioned taxonomic confusion. The sequencing of this genome will aid in discovering whether F. velutipes also shares these attributes and will further resolve the taxonomic uncertainties of this cryptic genus.
Flammulina velutipes is a model organism and other genome assemblies are available (Kurata et al., 2016;Park et al., 2014). The genome of F. velutipes was sequenced here as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for F. velutipes, grown in a pure culture obtained from a single sporocarp collected at Leigh Woods, Bristol, UK.

Genome sequence report
The genome was sequenced from a culture of Flammulina velutipes (Figure 1) grown from a single sporocarp collected in Leigh Woods, Bristol, UK (51.48, -2.49). A total of 255-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 109 missing joins or mis-joins and removed 10 haplotypic duplications, reducing the scaffold number by 79.73%, and increasing the scaffold N50 by 9.77%.
The final assembly has a total length of 37.4 Mb in 15 sequence scaffolds with a scaffold N50 of 3.5 Mb (Table 1). Most (99.72%) of the assembly sequence was assigned to 12 chromosomal-level scaffolds. Chromosome-scale scaffolds confirmed by the Hi-C data are named by synteny (Figure 2- Figure 5; Table 2). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/38945.
The specimen was identified and grown in pure culture obtained from a single sporocarp by Richard Wright. Culture handling and initial barcoding to confirm identity was carried out by Kieran Woof (RBGK) and samples taken from it were preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The gfFlaVelt1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. The tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. High molecular weight (HMW) DNA was   instructions. Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit. DNA and RNA sequencing were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from a sample of gfFlaVelt1 using the Arimav2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hicanu (Nurk et al., 2020) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023). The assembly was checked for as described previously (Howe et al., 2021). Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.  A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020). This work was done using Nextflow (Di Tommaso et al., 2017) DSL2 pipelines "sanger-tol/readmapping" (Surana et al., 2023a) and "sangertol/genomenote" (Surana et al., 2023b). The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated. Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use. The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible. The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material The genome sequence is released openly for reuse. The Flammulina velutipes genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.