GENOME SEQUENCES Whole-Genome Sequencing and De Novo Assembly of 61 Staphylococcus pseudintermedius Isolates from Healthy Dogs and Dogs with Pyoderma O. Francino,a D. Pérez,a J. Viñes,a,c R. Fonticoba,b S. Madroñero,a G. Meroni,d P. Martino,d S. Martínez,e A. Cusco,c N. Fàbregas,c L. Migura-García,f L. Ferrerb,g a SVGM, Molecular Genetics Veterinary Service, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain b Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain c Vetgenomics, Edifici EUREKA, PRUAB, Bellaterra, Barcelona, Spain d Department of Biomedical, Surgical and Dental Sciences–One Health Unit, Milan, Italy e Hospital Escuela de Pequeños Animales (HEPA), Facultad de Ciencias Veterinarias de la Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Buenos Aires, Argentina IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Bellaterra, Barcelona, Spain f g Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain ABSTRACT We have de novo assembled and polished 61 Staphylococcus pseudinterme- dius genome sequences with Nanopore-only long reads. Completeness was 99.25%. The average genome size was 2.70 Mbp, comprising 2,506 coding sequences, 19 complete rRNAs, 56 to 59 tRNAs, and 4 noncoding RNAs (ncRNAs), as well as CRISPR arrays. S taphylococcus pseudintermedius is a common microorganism of canine skin (1, 2) and the leading cause of pyoderma in dogs (3, 4). In this study, we aimed to retrieve S. pseudintermedius high-quality genome sequences from healthy dogs and dogs with pyoderma using a de novo assembly and polishing strategy with Nanopore- only long reads. Samples were obtained by rubbing a sterile swab on the skin of healthy dogs (n = 22; H) and the nonlesional skin of a dog with pyoderma (n = 6; DH) or from the pustules of dogs with pyoderma (n = 33; D). After culture in blood agar at 37°C for 24 hours, colonies were seeded in 3 ml of brain heart infusion (BHI) broth at 37°C for 16 hours. DNA was extracted with a ZymoBIOMICS DNA miniprep kit (Zymo Research, Irvine, CA, USA). DNA quality and quantity were determined using a Nanodrop 2000 spectrophotometer and Qubit double- Citation Francino O, Pérez D, Viñes J, stranded DNA (dsDNA) broad-range (BR) assay kit (Fisher Scientific SL, Madrid, Spain). The Fonticoba R, Madroñero S, Meroni G, Martino P, sequencing libraries were prepared with the rapid barcoding sequencing kit (SQK-RBK004; Martínez S, Cusco A, Fàbregas N, Migura-García L, Ferrer L. 2021. Whole-genome sequencing Oxford Nanopore Technologies, UK). Twelve barcoded samples were loaded into a MinION and de novo assembly of 61 Staphylococcus FLO-MIN106 v9.4.1 flow cell (Oxford Nanopore Technologies Ltd.) and sequenced into a pseudintermedius isolates from healthy dogs and dogs with pyoderma. Microbiol Resour MinION Mk1B instrument. The fast5 files were basecalled and demultiplexed and adapters Announc 10:e00152-21. https://doi.org/10 trimmed with Guppy 4.0.11 (Oxford Nanopore Technologies) (--config dna_r9.4.1_450bp- .1128/MRA.00152-21. s_hac.cfg) (--config configuration.cfg --barcode_kits SQK-RBK004 --trim_barcodes; min_score Editor Irene L. G. Newton, Indiana University, threshold default 60). Reads with a quality score lower than 7 were discarded. Run summary Bloomington statistics were obtained with Nanoplot 1.27 (5) (--N50 --fastq). Copyright © 2021 Francino et al. This is an open-access article distributed under the terms Samples assigned to S. pseudintermedius by WIMP (6) were de novo assembled using of the Creative Commons Attribution 4.0 Flye 2.7.1 (7) (--nano-raw --genome-size 2.6m --plasmids --trestle). After minimap 2.17 International license. alignment (8), the resulting contigs were polished with Racon 1.4.13 (https://github.com/ Address correspondence to O. Francino, olga.francino@uab.cat. lbcb-sci/racon) and Medaka 1.0.3 (https://nanoporetech.github.io/medaka/) (medaka_con- Received 9 February 2021 sensus; -m r941_min_high_g360). Genome completeness was assessed with CheckM 1.1.1 Accepted 22 March 2021 (lineage_wf) (9). Circlator 1.5.5 was used to identify the origin (10) (fixstart --min_id 70). Published 22 April 2021 Genomes were annotated with NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (11). Volume 10 Issue 16 e00152-21 mra.asm.org 1 TABLE 1 Characteristics and accession numbers for high-quality genome assemblies from 61 Staphylococcus pseudintermedius isolatesa Genome Assembly ID N50 size (all Francino et al. Yr of Country of (accession Assembly Genome accession Genome No. of (contigs) contigs) No. of CDS COVf Compf Contf Isolate IDb MLSTc Sourced isolation isolation version no.) levele no. assembly contigs (bp) (bp) (total) (×) (%) (%) DG040 Unknown Cf, A, SP 2017 Italy GCF_016482145.1 Contig JAENDF000000000 Circular 3 2,527,299 2,564,892 2,369 664 99.43 0.00 DG050 Unknown Cf, A, SP 2016 Italy GCF_016482445.1 Contig JAENDE000000000 Circular 4 2,594,061 2,637,713 2,429 326 99.43 0.57 DG059 Unknown Cf, A, SP 2017 Italy GCF_016481825.1 Contig JAENDD000000000 Circular 2 2,568,059 2,573,568 2,364 223 99.29 0.00 Volume 10 Issue 16 e00152-21 DG062 ST71 Cf, A, SP 2016 Italy GCF_016482085.1 Contig JAENDC000000000 Linear 10 2,096,046 2,991,046 2,901 253 99.43 2.84 DG063 ST71 Cf, A, SP 2016 Italy GCF_016482045.1 Contig JAENDB000000000 Linear 10 2,099,753 2,981,523 2,891 256 99.43 0.57 DG064 ST71 Cf, A, SP 2017 Italy GCF_016455205.1 Compl CP066718 Circular 1 2,895,060 2,895,060 2,764 361 99.43 0.57 DG066 ST71 Cf, A, SP 2017 Italy GCF_016482585.1 Contig JAENDA000000000 Circular 3 2,804,246 2,808,032 2,639 213 99.43 0.57 DG067 ST71 Cf, A, SP 2017 Italy GCF_016482535.1 Contig JAENCZ000000000 Linear 3 2,890,387 2,896,399 2,776 533 99.43 0.57 DG071 ST71 Cf, A, SP 2012 Italy GCF_016482425.1 Contig JAENCY000000000 Linear 3 2,800,617 2,807,986 2,644 85 99.43 0.57 DG072 ST71 Cf, A, SP 2012 Italy GCF_016455165.1 Compl CP066717 Circular 1 2,837,133 2,837,133 2,690 304 99.43 0.57 DG076 ST258 Cf, A, SP 2012 Italy GCF_016482525.1 Contig JAENCX000000000 Circular 5 2,732,273 2,780,144 2,573 323 99.43 0.85 DG077 ST71 Cf, A, SP 2016 Italy GCF_016481985.1 Contig JAENCW000000000 Circular 4 2,789,419 2,791,496 2,624 479 99.43 0.57 DG078 ST71 Cf, A, SP 2012 Italy GCF_016481935.1 Contig JAENCV000000000 Circular 3 2,792,180 2,799,396 2,637 208 99.43 0.57 DG081 ST301 Cf, A, SP 2012 Italy GCF_016482005.1 Contig JAENCU000000000 Circular 3 2,676,449 2,694,747 2,519 353 99.43 0.00 DG082 ST258 Cf, A, SP 2017 Italy GCF_016481945.1 Contig JAENCT000000000 Circular 2 2,623,528 2,626,557 2,396 259 99.43 0.00 DG089 ST71 Cf, A, SP 2012 Italy GCF_016481925.1 Contig JAENCS000000000 Circular 2 2,793,862 2,797,937 2,628 281 99.43 0.57 DG093 ST258 Cf, A, SP 2017 Italy GCF_016481905.1 Contig JAENCR000000000 Circular 2 2,640,063 2,648,891 2,433 39 99.43 0.00 DG094 ST71 Cf, A, SP 2017 Italy GCF_016481855.1 Contig JAENCQ000000000 Circular 2 2,840,672 2,849,103 2,675 191 99.43 0.57 DG099 Unknown Cf, A, SP 2017 Italy GCF_016455185.1 Chrom CP066716 Linear 1 2,623,014 2,623,014 2,416 168 99.43 0.57 DSP020 ST71 Cf, A, SP 2013 Spain GCF_016455145.1 Compl CP066715 Circular 1 2,793,830 2,793,830 2,637 213 99.15 0.57 DSP021 ST71 Cf, A, SP 2013 Spain GCF_016455225.1 Compl CP066714 Circular 1 2,795,724 2,795,724 2,637 197 99.43 0.57 DSP022 Unknown Cf, A, SP 2014 Spain GCF_016481865.1 Contig JAENCP000000000 Circular 3 2,718,542 2,767,901 2,555 174 98.86 0.28 DSP024 ST611 Cf, A, SP 2014 Spain GCF_016482205.1 Contig JAENCO000000000 Circular 2 2,758,929 2,762,026 2,595 289 99.43 0.57 DSP025 ST71 Cf, A, SP 2014 Spain GCF_016482155.1 Contig JAENCN000000000 Circular 6 2,801,923 2,805,515 2,641 295 99.43 0.57 DSP026 ST503 Cf, A, SP 2014 Spain GCF_016455085.1 Compl CP066713 Circular 1 2,567,628 2,567,628 2,387 303 99.43 0.00 DSP027 Unknown Cf, A, SP 2014 Spain GCA_016455285.1 Compl CP066712 Circular 1 2,717,194 2,717,194 2,537 352 99.43 0.28 DSP028 Unknown Cf, A, SP 2019 Spain GCF_016482125.1 Contig JAENCM000000000 Circular 2 2,569,147 2,575,420 2,367 238 98.86 0.00 DSP029 ST258 Cf, A, SP 2019 Spain GCF_016481265.1 Contig JAENBQ000000000 Linear 8 2,413,604 2,723,805 2,517 270 99.43 0.00 DSP030 Unknown Cf, A, SP 2018 Argentina GCF_016455265.1 Compl CP066711 Circular 1 2,612,059 2,612,059 2,386 164 99.43 0.00 DSP032 ST1631 Cf, A, SP 2019 Argentina GCA_016482035.1 Contig JAENCL000000000 Linear 3 1,369,210 2,670,199 2,449 119 98.66 0.57 DSP034 ST1827 Cf, A, SP 2018 Argentina GCF_016455025.1 Compl CP066710 Circular 1 2,550,368 2,550,368 2,319 204 99.43 0.00 DSP035 Unknown Cf, A, SP 2019 Argentina GCF_016481845.1 Contig JAENCK000000000 Linear 2 2,640,488 2,642,865 2,489 117 99.43 0.00 DSP036 ST71 Cf, A, SP 2019 Spain GCF_016482105.1 Contig JAENCJ000000000 Circular 7 2,849,760 2,927,015 2,755 129 99.43 0.57 DHSP041 ST71 Cf, A, H 2019 Spain GCF_016482265.1 Contig JAENBW000000000 Linear 3 2,849,717 2,909,751 2,748 123 99.41 0.57 DHSP042 ST71 Cf, A, H 2019 Spain GCF_016482345.1 Contig JAENBV000000000 Linear 6 2,822,064 2,930,017 2,768 72 99.43 0.57 DHSP043 ST71 Cf, A, H 2019 Spain GCF_016482305.1 Contig JAENBU000000000 Linear 12 1,649,358 2,931,289 2,776 66 99.43 0.57 DHSP044 ST71 Cf, A, H 2019 Spain GCF_016482225.1 Contig JAENBT000000000 Circular 4 2,849,602 2,907,542 2,748 132 99.43 0.57 DHSP045 ST71 Cf, A, H 2019 Spain GCF_016482025.1 Contig JAENBS000000000 Circular 5 2,849,736 2,915,461 2,742 127 99.41 0.57 DHSP046 ST496 Cf, P, H 2019 Spain GCF_016481295.1 Contig JAENBR000000000 Circular 2 2,695,721 2,710,903 2,475 100 99.43 0.00 HSP079 Unknown Cf, P, H 2019 Spain GCF_016598895.1 Compl CP066885 Circular 1 2,585,691 2,587,693 2,337 301 99.43 0.00 HSP080 Unknown Cf, P, H 2019 Spain GCF_016598915.1 Compl CP066884 Circular 1 2,585,570 2,587,506 2,336 199 99.41 0.00 HSP081 Unknown Cf, P, H 2019 Spain GCA_016482135.1 Contig JAENCI000000000 Circular 4 2,617,897 2,621,254 2,467 94 98.58 1.14 (Continued on next page) mra.asm.org 2 TABLE 1 (Continued) Genome Assembly ID N50 size (all Yr of Country of (accession Assembly Genome accession Genome No. of (contigs) contigs) No. of CDS COVf Compf Contf b c d Isolate ID MLST Source isolation isolation version no.) levele no. assembly contigs (bp) (bp) (total) (×) (%) (%) HSP082 Unknown Cf, P, H 2019 Spain GCA_016481725.1 Contig JAENCH000000000 Circular 2 2,617,857 2,620,663 2,466 149 96.73 1.14 HSP093 Unknown Cf, A, H 2019 Spain GCF_016481735.1 Contig JAENCG000000000 Circular 3 2,571,836 2,590,335 2,361 274 99.43 0.57 HSP094 Unknown Cf, A, H 2019 Spain GCF_016481805.1 Contig JAENCF000000000 Circular 2 2,567,494 2,570,595 2,337 550 99.43 0.57 HSP095 Unknown Cf, A, H 2019 Spain GCF_016482325.1 Contig JAENCE000000000 Circular 2 2,570,328 2,575,879 2,360 341 99.43 0.57 Volume 10 Issue 16 e00152-21 HSP096 Unknown Cf, A, H 2019 Spain GCF_016482245.1 Contig JAENCD000000000 Circular 3 2,570,206 2,578,330 2,359 89 99.43 0.57 HSP097 Unknown Cf, A, H 2019 Spain GCF_016481665.1 Contig JAENCC000000000 Circular 3 2,570,223 2,575,223 2,356 136 99.43 0.57 Microbiology Resource Announcement HSP118 Unknown Cf, A, H 2019 Spain GCF_016455005.1 Compl CP066709 Circular 1 2,512,855 2,512,855 2,277 313 99.43 0.00 HSP125 ST1248 Cf, A, H 2019 Spain GCF_016455245.1 Compl CP066708 Circular 1 2,551,473 2,551,473 2,330 327 98.86 0.00 HSP127 ST1061 Cf, P, H 2019 Spain GCF_016481685.1 Contig JAENCB000000000 Circular 2 2,660,509 2,690,618 2,507 162 98.86 1.14 HSP132 Unknown Cf, P, H 2019 Spain GCA_016455125.1 Chrom CP066707 Linear 1 2,515,164 2,515,164 2,275 132 98.72 0.00 HSP134 Unknown Cf, P, H 2019 Spain GCA_016455105.1 Compl CP066706 Circular 1 2,514,594 2,514,594 2,274 146 98.86 0.00 HSP135 Unknown Cf, A, H 2019 Spain GCF_016455065.1 Compl CP066705 Circular 1 2,512,727 2,512,727 2,277 154 98.86 0.00 HSP136 Unknown Cf, A, H 2019 Spain GCA_016455045.1 Chrom CP066704 Linear 1 2,512,726 2,512,726 2,274 104 98.86 0.00 HSP137 Unknown Cf, A, H 2019 Spain GCF_016454985.1 Compl CP066703 Circular 1 2,512,757 2,512,757 2,271 224 99.43 0.00 HSP138 Unknown Cf, A, H 2019 Spain GCF_016454965.1 Compl CP066702 Circular 1 2,512,830 2,512,830 2,277 227 98.86 0.00 HSP140 Unknown Cf, P, H 2019 Spain GCF_016482365.1 Contig JAENCA000000000 Circular 2 2,594,004 2,597,272 2,387 296 99.24 0.00 HSP141 ST257 Cf, P, H 2019 Spain GCF_016482355.1 Contig JAENBZ000000000 Circular 4 2,615,633 2,622,529 2,450 150 99.43 0.57 HSP142 Unknown Cf, P, H 2019 Spain GCF_016482405.1 Contig JAENBY000000000 Circular 2 2,594,059 2,597,098 2,393 461 99.43 0.00 HSP143 Unknown Cf, P, H 2019 Spain GCF_016482235.1 Contig JAENBX000000000 Linear 6 2,537,079 2,779,670 2,589 293 98.86 0.57 a Obtained from the lesional skin of 33 dogs with pyoderma (33 D), the nonlesional skin of a dog with pyoderma (6 DH), and 6 healthy dogs (22 H). b ID, identifier. c MLST, multilocus sequence type; ST, sequence type. d Cf, Canis lupus familiaris; A, abdominal skin swab; P, perioral skin swab; SP, superficial pyoderma; H, healthy. e Compl, complete genome; Chrom, chromosome. f COV, coverage; Comp, completeness; Cont, contamination. mra.asm.org 3 Francino et al. Multilocus sequence types (MLSTs) were assigned with software MLST 2.0. and database 2.0.0 (https://cge.cbs.dtu.dk/services/MLST-2.0/) (12). Nanopore sequencing allowed successful de novo assembly and polishing of 61 S. pseudintermedius isolates (Table 1). The average read N50 value was 4,223.38 bp for 2,848,339.50 reads per flow cell (237,361.63 per barcoded sample). The mean genome coverage was 235 (39 to 664), with an average contig N50 value of 2.6 Mbp (1.6 Mbp to 2.9 Mbp). The average genome completeness was 99.25% (98.6% to 99.4%, except for HSP082), which is close to previous results with hybrid assemblies (13). The number of contigs per isolate ranged from 1 to 12 (median, 2). The main contig was cir- cular for 47 isolates. The average genome size of S. pseudintermedius was 2.70 Mbp (2.51 to 2.99 Mb), comprising 2,506 coding sequences (CDS; 2,271 to 2,901), 19 com- plete rRNAs (6 to 7 5S, 6 16S, and 6 23S rRNA genes), 56 to 59 tRNAs, and 4 noncoding RNAs (ncRNAs), as well as CRISPR arrays (0.5; range from 0 to 2). Pangenome analyses of isolates from healthy and diseased individuals will help unravel the differences, if any, that exist between commensal and pathogenic S. pseudintermedius populations. Data availability. The standardized strain descriptions and accession numbers are presented in Table 1; the genome sequence assemblies and genomic data are publicly available in DDBJ/ENA/GenBank under BioProject no. PRJNA685966 with the accession numbers CP066702 to CP066718, CP066884, CP066885, and JAENBQ000000000 to JAENDF000000000. The versions described in this paper are the first versions. The raw data are available from the Sequence Read Archive (SRA) under the same BioProject no., PRJNA685966. ACKNOWLEDGMENTS Funding sources were as follows: Research Project RTI2018-101991-B-100 (“From whole genome sequencing to clinical metagenomics: investigations on the pathogenesis of Staphylococcus pseudintermedius pyoderma in the dog”); Spanish Ministry of Science, Innovation and Universities; Industrial Doctorate Program grant 2017DI037 AGAUR; and Generalitat de Catalunya, Spain. The Spanish Ministry of Science and Innovation granted a Torres Quevedo Project to Vegenomics, S.L. with reference PTQ2018-009961 that is cofinanced by the European Social Fund. L.F. has received unrelated honoraria for lecturing from Zoetis, Bayer, LETI, and Affinity Petcare. O.F. and A.C. have received unrelated honoraria for lecturing from Oxford Nanopore Technologies. REFERENCES 1. Paul NC, Bärgman SC, Moodley A, Nielsen SS, Guardabassi L. 2012. Staphy- 8. 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