We present the draft genome sequences of two clinical strains of Aspergillus turcosus, one azole-susceptible (strain HMR-AF-23/LSPQ-01275) and the other azole-resistant (strain HMR-AF-1038/LSPQ-01280), isolated from bronchoalveolar lavage fluid of two adult patients. These two strains are the first reported clinical isolates of A. turcosus.
ABSTRACT
We present the draft genome sequences of two clinical strains of Aspergillus turcosus, one azole-susceptible (strain HMR-AF-23/LSPQ-01275) and the other azole-resistant (strain HMR-AF-1038/LSPQ-01280), isolated from bronchoalveolar lavage fluid of two adult patients. These two strains are the first reported clinical isolates of A. turcosus.
ANNOUNCEMENT
Invasive aspergillosis is a serious fungal infection of major concern in immunocompromised patients and is associated with a high mortality rate (1–4). Aspergillus fumigatus is the most frequent species causing invasive aspergillosis. Its genome was completely sequenced in 2005, along with A. nidulans and A. oryzae (5–7). Aspergillus section Fumigati comprises about 60 species (8), often misidentified as A. fumigatus by morphotyping (9–12). First described in 2008, Aspergillus turcosus is a rare species belonging to this section (13). The two isolates whose complete genomes are presented herein are the first reported clinical isolates and the first occurrence of this species outside South Korea.
Four A. turcosus were identified among a collection of 1,186 presumed A. fumigatus clinical isolates collected at Maisonneuve-Rosemont Hospital in Montréal, Québec, Canada. Identification was based on phenotypic characteristics and confirmed by sequencing the following three relevant loci: beta-tubulin, calmodulin, and internal transcribed spacer (13, 14). Antifungal susceptibility testing, performed according to the Clinical and Laboratory Standards Institute (CLSI) broth microdilution procedure (15), revealed reduced susceptibility of strain HMR-AF-1038 to voriconazole, itraconazole, and posaconazole, compared to wild-type A. fumigatus (16). We sequenced its whole genome, along with that of a randomly selected azole-susceptible A. turcosus isolate (HMR-AF-23), for comparison. HMR-AF-23 and HMR-AF-1038 were isolated from bronchoalveolar lavage fluid samples in 2000 and 2012, respectively.
Whole-genome sequencing was performed at the provincial public health laboratory. Isolates were grown on potato dextrose agar at 30°C for 4 to 7 days, then DNA was extracted from conidia with the ZR fungal/bacterial DNA miniprep kit (Zymo Research Corp.). The Nextera XT library preparation kit (Illumina, Inc.) was used to prepare sequencing libraries for both strains. They were then sequenced with a MiSeq 600-cycle sequencing kit v3 as paired-end reads (2 × 300 bp) on a MiSeq sequencing platform (Illumina). HMR-AF-23 generated 25,658,892 raw reads, and the mean per-base sequence depth reached 85×. HMR-AF-1038 generated 25,586,278 raw reads for a 42× per-base mean coverage. Reads were quality and quantity controlled with FastQC version 0.11.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then cleaned with Trimmomatic v0.36 (parameters, LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:50 ILLUMINACLIP:NexteraPE-PE.fa:2:30:10) (17). Mitochondrial sequences were discarded by mapping the cleaned reads onto the A. fumigatus mitochondrial genome (reference genome JQ346807) with SMALT v0.7.6. Remaining nuclear reads were assembled with SPAdes v3.7.0 (18) on Calcul Québec servers (http://www.calculquebec.ca/). An in-house Python script (https://github.com/EricFournier3/PythonGenomicTools) was used to eliminate contigs with coverage lower than 5× and length less than 2 kb. The quality of the assembly was further assessed with QUAST 4.1 (19). The absence of duplicated contigs was ensured by running dedup.sh (https://jgi.doe.gov/data-and-tools/bbtools/), and the absence of Illumina adapters was checked with the Linux grep command. A total of 939 contigs for HMR-AF-23 (N50 = 110,871 bp) and 1,043 contigs for HMR-AF-1038 (N50 = 95,868 bp) were found. The overall genome size and G+C content for HMR-AF-23 and HMR-AF-1038 are 32,352,689 bp and 48% and 34,076,230 bp and 46%, respectively.
Genome annotation was performed with funannotate v1.4.2 (https://github.com/nextgenusfs/funannotate). We used an Augustus (20) pretrained gene model of A. fumigatus to predict 9,223 genes for HMR-AF-23, including 833 with functional annotation. For HMR-AF-1038, 9,174 genes were predicted, from which 830 have functional annotation. The completeness of these annotations was assessed by comparing the number of conserved orthologs to those of A. fumigatus, the closest reference species, using the tool BUSCO v2.0 (21) run with the Dikarya data set. Approximately 94% orthologs were found for both isolates, which is nearly as many as for A. fumigatus (96%). We found 1,232 and 36 complete and fragmented orthologs, respectively, for HMR-AF-23, compared to 1,227 and 39, respectively, for HMR-AF-1038.
These first draft genome sequences of A. turcosus may be useful in deciphering the genetic differences which result in elevated azole resistance for this species and allow a better understanding of virulence through genomic comparison with other related clinically relevant Aspergillus species.
Data availability.
These whole-genome shotgun projects have been deposited in GenBank under the accession numbers NKHV00000000 for strain HMR-AF-23 and NIDN00000000 for strain HMR-AF-1038. The versions described in this paper are the second versions, NKHV02000000 and NIDN02000000, respectively. Raw sequence data can be accessed using the following run numbers: SRR8165489 (HMR-AF-23) and SRR8165475 (HMR-AF-1038).
ACKNOWLEDGMENT
M.P.-M. received the Gabriel-Marquis scholarship from the University of Montréal.
REFERENCES
- 1.Neofytos D, Horn D, Anaissie E, Steinbach W, Olyaei A, Fishman J, Pfaller M, Chang C, Webster K, Marr K. 2009. Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of multicenter Prospective Antifungal Therapy (PATH) Alliance registry. Clin Infect Dis 48:265–273. doi: 10.1086/595846. [DOI] [PubMed] [Google Scholar]
- 2.Pappas PG, Alexander BD, Andes DR, Hadley S, Kauffman CA, Freifeld A, Anaissie EJ, Brumble LM, Herwaldt L, Ito J, Kontoyiannis DP, Lyon GM, Marr KA, Morrison VA, Park BJ, Patterson TF, Perl TM, Oster RA, Schuster MG, Walker R, Walsh TJ, Wannemuehler KA, Chiller TM. 2010. Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Clin Infect Dis 50:1101–1111. doi: 10.1086/651262. [DOI] [PubMed] [Google Scholar]
- 3.Kontoyiannis DP, Marr KA, Park BJ, Alexander BD, Anaissie EJ, Walsh TJ, Ito J, Andes DR, Baddley JW, Brown JM, Brumble LM, Freifeld AG, Hadley S, Herwaldt LA, Kauffman CA, Knapp K, Lyon GM, Morrison VA, Papanicolaou G, Patterson TF, Perl TM, Schuster MG, Walker R, Wannemuehler KA, Wingard JR, Chiller TM, Pappas PG. 2010. Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001–2006: overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) database. Clin Infect Dis 50:1091–1100. doi: 10.1086/651263. [DOI] [PubMed] [Google Scholar]
- 4.Steinbach WJ, Marr KA, Anaissie EJ, Azie N, Quan SP, Meier-Kriesche H-U, Apewokin S, Horn DL. 2012. Clinical epidemiology of 960 patients with invasive aspergillosis from the PATH Alliance registry. J Infect 65:453–464. doi: 10.1016/j.jinf.2012.08.003. [DOI] [PubMed] [Google Scholar]
- 5.Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, Arroyo J, Berriman M, Abe K, Archer DB, Bermejo C, Bennett J, Bowyer P, Chen D, Collins M, Coulsen R, Davies R, Dyer PS, Farman M, Fedorova N, Fedorova N, Feldblyum TV, Fischer R, Fosker N, Fraser A, García JL, García MJ, Goble A, Goldman GH, Gomi K, Griffith-Jones S, Gwilliam R, Haas B, Haas H, Harris D, Horiuchi H, Huang J, Humphray S, Jiménez J, Keller N, Khouri H, Kitamoto K, Kobayashi T, Konzack S, Kulkarni R, Kumagai T, Lafon A, Lafton A, Latgé J-P, Li W, Lord A, et al. . 2005. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151–1156. doi: 10.1038/nature04332. [DOI] [PubMed] [Google Scholar]
- 6.Galagan JE, Calvo SE, Cuomo C, Ma L-J, Wortman JR, Batzoglou S, Lee S-I, Baştürkmen M, Spevak CC, Clutterbuck J, Kapitonov V, Jurka J, Scazzocchio C, Farman M, Butler J, Purcell S, Harris S, Braus GH, Draht O, Busch S, D'Enfert C, Bouchier C, Goldman GH, Bell-Pedersen D, Griffiths-Jones S, Doonan JH, Yu J, Vienken K, Pain A, Freitag M, Selker EU, Archer DB, Peñalva MÁ, Oakley BR, Momany M, Tanaka T, Kumagai T, Asai K, Machida M, Nierman WC, Denning DW, Caddick M, Hynes M, Paoletti M, Fischer R, Miller B, Dyer P, Sachs MS, Osmani SA, Birren BW. 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438:1105–1115. doi: 10.1038/nature04341. [DOI] [PubMed] [Google Scholar]
- 7.Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K-I, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J-i, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H. 2005. Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161. doi: 10.1038/nature04300. [DOI] [PubMed] [Google Scholar]
- 8.Samson RA, Hong S, Peterson SW, Frisvad JC, Varga J. 2007. Polyphasic taxonomy of Aspergillus section Fumigati and its teleomorph Neosartorya. Stud Mycol 59:147–203. doi: 10.3114/sim.2007.59.14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Balajee SA, Gribskov J, Brandt M, Ito J, Fothergill A, Marr KA. 2005. Mistaken identity: Neosartorya pseudofischeri and its anamorph masquerading as Aspergillus fumigatus. J Clin Microbiol 43:5996–5999. doi: 10.1128/JCM.43.12.5996-5999.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Balajee SA, Nickle D, Varga J, Marr KA. 2006. Molecular studies reveal frequent misidentification of Aspergillus fumigatus by morphotyping. Eukaryot Cell 5:1705–1712. doi: 10.1128/EC.00162-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Balajee SA, Kano R, Baddley JW, Moser SA, Marr KA, Alexander BD, Andes D, Kontoyiannis DP, Perrone G, Peterson S, Brandt ME, Pappas PG, Chiller T. 2009. Molecular identification of Aspergillus species collected for the Transplant-Associated Infection Surveillance Network. J Clin Microbiol 47:3138–3141. doi: 10.1128/JCM.01070-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Katz ME, Dougall AM, Weeks K, Cheetham BF. 2005. Multiple genetically distinct groups revealed among clinical isolates identified as atypical Aspergillus fumigatus. J Clin Microbiol 43:551–555. doi: 10.1128/JCM.43.2.551-555.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Hong S-B, Shin H-D, Hong J, Frisvad JC, Nielsen PV, Varga J, Samson RA. 2008. New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati. Antonie Van Leeuwenhoek 93:87–98. doi: 10.1007/s10482-007-9183-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Samson RA, Visagie CM, Houbraken J, Hong SB, Hubka V, Klaassen CHW, Perrone G, Seifert KA, Susca A, Tanney JB, Varga J, Kocsubé S, Szigeti G, Yaguchi T, Frisvad JC. 2014. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol 78:141–173. doi: 10.1016/j.simyco.2014.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.CLSI. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard, 2nd ed CLSI Document MM38-A2. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 16.Espinel-Ingroff A, Diekema DJ, Fothergill A, Johnson E, Pelaez T, Pfaller MA, Rinaldi MG, Canton E, Turnidge J. 2010. Wild-type MIC distributions and epidemiological cutoff values for the triazoles and six Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document). J Clin Microbiol 48:3251–3257. doi: 10.1128/JCM.00536-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Nurk S, Bankevich A, Antipov D, Gurevich A, Korobeynikov A, Lapidus A, Prjibelsky A, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, McLean J, Lasken R, Clingenpeel SR, Woyke T, Tesler G, Alekseyev MA, Pevzner PA. 2013. Assembling genomes and mini-metagenomes from highly chimeric reads, p 158–170. In Deng M, Jiang R, Sun F, Zhang X (ed), Research in computational molecular biology: 17th annual international conference, RECOMB 2013, Beijing, China, April 7–10, 2013 proceedings. Springer, Berlin, Heidelberg. doi: 10.1007/978-3-642-37195-0_13. [DOI] [Google Scholar]
- 19.Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Stanke M, Schöffmann O, Morgenstern B, Waack S. 2006. Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics 7:62. doi: 10.1186/1471-2105-7-62. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31:3210–3212. doi: 10.1093/bioinformatics/btv351. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
These whole-genome shotgun projects have been deposited in GenBank under the accession numbers NKHV00000000 for strain HMR-AF-23 and NIDN00000000 for strain HMR-AF-1038. The versions described in this paper are the second versions, NKHV02000000 and NIDN02000000, respectively. Raw sequence data can be accessed using the following run numbers: SRR8165489 (HMR-AF-23) and SRR8165475 (HMR-AF-1038).