GENOME SEQUENCES Whole-Genome Sequencing and De Novo Assembly of Malassezia pachydermatis Isolated from the Ear Canal of a Dog with Otitis S. D’Andreano,a,b J. Viñes,a,b O. Francinoa SVGM, Molecular Genetics Veterinary Service, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain a b Vetgenomics, Edifici EUREKA, PRUAB, Bellaterra, Barcelona, Spain ABSTRACT We have de novo assembled the genome sequence of Malassezia pachy- dermatis isolated from a canine otitis sample with Nanopore-only long reads. With 99 coverage and 8.23 Mbp, the genome sequence was assembled in 10 contigs, with 6 of them corresponding to chromosomes, improving the scaffolding of previ- ous genome assemblies for the species. M alassezia (Eukaryota; Fungi; Basidiomycetes) is a lipid-dependent yeast of the skin mycobiome in humans and other mammals (1–3). The genus includes 18 closely related species described to date (4), and M. globosa, M. restricta, and M. sympo- dialis are the most common human skin residents, whereas M. pachydermatis is the most common species related to skin disorders in veterinary medicine (3, 5). As of February 2021, there are only two assembled genome sequences for M. pachy- dermatis, under NCBI accession no. GCF_001278385.1 (6) and GCA_001264975.1 (7), which are both from the collection strain CBS 1879. There is a need to gather genomic information from Malassezia isolates associated with skin disorders important in animal and human health. We have assembled a high-quality M. pachydermatis genome sequence isolated from a dog with otitis using a de novo assembly and polishing strat- egy with Nanopore-only long reads. Leti Animal Health kindly provided an isolate identified as M. pachydermatis after microbiological culture of a swab sample collected from a dog’s ear canal with otitis in Barcelona, Spain. The culture was grown at 28°C in dermatophyte test medium (DTM) agar and Saboraud-chloramphenicol agar. DNA was extracted with a Biomics DNA min- iprep kit (Zymo Research, USA). DNA quality and quantity were determined using a Nanodrop 2000 spectrophotometer and Qubit double-stranded DNA (dsDNA) broad- range (BR) assay kit (Fisher Scientific SL, Spain). Before whole-genome sequencing, the Citation D’Andreano S, Viñes J, Francino O. isolate was confirmed as Malassezia pachydermatis by long-amplicon Nanopore 2021. Whole-genome sequencing and de novo sequencing of the fungal ribosomal operon and taxonomy assignment at the species assembly of Malassezia pachydermatis isolated from the ear canal of a dog with otitis. level with the cloud-based WIMP application from the EPI2ME platform (Oxford Microbiol Resour Announc 10:e00205-21. Nanopore Technologies [ONT] Ltd., UK), as previously reported (8). The sequencing https://doi.org/10.1128/MRA.00205-21. library was prepared with the 1D native barcoding genomic DNA kits (EXP-NBD103 Editor Jason E. Stajich, University of California, and SQK-LSK109; ONT) and sequenced in a MinION FLO-MIN106 v9.4.1 flow cell in a Riverside Copyright © 2021 D’Andreano et al. This is an MinION Mk1B instrument for 21 h, using the MinKNOW v18.12.9 software. open-access article distributed under the terms The fast5 files were base called with Guppy v2.3.7 (ONT), with the flip-flop algorithm for of the Creative Commons Attribution 4.0 improved accuracy. Reads with a quality score lower than 7 were discarded. The fastq files International license. were demultiplexed and trimmed with Deepbinner (9). We further de novo assembled the Address correspondence to O. Francino, olga.francino@uab.cat. genome sequence with Flye 2.6 (10) and corrected the contigs with racon (https://github Received 23 February 2021 .com/lbcb-sci/racon) and Medaka (https://github.com/nanoporetech/medaka). We assessed Accepted 28 April 2021 the genome completeness with Benchmarking Universal Single-Copy Orthologs (BUSCO) Published 27 May 2021 v4.0.1 (https://gitlab.com/ezlab/busco) (11) in mode genome with basidiomycota_odb10 Volume 10 Issue 21 e00205-21 mra.asm.org 1 D’Andreano et al. TABLE 1 De novo assembly of the Malassezia pachydermatis genome sequence isolated from a canine otitis samplea Assigned Assigned molecule GenBank Sequence Sequence name Sequence role molecule location type accession no. length (bp) Contig_1 Assembled molecule 1 Chromosome CM025284.1 1,799,555 Contig_7 Assembled molecule 2 Chromosome CM025285.1 1,692,251 Contig_13 Assembled molecule 3 Chromosome CM025286.1 1,423,205 Contig_8 Assembled molecule 4 Chromosome CM025287.1 1,405,702 Contig_4 Assembled molecule 5 Chromosome CM025288.1 1,144,030 Contig_9 Assembled molecule 6 Chromosome CM025289.1 645,491 Contig_11 Unplaced scaffold NA NA JABNMX010000001.1 28,388 Contig_12 Unplaced scaffold NA NA JABNMX010000002.1 9,493 Contig_3 Unplaced scaffold NA NA JABNMX010000005.1 2,740 Contig_5 Unplaced scaffold NA NA JABNMX010000007.1 74,481 a BioProject PRJNA631787, assembly GCA_014493585.1, accession no. JABNMX01, and isolate M13-UAB2019. NA, not applicable. (20 November 2019; no. of species, 133; no. of BUSCOs, 1,764). We used Bandage (12) for contig visualization and comparison with the CBS 1879 M. pachydermatis reference genome (GCF_001278385.1) (6). Nanopore sequencing generated 265,298 reads with an N50 value of 5,648 bp and allowed successful de novo assembly and polishing of the genome sequence of M. pachydermatis (Table 1). Coverage was 99, with a GC content of 54.54%. The genome was 8.23 Mbp in 10 contigs (longest contig, 1.799 Mbp; N50, 1.423 Mbp), with the lon- gest ones corresponding to the 6 expected chromosomes (13) (Fig. 1). The average nu- cleotide identity (ANI) (14) was 96.3% with the M. pachydermatis reference genome (GCF_001278385.1), confirming the isolate as the same species (15, 16). The 76.6% BUSCO completeness (C,76.6% [S,76.5%; D,0.1%]; F,3.8%; M,19.6%; n,1,764) is similar to the M. pachydermatis reference genome with short reads (6, 7) and with the same duplication of the glutathione reductase gene as the reference genome. Data availability. The standardized strain descriptions and accession numbers are presented in Table 1; the genome assemblies and genomic data are publicly available in DDBJ/ENA/GenBank under BioProject no. PRJNA631787 with the assembly accession no. GCA_014493585.1 and genome accession no. JABNMX01. The version described in this paper is the first version. The raw data are available from the Sequence Read Archive (SRA) under the accession no. SRR13764144. Raw data (fast5) are available at Zenodo (https://zenodo.org/record/3874381#.YJQ1rLVKi70). FIG 1 Bandage visualization of the genome assembly for Malassezia pachydermatis (GenBank accession no. GCA_014493585.1). Contig_1, Contig_13, Contig_7, Contig_8, Contig_4, and Contig_9 correspond to the six chromosomes of M. pachydermatis. Contigs are colored relative to the reference assembly for the CBS 1879 strain (GCF_001278385.1) (6) and indicate the merge of contigs that are separate from the CBS reference genome assembly with short reads. Volume 10 Issue 21 e00205-21 mra.asm.org 2 Microbiology Resource Announcement ACKNOWLEDGMENTS Funding was provided by Industrial Doctorate Program grants 2015DI044 to S.D. and 2017DI037 to J.V., AGAUR, Generalitat de Catalunya, Spain. O.F. has received unrelated honoraria for lecturing from Oxford Nanopore Technologies. REFERENCES 1. Findley K, Oh J, Yang J, Conlan S, Deming C, Meyer JA, Schoenfeld D, clinical samples. Biol Methods Protoc 6:bpaa026. https://doi.org/10.1093/ Nomicos E, Park M, Kong HH, Segre JA, NIH Intramural Sequencing Center biomethods/bpaa026. Comparative Sequencing Program. 2013. Topographic diversity of fungal 9. Wick RR, Judd LM, Holt KE. 2018. Deepbinner: demultiplexing barcoded and bacterial communities in human skin. Nature 498:367–370. https:// Oxford Nanopore reads with deep convolutional neural networks. PLoS doi.org/10.1038/nature12171. Comput Biol 14:e1006583. https://doi.org/10.1371/journal.pcbi.1006583. 2. Oh J, Byrd A, Deming C, Conlan S, Kong H, Segre J, NISC Comparative Sequenc- 10. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error- ing Program. 2014. Biogeography and individuality shape function in the human prone reads using repeat graphs. Nat Biotechnol 37:540–549. https://doi skin metagenome. Nature 514:59–64. https://doi.org/10.1038/nature13786. .org/10.1038/s41587-019-0072-8. 3. Cabañes FJ. 2014. Malassezia yeasts: how many species infect humans and ani- 11. Seppey M, Manni M, Zdobnov EM. 2019. BUSCO: assessing genome as- mals? PLoS Pathog 10:e1003892. https://doi.org/10.1371/journal.ppat.1003892. sembly and annotation completeness. In Kollmar M (ed), Gene prediction: 4. Theelen B, Cafarchia C, Gaitanis G, Bassukas ID, Boekhout T, Dawson TL. methods in molecular biology, vol 1962. Humana, New York, NY. 2018. Malassezia ecology, pathophysiology, and treatment. Med Mycol 12. Wick RR, Schultz MB, Zobel J, Holt KE. 2015. Bandage: interactive visualization 56:S10–S25. https://doi.org/10.1093/mmy/myx134. of de novo genome assemblies. Bioinformatics 31:3350–3352. https://doi.org/ 5. Coelho MA, Sampaio JP, Gonçalves P. 2013. Living and thriving on the 10.1093/bioinformatics/btv383. skin: Malassezia genomes tell the story. mBio 4:e00117-13. https://doi 13. Sankaranarayanan SR, Ianiri G, Coelho MA, Reza MH, Thimmappa BC, .org/10.1128/mBio.00117-13. Ganguly P, Vadnala RN, Sun S, Siddharthan R, Tellgren-Roth C, Dawson 6. Triana S, González A, Ohm RA, Wösten HA, de Cock H, Restrepo S, Celis A. 2015. Draft genome sequence of the animal and human pathogen Malas- TL, Heitman J, Sanyal K. 2020. Loss of centromere function drives karyo- sezia pachydermatis strain CBS 1879. Genome Announc 3:e01197-15. type evolution in closely related Malassezia species. Elife 9:e53944. https://doi.org/10.1128/genomeA.01197-15. https://doi.org/10.7554/eLife.53944. 7. Wu G, Zhao H, Li C, Rajapakse MP, Wong WC, Xu J, Saunders CW, Reeder 14. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. 2018. High NL, Reilman RA, Scheynius A, Sun S, Billmyre BR, Li W, Averette AF, throughput ANI analysis of 90K prokaryotic genomes reveals clear species Mieczkowski P, Heitman J, Theelen B, Schröder MS, De Sessions PF, Butler boundaries. Nat Commun 9:5114. https://doi.org/10.1038/s41467-018-07641-9. G, Maurer-Stroh S, Boekhout T, Nagarajan N, Dawson TL, Jr. 2015. Genus- 15. Lachance MA, Lee DK, Hsiang T. 2020. Delineating yeast species with ge- wide comparative genomics of Malassezia delineates its phylogeny, phys- nome average nucleotide identity: a calibration of ANI with haplontic, heter- iology, and niche adaptation on human skin. PLoS Genet 11:e1005614. othallic Metschnikowia species. Antonie Van Leeuwenhoek 113:2097–2106. https://doi.org/10.1371/journal.pgen.1005614. https://doi.org/10.1007/s10482-020-01480-9. 8. D’Andreano S, Cusco A, Francino O. 2020. Rapid and real-time identification 16. Gostincar C. 2020. Towards genomic criteria for delineating fungal spe- of fungi up to species level with long amplicon nanopore sequencing from cies. J Fungi 6:246. https://doi.org/10.3390/jof6040246. Volume 10 Issue 21 e00205-21 mra.asm.org 3