GENOME SEQUENCES Whole-Genome Sequencing and De Novo Assembly of 67 Staphylococcus pseudintermedius Strains Isolated from the Skin of Healthy Dogs Norma Fàbregas,a Daniel Pérez,b Joaquim Viñes,a Rocío Fonticoba,b Anna Cuscó,a Lourdes Migura-García,c Lluís Ferrer,b Olga Francinod a Vetgenomics, Edifici EUREKA, PRUAB, Campus UAB, Barcelona, Spain b Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Barcelona, Spain c IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Barcelona, Spain d SVGM, Molecular Genetics Veterinary Service, Universitat Autònoma de Barcelona, Barcelona, Spain Norma Fàbregas and Daniel Pérez contributed equally to this work. Author order was determined by seniority. ABSTRACT We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 Mbp size and 99.43% completeness, 2,386 coding sequences, 19 complete rRNAs, 59 tRNAs, and 4 noncoding RNAs. We released 51 single-contig com- plete genomes and 16 genomes with a circular main contig using Nanopore sequencing. S taphylococcus pseudintermedius is a commensal of the skin of dogs and the main causative agent of canine pyoderma (1), but populations inhabiting the skin of healthy dogs are largely unknown. The current paradigm indicates that infection arises when the skin barrier is altered by predisposing factors (2). We previously demon- strated that Nanopore sequencing allows de novo assembly of the entire genome of S. pseudintermedius (3). Here, we retrieved 67 S. pseudintermedius genomes from isolates from the skin of healthy dogs using long-read Nanopore sequencing. Samples were obtained by rubbing sterile swabs on four skin sites from nine healthy dogs—perinasal, perioral, inguinal, and perianal. After culture in blood agar at 37°C for 24 h, colonies grown with the morphology of S. pseudintermedius (small silver colonies) were seeded in 3 mL of brain heart infusion (BHI) at 37°C for 16 h. DNA was extracted with a ZymoBIOMICS DNA miniprep kit (Zymo Research). DNA quality and quantity were determined using a NanoDrop 2000 instrument and a Qubit double-stranded DNA (dsDNA) broad range (BR) assay kit (Fisher Scientific). The sequencing libraries were prepared with the rapid barcoding sequencing kit (SQK-RBK004; Oxford Nanopore Technologies [ONT]). Up to 12 bar- coded samples were loaded in a MinION FLO-MIN106 9.4.1 flow cell and sequenced in a MinION Mk1B or Mk1C device (ONT). The fast5 files were base- Editor Steven R. Gill, University of Rochester called and demultiplexed, and adapters were trimmed with Guppy 5.0.11 (4) (ONT) School of Medicine and Dentistry (--dna_r9.4.1_450bps_sup.cfg) (--config configuration.cfg --barcode_kits SQK- Copyright © 2022 Fàbregas et al. This is an RBK004 --trim_barcodes; min_score threshold default 60). Reads with a quality open-access article distributed under the terms of the Creative Commons Attribution 4.0 score lower than 10 were discarded. Run summary statistics were obtained with International license. Nanoplot 1.38.1 (5) (--N50 --fastq). Address correspondence to Olga Francino, Isolates were confirmed as S. pseudintermedius by EPI2ME WIMP workflow (6). Genomes olga.francino@uab.cat, or Norma Fàbregas, norma.fabregas@vetgenomics.com. were de novo assembled using Flye 2.8.3 (7) (--nano-raw --plasmids --trestle), except HSP279 The authors declare a conflict of interest. and HSP281, which were assembled with Flye 2.9 (--nano-hq). Contigs were polished with Received 15 January 2022 medaka 1.4.3 (8) (medaka_consensus; -m r941_min_sup_g507). Genome completeness and Accepted 15 February 2022 contamination were assessed with CheckM 1.1.3 (lineage_wf) (9). Circlator 1.5.5 was used to Published 2 March 2022 rotate the genomes, fixing the start with the dnaA gene (10) (fixstart --min_id 70). Genomes March 2022 Volume 11 Issue 3 10.1128/mra.00039-22 1 TABLE 1 Characteristics and accession numbers of high-quality genome assemblies from 67 Staphylococcus pseudintermedius isolates from 4 different skin sites of 9 healthy dogsa Assembly ID Genome size No. of Yr of Country of (accession version Assembly Genome Genome No. of N50 contigs of all contigs CDS Cov Comp Cont GC content ID MLST Source isolation isolation no.) level accession no. assembly contigs (bp) (bp) (total) (×) (%) (%) (%) Announcement HSP147 45b Cf, I, H 2020 Spain GCA_019997105.1 Compl CP083230 Circular 1 2,596,738 2,596,738 2,379 199 97.96 0.57 37.6 HSP149 45b Cf, I, H 2020 Spain GCA_019997905.1 Compl CP083229 Circularc 1 2,592,238 2,592,238 2,384 90 97.82 0.57 37.6 HSP151 45b Cf, Pa, H 2020 Spain GCA_019997185.1 Compl CP083231 Circularc 1 2,596,528 2,596,528 2,380 134 97.96 0.57 37.6 HSP152 45b Cf, Pa, H 2020 Spain GCA_019997145.1 Compl CP083228 Circular 1 2,596,499 2,596,499 2,386 78 96.92 0.57 37.6 HSP153 402b Cf, Pa, H 2020 Spain GCA_019997125.1 Compl CP083227 Circularc 1 2,596,563 2,596,563 2,387 113 97.68 0.57 37.6 March 2022 Volume 11 Issue 3 b HSP154 45 Cf, Pa, H 2020 Spain GCA_019997265.1 Compl CP083226 Circular 1 2,596,624 2,596,624 2,376 53 97.96 0.57 37.6 HSP155 45b Cf, Pa, H 2020 Spain GCA_019998705.1 Compl CP083225 Circular 1 2,596,628 2,596,628 2,371 158 98.77 0.57 37.6 HSP156 45b Cf, P, H 2020 Spain GCA_019997565.1 Compl CP083224 Circularc 1 2,596,695 2,596,695 2,374 132 98.11 0.57 37.6 HSP157 45 Cf, P, H 2020 Spain GCA_019998065.1 Compl CP083223 Circularc 1 2,596,646 2,596,646 2,381 151 98.25 0.57 37.6 HSP158 45b Cf, P, H 2020 Spain GCA_019997985.1 Compl CP083222 Circular 1 2,596,630 2,596,630 2,391 120 97.85 0.57 37.6 HSP159 45b Cf, P, H 2020 Spain GCA_019998395.1 Compl CP083221 Circular 1 2,596,316 2,596,316 2,377 90 97.66 0.57 37.6 HSP160 45b Cf, P, H 2020 Spain GCA_019998665.1 Compl CP083220 Circular 1 2,596,481 2,596,481 2,388 38 97.57 0.57 37.6 HSP166 45b Cf, Pn, H 2020 Spain GCA_019997325.1 Compl CP083219 Circular 1 2,596,789 2,596,789 2,375 148 97.82 0.57 37.6 HSP199 2189 Cf, Pa, H 2020 Spain GCA_019998005.1 Compl CP083218 Circular 1 2,574,155 2,574,155 2,380 33 99.29 0.00 37.6 HSP204 2190 Cf, Pa, H 2020 Spain GCA_019997445.1 Compl CP083217 Circular 1 2,470,602 2,470,602 2,260 33 98.30 0.00 37.8 HSP210 2191 Cf, P, H 2020 Spain GCA_019998825.1 Compl CP083216 Circular 1 2,472,661 2,472,661 2,249 191 99.43 0.00 37.8 HSP211 2175 Cf, P, H 2020 Spain GCA_019998805.1 Compl CP083215 Circular 1 2,472,653 2,472,653 2,249 136 99.43 0.00 37.8 HSP212 2175 Cf, P, H 2020 Spain GCA_019998765.1 Compl CP083214 Circular 1 2,472,754 2,472,754 2,257 158 99.43 0.00 37.8 HSP213 2175 Cf, P, H 2020 Spain GCA_019998525.1 Compl CP083213 Circular 1 2,472,662 2,472,662 2,247 398 99.43 0.00 37.8 HSP214 2192 Cf, Pn, H 2020 Spain GCA_019998185.1 Compl CP083212 Circular 1 2,524,384 2,524,384 2,319 119 99.43 0.00 37.8 HSP216 2176 Cf, Pn, H 2020 Spain GCA_019998745.1 Compl CP083211 Circular 1 2,611,897 2,611,897 2,403 97 99.43 0.57 37.6 HSP224 2177 Cf, P, H 2020 Spain GCA_019998685.1 Compl CP083210 Circular 1 2,711,397 2,711,397 2,501 88 99.43 0.57 37.6 HSP225 2177 Cf, P, H 2020 Spain GCA_019998785.1 Compl CP083209 Circular 1 2,752,898 2,752,898 2,571 289 99.43 0.57 37.6 HSP226 2177 Cf, P, H 2020 Spain GCA_019997165.1 Compl CP083208 Circular 1 2,750,631 2,750,631 2,567 227 99.43 0.57 37.6 HSP227 2177 Cf, P, H 2020 Spain GCA_019996845.1 Compl CP083207 Circular 1 2,750,563 2,750,563 2,568 212 99.43 0.57 37.6 HSP228 2177 Cf, P, H 2020 Spain GCA_019997925.1 Compl CP083206 Circular 1 2,750,560 2,750,560 2,568 195 99.43 0.57 37.6 HSP232 2178 Cf, Pa, H 2020 Spain GCA_020685925.1 Contig JAJEKF000000000 Circular 2 2,598,204 2,607,192 2,403 220 99.43 0.00 37.6 HSP235 551 Cf, Pn, H 2020 Spain GCA_020685885.1 Contig JAJEKT000000000 Circular 2 2,870,862 2,873,604 2,791 312 99.43 1.14 37.2 HSP236 551 Cf, Pn, H 2020 Spain GCA_020685905.1 Contig JAJEKS000000000 Circular 2 2,876,047 2,878,792 2,790 126 99.43 1.14 37.3 HSP237 551 Cf, Pn, H 2020 Spain GCA_020685685.1 Contig JAJEKR000000000 Circular 2 2,829,605 2,835,087 2,727 157 99.43 1.14 37.3 HSP238 45b Cf, Pn, H 2020 Spain GCA_019997285.1 Compl CP083205 Circular 1 2,646,644 2,646,644 2,453 272 96.88 0.57 37.5 HSP239 45b Cf, Pn, H 2020 Spain GCA_019997805.1 Compl CP083204 Circular 1 2,647,982 2,647,982 2,450 265 98.06 0.57 37.5 HSP240 551 Cf, P, H 2020 Spain GCA_019998365.1 Compl CP083203 Circular 1 2,870,858 2,870,858 2,760 169 99.43 1.14 37.2 HSP241 551 Cf, P, H 2020 Spain GCA_020685845.1 Contig JAJEKQ000000000 Circular 2 2,870,853 2,873,594 2,784 181 99.43 1.14 37.2 HSP242 551 Cf, P, H 2020 Spain GCA_020685645.1 Contig JAJEKP000000000 Circular 2 2,870,853 2,876,339 2,794 182 99.43 1.14 37.2 HSP243 551 Cf, P, H 2020 Spain GCA_020685745.1 Contig JAJEKO000000000 Circular 2 2,870,749 2,876,235 2,779 266 99.43 1.14 37.2 HSP244 551 Cf, P, H 2020 Spain GCA_020685805.1 Contig JAJEKN000000000 Circular 2 2,870,863 2,876,349 2,805 204 99.43 1.14 37.2 HSP245 2178 Cf, Pa, H 2020 Spain GCA_020685785.1 Contig JAJEKM000000000 Circular 3 2,598,196 2,609,478 2,399 198 99.43 0.00 37.6 HSP246 2178 Cf, Pa, H 2020 Spain GCA_020685705.1 Contig JAJEKL000000000 Circular 2 2,597,938 2,600,981 2,397 137 99.43 0.00 37.6 HSP249 2178 Cf, Pa, H 2020 Spain GCA_020685725.1 Contig JAJEKK000000000 Circular 2 2,597,934 2,600,977 2,396 148 99.43 0.00 37.6 HSP250 2178 Cf, I, H 2020 Spain GCA_020685625.1 Contig JAJEKJ000000000 Circular 2 2,598,182 2,601,232 2,405 175 99.43 0.00 37.6 HSP251 2178 Cf, I, H 2020 Spain GCA_020685865.1 Contig JAJEKI000000000 Circular 2 2,598,180 2,602,649 2,400 158 99.43 0.00 37.6 HSP252 2178 Cf, I, H 2020 Spain GCA_019997885.1 Compl CP083202 Circular 1 2,598,184 2,598,184 2,380 176 99.43 0.00 37.6 10.1128/mra.00039-22 HSP253 2178 Cf, I, H 2020 Spain GCA_020685765.1 Contig JAJEKH000000000 Circular 2 2,598,192 2,604,278 2,398 206 99.43 0.00 37.6 HSP255 1026 Cf, Pn, H 2020 Spain GCA_019997765.1 Compl CP083201 Circular 1 2,599,010 2,599,010 2,377 131 98.86 0.00 37.6 Microbiology Resource Announcements 2 (Continued on next page) TABLE 1 (Continued) Assembly ID Genome size No. of Yr of Country of (accession version Assembly Genome Genome No. of N50 contigs of all contigs CDS Cov Comp Cont GC content ID MLST Source isolation isolation no.) level accession no. assembly contigs (bp) (bp) (total) (×) (%) (%) (%) Announcement HSP258 1026 Cf, P, H 2020 Spain GCA_019997205.1 Compl CP083200 Circular 1 2,599,012 2,599,012 2,370 120 98.30 0.00 37.6 HSP259 1026 Cf, P, H 2020 Spain GCA_019997005.1 Compl CP083199 Circular 1 2,599,031 2,599,031 2,383 155 98.86 0.00 37.6 HSP260 1026 Cf, P, H 2020 Spain GCA_019997245.1 Compl CP083198 Circular 1 2,599,024 2,599,024 2,375 71 99.24 0.00 37.6 HSP261 1026 Cf, P, H 2020 Spain GCA_019997785.1 Compl CP083197 Circular 1 2,599,041 2,599,041 2,372 145 99.43 0.00 37.6 HSP262 1026 Cf, P, H 2020 Spain GCA_019997825.1 Compl CP083196 Circular 1 2,599,036 2,599,036 2,382 147 99.43 0.00 37.6 March 2022 Volume 11 Issue 3 HSP263 1026 Cf, I, H 2020 Spain GCA_019997065.1 Compl CP083195 Circular 1 2,599,019 2,599,019 2,371 125 99.43 0.00 37.6 HSP264 1026 Cf, I, H 2020 Spain GCA_019997705.1 Compl CP083194 Circular 1 2,599,017 2,599,017 2,384 155 98.86 0.00 37.6 HSP265 1026 Cf, I, H 2020 Spain GCA_019997085.1 Compl CP083193 Circular 1 2,599,016 2,599,016 2,369 109 99.43 0.00 37.6 HSP266 1026 Cf, I, H 2020 Spain GCA_019997845.1 Compl CP083192 Circular 1 2,599,010 2,599,010 2,371 204 99.43 0.00 37.6 HSP267 1026 Cf, I, H 2020 Spain GCA_019997625.1 Compl CP083191 Circular 1 2,598,988 2,598,988 2,374 91 99.43 0.00 37.6 HSP274 2179 Cf, P, H 2020 Spain GCA_019998045.1 Compl CP083190 Circular 1 2,587,513 2,587,513 2,394 230 97.82 0.00 37.6 HSP276 2179 Cf, P, H 2020 Spain GCA_019998025.1 Compl CP083189 Circular 1 2,587,526 2,587,526 2,386 219 98.67 0.00 37.6 HSP277 2179 Cf, P, H 2020 Spain GCA_019997945.1 Compl CP083188 Circular 1 2,587,535 2,587,535 2,391 316 98.30 0.00 37.6 HSP278 2179 Cf, Pa, H 2020 Spain GCA_019997225.1 Compl CP083187 Circular 1 2,587,540 2,587,540 2,386 196 98.30 0.00 37.6 HSP279 Unknownb Cf, Pa, H 2020 Spain GCA_020693965.1 Compl CP085724 Circularc 1 2,575,504 2,575,504 2,436 139 98.56 0.00 37.8 HSP280 Unknownb Cf, Pa, H 2020 Spain GCA_019997045.1 Compl CP083186 Circularc 1 2,575,827 2,575,827 2,374 137 99.41 0.00 37.8 HSP281 Unknownb Cf, Pa, H 2020 Spain GCA_020693985.1 Compl CP085723 Circular 1 2,575,390 2,575,390 2,441 274 99.13 0.00 37.8 HSP282 Unknownb Cf, Pa, H 2020 Spain GCA_019998285.1 Compl CP083185 Circularc 1 2,575,851 2,575,851 2,381 170 99.15 0.00 37.8 HSP283 294 Cf, I, H 2020 Spain GCA_019997965.1 Compl CP083184 Circular 1 2,607,096 2,607,096 2,399 154 99.43 0.00 37.6 HSP284 2180 Cf, I, H 2020 Spain GCA_019996945.1 Compl CP083183 Circular 1 2,568,734 2,568,734 2,361 113 99.43 1.14 37.6 HSP285 2181 Cf, Pn, H 2020 Spain GCA_020685605.1 Contig JAJEKG000000000 Circular 2 2,642,410 2,646,739 2,467 127 99.43 0.00 37.5 HSP286 2181 Cf, Pn, H 2020 Spain GCA_020686685.1 Contig JAJEKF000000000 Circular 4 2,642,415 2,650,568 2,463 144 99.43 0.00 37.5 a ID, identifier; MLST, multilocus sequence type; Cf, Canis lupus familiaris; P, perioral; Pa, perianal; I, inguinal; Pn, perinasal; H, healthy; Compl, complete genome; CDS (total), total number of coding sequences; Cov, coverage; Comp, completeness; Cont, contamination. b MLST alleles with mutations that have not been reported. c Genomes where the dnaA gene is not detected start with a random ATG gene. 10.1128/mra.00039-22 Microbiology Resource Announcements 3 Announcement Microbiology Resource Announcements were annotated with the NCBI Prokaryotic Genome Annotation Pipeline 5.2 and 5.3 (11). Multilocus sequence types (MLSTs) were assigned with PubMLST (https://pubmlst.org/; accessed November 2021) (12). Nanopore sequencing allowed successful de novo assembly and polishing of 67 S. pseudintermedius genomes (Table 1). The average read N50 value was 5,270.77 bp with 133,226.86 reads per sample. The median values were 154 coverage (33 to 398), 99.43% completeness (96.9 to 99.4%) as in previous hybrid assemblies (13), 2.6 Mbp for contig N50 value and genome size (2.5 to 2.9 Mbp), 37.6% GC content (37.2 to 37.8%), 2,386 coding DNA sequences (CDS) (2,247 to 2,805), 19 complete rRNAs (6 to 8 5S, 5 to 7 26S, and 5 to 8 23S), 59 tRNAs (58 to 61), and 4 noncoding RNAs (ncRNAs). A total of 51 bacterial isolates were assembled into single-circular-contig complete genomes; 16 isolates were assembled into a main circular contig corresponding to the bacterial chromosome and smaller contigs containing replicon proteins supporting their plasmid origin. Together with our previous study (3), we have announced 95 S. pseudintermedius genomes from the skin of healthy dogs, contributing to a better understanding of S. pseudintermedius pathogenesis. Data availability. The standardized strain descriptions and accession numbers are pre- sented in Table 1; the genome assemblies, genomic data, and raw data are publicly avail- able in GenBank under BioProject PRJNA685966 and genome accession numbers CP083183 to CP083231, CP085723, CP085724, and JAJEKF000000000 to JAJEKU000000000. ACKNOWLEDGMENTS Funding sources were as follows: Research Project RTI2018-101991-B-I00 (“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; Torres Quevedo Project, PTQ2018-009961, cofinanced by the European Social Fund and the Spanish Ministry of Science and Innovation; and Industrial Doctorate Program grant 2017DI037 AGAUR, Generalitat de Catalunya, Spain. 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. Bannoehr J, Guardabassi L. 2012. Staphylococcus pseudintermedius in the dog: prone reads using repeat graphs. Nat Biotechnol 37:540–549. https://doi taxonomy, diagnostics, ecology, epidemiology and pathogenicity. 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