Abstract
This study reports the genomic insights into a Streptococcus pneumoniae serotype 17F isolate in Malaysia (IMRCC/SS430/25), collected from an adult patient with aspiration pneumonia in 2025. Phenotypic identification revealed characteristic alpha-haemolytic colonies and optochin susceptibility. Species identity as S. pneumoniae was further verified by bile solubility testing and the 17F serotype was confirmed using the Quellung reaction. The isolate was subjected to whole-genome sequencing using the Oxford Nanopore platform. The assembly yielded a highly accurate, single circular contig of 2,065,967 bp with 100% completeness and 48× coverage. In silico typing assigned the genome to ST2355 within the globally disseminated GPSC10 cluster. Antimicrobial susceptibility testing demonstrated resistance to erythromycin, azithromycin, clindamycin, and tetracycline. This phenotypic profile was concordant with genomic screening which identified the ermB and tetM_8 genes responsible for macrolide-lincosamide-streptogramin B (MLSB) and tetracycline resistance respectively. The penicillin-binding protein allelic profile (pbp1a: 79, pbp2b: 26, pbp2x: 194) predicted uniform susceptibility to beta-lactam antibiotics shown by phenotypic testing of amoxicillin-clavulanate and penicillin. Phylogenomic characterization identified the Malaysian isolate within the predominant global ST2355 clade of GPSC10, clustering alongside isolates from Nepal, the United Kingdom, Bangladesh, and the United States. This study demonstrates the utility of combining routine clinical microbiology with high-resolution genomic surveillance to resolve lineage relationship, reveal underlying resistance mechanisms, and confirm genotype-phenotype concordance.
Keywords: Streptococcus pneumoniae, Serotype 17F, Whole genome sequencing, Genomic characterization, Malaysia
Specifications Table
| Subject | Biology |
| Specific subject area | Genomics. |
| Type of data | Raw genome sequence data in FASTQ format. |
| Data collection | Genome sequencing platform: Oxford Nanopore Genomic assembly: EPI2ME wf-bacterialgenomes Genomic analysis: Pathogenwatch (https://pathogen.watch/) Parameters for data collection: Genomic DNA was extracted from purified cultures of S. pneumoniae. Description of data collection: Whole genome sequencing, assembly, annotation, genomic analysis |
| Data source location | Institute for Medical Research, National Institutes of Health Malaysia, Persiaran Setia Murni U13/52, Setia Alam, 40170 Shah Alam, Malaysia. |
| Data accessibility | Repository name: National Center of Biotechnology Information (NCBI) Data identification number: BioProject database: PRJNA1245582, Sequence Read Archive (SRA) database: SRR37999637 Direct URL to data: https://trace.ncbi.nlm.nih.gov/Traces?run=SRR37999637 |
| Related research article | None |
1. Value of the Data
-
•
Data provides the genomic information of S. pneumoniae serotype 17F from a clinical isolate in Malaysia.
-
•
Data characterise the antibiotic resistome of the S. pneumoniae serotype 17F strain and provide baseline information on its antimicrobial resistance burden in Malaysia.
-
•
Data support broader genome studies of S. pneumoniae circulating among patients in Malaysia.
2. Background
Streptococcus pneumoniae (pneumococcus) is a major human pathogen that colonizes the upper respiratory tract, and causes both invasive and non-invasive diseases, contributing significantly to global morbidity and mortality [1]. S. pneumoniae was previously estimated to be the leading cause of non-COVID lower respiratory tract infections (LRTI) globally and in Malaysia [2]. To reduce disease burden, pneumococcal conjugate vaccines (PCVs) have been administered to children born since 2020 through the Malaysian National Immunization Programme (NIP) [3]. These vaccines target specific capsular serotypes, with PCV10 introduced in late 2020 and subsequently replaced by PCV13 in 2023. On the other hand, vaccination for adults involve both the 23-valent pneumococcal polysaccharide vaccine (PPV23) and 13-valent pneumococcal conjugate vaccine (PCV13) [3,4]. However, adult immunization is not mandatory, but only recommended based on age (adults aged 60 and older) or specific risk factors, including chronic or immunocompromising conditions and religious pilgrimages such as Hajj [3]. As part of the surveillance of circulating serotypes, this study reports the genomic characterization of a S. pneumoniae serotype 17F isolate from Malaysia, which is a serotype specifically covered by PPV23.
3. Data Description
A clinical isolate of Streptococcus pneumoniae serotype 17F (IMRCC/SS430/25) was identified in 2025 at the Bacteriology Unit, Institute for Medical Research (IMR), following recent observations indicating the emergence of serotype 17F in Malaysia. This isolate was subsequently selected for genomic characterization. The isolate was collected from a 46-year-old Malay male who suffered from aspiration pneumonia and was admitted to Hospital Sungai Buloh, Selangor, Malaysia. The pneumococcal isolate (IMRCC/SS430/25) was isolated from a tracheal aspirate specimen collected from the patient. The isolate displayed typical S. pneumoniae morphology when cultured on 5% Columbia Sheep Blood Agar, presenting as small, round, alpha-haemolytic colonies with an inhibition zone >14mm around an optochin disk (Fig. 1). Subsequent bile solubility testing confirmed the identity of S. pneumoniae while serotyping via the Quellung reaction using antisera from the Statens Serum Institut verified the isolate as serotype 17F.
Fig. 1.
Streptococcus pneumoniae serotype 17F isolate colonies grown on 5% Columbia Sheep Blood Agar exhibited a greenish-brown, small, round appearance. A clear zone observed around optochin disc (>14mm) is a typical characteristic to identify S. pneumoniae.
Table 1 summarizes the Sequencing, assembly, annotation, and lineage information of the isolate. The pneumococcal isolate was subjected to genomic DNA extraction using the DNeasy® UltraClean® Microbial Kit (Qiagen, Germany) in accordance with the manufacturer’s protocol (HB-2248-003). Genomic DNA was subjected to quality control using the QubitTM dsDNA BR (Broad Range) Assay kit (Thermo Fisher Scientific, USA). Library preparation of the genomic DNA was performed using the Native Barcoding Kit 24 V14 (SQK-NBD114.24) following manufacturer’s protocol. The DNA library of the isolate was then subjected to whole genome sequencing (WGS) using the Oxford Nanopore MinION Mk1B platform. FASTQ data was subjected to quality control, which revealed a total of 108,286,605 bases were generated from 77,255 reads in the sequencing. The mean Phred score of the raw sequencing reads was 15.6, and the read length N50 for the sequenced data was 4,541 bp. The raw FASTQ reads were then used to assemble the pneumococcal genome through EPI2ME integrated workflow wf-bacterialgenomes. The assembly produced a single circular contig of pneumococcal genome (Fig. 2), with length of 2,065,967 bp and coverage of 48. GC content of the genomes was 39.71%. Assessment of genomic quality through CheckM2 identified a 100% completeness and contamination of 0.23%. The genome annotation from Bakta revealed 2093 genes, including 2022 protein coding sequences (CDS), 12 rRNAs, 58 tRNAs, and 1 tmRNA. Genomic screening via Pathogenwatch confirmed the isolate IMRCC/SS430/25 was assigned to ST2355 and clustered within GPSC10.
Table 1.
Sequencing, assembly, annotation, and lineage information of S. pneumoniae isolate IMRCC/SS430/25.
| Isolate | IMRCC/SS430/25 | |
| Nucleotide Accession Number | SRR37999637 | |
| Raw FASTQ | Number of sequences | 77,255 |
| Total data yield (bp) | 108,286,605 | |
| Average Phred score | 15.6 | |
| N50 (bp) | 4,541 | |
| Genome assembly | Contigs | 1 |
| Genome Length (bp) | 2,065,967 | |
| Sequencing coverage | 48 | |
| GC content (%) | 39.71 | |
| Completeness (%) | 100 | |
| Contamination (%) | 0.23 | |
| Genome annotation features | Protein coding sequences (CDS) | 2022 |
| tRNA | 58 | |
| rRNA | 12 | |
| tmRNA | 1 | |
| Lineage information | Sequence type (ST) | 2355 |
| Global Pneumococcal Sequence Cluster (GPSC) | 10 | |
Fig. 2.
Circular genome map of S. pneumoniae serotype 17F isolate IMRCC/SS430/25. Tracks displayed from outermost to innermost: Bakta annotation on the forward and reverse strands for predicted coding sequences (CDS) (gray), ncRNA (blue), rRNA (red), tRNA (purple), tmRNA (orange), and predicted origin of replication (oriC, light green); GC content (black); and GC skew, with positive skew shown in green and negative skew in purple.
Antimicrobial susceptibility testing (AST) identified the serotype 17F isolate was resistant to erythromycin, azithromycin, clindamycin, and tetracycline (Table 2). Subsequent genomic analyses of resistance determinants through Pathogenwatch identified the ermB gene, which is responsible for macrolide-lincosamide-streptogramin B (MLSB) resistance, as well as the tetM_8 gene which confers tetracycline resistance. Interestingly, ST2355 genomes from Thailand and Israel were not detected with ermB gene in contrast to genomes of the same lineage from the USA and Canada [5]. Furthermore, IMRCC/SS430/25 was found to be susceptible to all beta-lactams tested in this study (penicillin, amoxicillin-clavulanate, cefotaxime) (Table 2). The penicillin-binding protein (pbp) allelic profile (pbp1a: 79, pbp2b: 26, pbp2x: 194) identified through Pathogenwatch analysis was predicted to confer uniform susceptibility to beta-lactams for IMRCC/SS430/25. This in silico prediction aligns with our phenotypic AST results, which demonstrated susceptibility to penicillin, cefotaxime, and amoxicillin-clavulanate [5].
Table 2.
Antimicrobial susceptibility testing (AST), prediction of antimicrobial resistance determinants, and penicillin-binding protein (pbp) gene profiles of S. pneumoniae serotype 17F isolate IMRCC/SS430/25.
| Isolate Genome | Antimicrobial susceptibility testing (AST) |
Genotypic prediction |
||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PEN |
CTX |
AMC | ERY | AZI | CLI | TET | CHL | SXT | VAN | Resistance Determinants |
pbp gene profile |
|||||
| men | non-men | men | non-men | pbp1a | pbp2b | pbp2x | ||||||||||
| IMRCC/SS430/25 | S | S | S | S | S | R | R | R | R | S | S | S | ermB, tetM_8 | 79 | 26 | 194 |
PEN G: Penicillin G, CTX: Cefotaxime, AMC: Amoxicillin-clavulanate, ERY: Erythromycin, AZI: Azithromycin, CLI: Clindamycin, TET: Tetracycline, CHL: chloramphenicol, SXT: trimethoprim-sulfamethoxazole, VAN: Vancomycin
men.: meningitis, non-men.: non-meningitis, S: sensitive, R: resistant
A maximum-likelihood phylogenomic tree rooted against reference strain TIGR4 positioned the Malaysian serotype 17F isolate (IMRCC/SS430/25) alongside 15 GPSC10 serotype 17F genomes (Fig. 3). The GPSC10 population was dominated by ST2355 (10/16 isolates), alongside five less frequent sequence types (ST17098, ST230, ST16399, ST11922, and ST8812). The Malaysian isolate, which was assigned to ST2355, clustered within the predominant ST2355 group together with isolates from several geographical regions. Other closely related sequence types, including ST11922, ST16399, and ST8812, were positioned within the broader ST2355-associated grouping, whereas ST17098 and ST230 formed a separate branch from the predominant ST2355 group. To further assess the allelic relatedness of these sequence types to the Malaysian isolate, the seven-locus MLST profiles were compared relative to ST2355 (Table 3). ST11922 and ST8812 were single-locus variants (SLVs) of ST2355, differing at one MLST locus each, while ST16399 was a double-locus variant (DLV), differing from ST2355 at two loci. In contrast, ST230 and ST17098 differed from ST2355 at three and five MLST loci, respectively, indicating greater allelic divergence from the Malaysian ST2355 isolate. These findings provide additional allelic context to the phylogenomic relationships observed within the GPSC10 serotype 17F population.
Fig. 3.
Maximum-likelihood phylogenetic tree comprising the Malaysian serotype 17F isolate IMRCC/SS430/25 in comparison to 15 global serotype 17F S. pneumoniae genomes. The tree was rooted using the TIGR4 reference genome. Numbers at internal nodes represents ultrafast bootstrap (UFBoot) support value. Tip labels are annotated from left to right with the year of isolation, country, Global Pneumococcal Sequence Cluster (GPSC), and sequence type (ST). Country and GPSC are indicated by color, while STs are indicated numerically. The Malaysian isolate IMRCC/SS430/25 from this study is highlighted in bold.
Table 3.
Seven-locus MLST allelic profiles and relationships among GPSC10 serotype 17F sequence types (STs) and their relationship to ST2355.
| ST | aroE | gdh | gki | recP | spi | xpt | ddl | Relationship to ST2355 |
|---|---|---|---|---|---|---|---|---|
| 2355 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
ST, sequence type; MLST, multilocus sequence typing; SLV, single-locus variant; DLV, double-locus variant. Allelic relationships are shown relative to ST2355 (IMRCC/SS430/25). SLVs and DLVs differ from ST2355 at one and two MLST loci, respectively.
4. Experimental Design, Materials and Methods
4.1. Bacterial culture, identification, and serotyping
The S. pneumoniae serotype 17F isolate (IMRCC/SS430/25) was previously archived by the Institute for Medical Research (IMR) Malaysia. The archived isolate was first cultured on 5% Columbia Sheep Blood Agar and incubated in 5% CO2 at 37°C for 18 to 24 hours. The isolate was evaluated for colony morphology, alpha-hemolysis pattern, and optochin susceptibility. A bile solubility test using 10% sodium deoxycholate was performed to observe the clearing of turbid suspension which indicated the identity of S. pneumoniae. The serotype of isolate was then confirmed via Quellung reaction using serotype/serogroup specific antisera (SSI Diagnostica, Denmark).
4.2. Antimicrobial susceptibility testing
The pneumococcal isolate was subjected to antimicrobial susceptibility using the Epsilometer test for penicillin G, cefotaxime, and amoxicillin-clavulanate, while disk diffusion method was used for, erythromycin, azithromycin, clindamycin, tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole, and vancomycin. Susceptibility categories for each antimicrobial agents were interpreted in accordance with the 36th edition Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing guidelines [6].
4.3. DNA extraction and library preparation
Genomic DNA of the S. pneumoniae isolate was extracted using the DNeasy® UltraClean® Microbial Kit (Qiagen, Germany) following the manufacturer’s protocol (HB-2248-003). DNA concentration was measured using the Qubit dsDNA Broad Range assay (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol (Pub. No. MAN0002325 Rev. B.0). Library preparation of the genomic DNA was performed using the Oxford Nanopore Native Barcoding Kit 24 V14 (SQK-NBD114.24) using 400 ng of input DNA, according to the manufacturer’s protocol (NBA_9168_v114_revT_02Oct2025). Long read sequencing was performed on the MinION Mk1B sequencing platform using the MinKNOW software (version 25.09.16), with at least 40 sequencing coverage of the genome size.
4.4. Sequencing analysis
Raw sequence data was basecalled in real-time using Dorado (version 1.3.0) and its integrated super-accurate basecalling (SUP) model (v5.2.0) (Oxford Nanopore Technologies Ltd., UK) [7]. The resulting raw FASTQ data was filtered during the basecalling to exclude reads with mean Phred quality score below Q10 to ensure an accurate, high-fidelity FASTQ output for downstream analysis. Quality of the FASTQ reads was then assessed using NanoPlot (version 1.46.2) [8]. De novo genome assembly was performed using the EPI2ME integrated workflow wf-bacterialgenomes (https://github.com/epi2me-labs/wf-bacterial-genomes). The pipeline automates the assembly of reads using Flye (version 2.9.6), contig reorientation via dnaapler (version 1.3.0), and alignment with mini_align (Pomoxis version 0.3.6, (https://nanoporetech.github.io/pomoxis/programs.html#mini-align) which is based on minimap2 (version 2.30). Subsequent polishing and coverage estimation were performed using Medaka (version 2.2.0) (https://github.com/nanoporetech/medaka), and mosdepth (version 0.3.12) (https://github.com/brentp/mosdepth) respectively [[9], [10], [11], [12]]. The quality of the assembled genome was examined through CheckM2 (version 1.1.0) to assess genome completeness and evaluate potential contamination [13]. Genome annotation was performed on Bakta (version 1.12.0) and was visualised using Proksee as a circular map [14,15]. The assembled S. pneumoniae genomes were then subjected to further analysis using Pathogenwatch (version 23.5.2) (https://pathogen.watch). This platform was utilized to determine Sequence Type (ST), Global Pneumococcal Sequence Cluster (GPSC), and the presence of antimicrobial resistance (AMR) genes, including specific variations in resistance genes and penicillin-binding protein (pbp) alleles [5]. A phylogenetic analysis was performed to compare the Malaysian serotype 17F isolate within the global GPSC10 serotype 17F population from Monocle (https://data-viewer.monocle.sanger.ac.uk/project/gps). The phylogenomic tree was constructed using Snippy (version 3.2.0) and IQ-TREE (version 3.1.1) and subsequently visualized using R package ggtree [[16], [17], [18]]. Based on the STs of the genomes included in the phylogenomic analysis, the allelic profiles of the seven MLST loci for each ST were retrieved from PubMLST (https://pubmlst.org), and the number of differing loci between STs was determined to assess pairwise genetic relatedness [19].
Limitations
The study provides a limited representation of Streptococcus pneumoniae serotype 17F in comparison to the Malaysian population. Besides, the estimation of sequencing depth may only represent the average genome coverage, which does not exclude the possibility of lower coverage across certain genomic regions.
Ethics Statement
This study was approved by the Malaysian Research Ethics Committee and National Medical Research Registry under the protocol number NMRR-24-01370-HHE, following The Code of Ethics of the World Medical Association (Declaration of Helsinki).
CRediT Author Statement
Revathy Arushothy: Supervision, Conceptualization, Methodology, Writing-Original Draft, Writing-Review and Editing, Cheng-Ngee Tan: Investigation, Formal analysis, Visualization, Writing-Original Draft, Nazirah Samsudin: Investigation, Resources, Rohaidah Hashim: Project administration, Supervision, Resources.
Acknowledgements
We gratefully acknowledge the Director General of Health Malaysia for granting permission to publish this article.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
- 1.Narciso A.R., Dookie R., Nannapaneni P., Normark S., Henriques-Normark B. Streptococcus pneumoniae epidemiology, pathogenesis and control. Nat. Rev. Microbiol. 2025;23:256–271. doi: 10.1038/s41579-024-01116-z. [DOI] [PubMed] [Google Scholar]
- 2.GBD 2023 Lower Respiratory Infections and Antimicrobial Resistance Collaborators Global burden of lower respiratory infections and aetiologies, 1990-2023: a systematic analysis for the global burden of disease study 2023. Lancet Infect. Dis. 2026;26:343–361. doi: 10.1016/S1473-3099(25)00689-9. [DOI] [PubMed] [Google Scholar]
- 3.Lister A.J.J., Dombay E., Cleary D.W., Sulaiman L.H., Clarke S.C. A brief history of and future prospects for pneumococcal vaccination in Malaysia. Pneumonia. 2023;15:12. doi: 10.1186/s41479-023-00114-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Malaysian Society of infectious Diseases and Chemotherapy . In: Guidelines for Adult Immunisation. 3rd ed. Victor K.E.L., editor. 2020. Available vaccines- pneumococcal; pp. 142–150. [Google Scholar]
- 5.Gladstone R.A., Lo S.W., Goater R., Yeats C., Taylor B., Hadfield J., Lees J.A., Croucher N.J., van Tonder A.J., Bentley L.J., Quah F.X., Blaschke A.J., Pershing N.L., Byington C.L., Balaji V., Hryniewicz W., Sigauque B., Ravikumar K.L., Almeida S.C.G., Ochoa T.J., Ho P.L., du Plessis M., Ndlangisa K.M., Cornick J.E., Kwambana-Adams B., Benisty R., Nzenze S.A., Madhi S.A., Hawkins P.A., Pollard A.J., Everett D.B., Antonio M., Dagan R., Klugman K.P., von Gottberg A., Metcalf B.J., Li Y., Beall B.W., McGee L., Breiman R.F., Aanensen D.M., Bentley S.D. Visualizing variation within global pneumococcal sequence clusters (GPSCs) and country population snapshots to contextualize pneumococcal isolates. Microb. Genom. 2020;6 doi: 10.1099/mgen.0.000357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.CLSI . Performance Standards for Antimicrobial Susceptibility Testing. CLSI M100TM. 36th ed. Clinical and Laboratory Standards Institute; 2026. Table 2G. Zone diameter and MIC breakpoints for Streptococcus pneumoniae; pp. 122–127. [Google Scholar]
- 7.Dorado, Oxford Nanopore Technologies 2025. https://github.com/nanoporetech/dorado (accessed December 20, 2025)
- 8.De Coster W., Rademakers R. NanoPack2: population-scale evaluation of long-read sequencing data. Bioinformatics. 2023;39 doi: 10.1093/bioinformatics/btad311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Bouras G., Grigson S.R., Papudeshi B., Mallawaarachchi V., Roach M.J. Dnaapler: a tool to reorient circular microbial genomes. J. Open Source Softw. 2024;9:5968. doi: 10.21105/joss.05968. [DOI] [Google Scholar]
- 10.Kolmogorov M., Yuan J., Lin Y., Pevzner P.A. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 2019;37:540–546. doi: 10.1038/s41587-019-0072-8. [DOI] [PubMed] [Google Scholar]
- 11.Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–3100. doi: 10.1093/bioinformatics/bty191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Pedersen B.S., Quinlan A.R. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics. 2018;34:867–868. doi: 10.1093/bioinformatics/btx699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Chklovski A., Parks D.H., Woodcroft B.J., Tyson G.W. CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning. Nat. Methods. 2023;20:1203–1212. doi: 10.1038/s41592-023-01940-w. [DOI] [PubMed] [Google Scholar]
- 14.Grant J.R., Enns E., Marinier E., Mandal A., Herman E.K., Chen C., Graham M., Van Domselaar G., Stothard P. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023;51:W484–W492. doi: 10.1093/nar/gkad326. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Schwengers O., Jelonek L., Dieckmann M.A., Beyvers S., Blom J., Goesmann A. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb. Genom. 2021;7 doi: 10.1099/mgen.0.000685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Jolley K.A., Bray J.E., Maiden M.C.J. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018;3:124. doi: 10.12688/wellcomeopenres.14826.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Seemann T. Snippy: rapid haploid variant calling and core genome alignment. 2020. https://github.com/tseemann/snippy (accessed December 5, 2025)
- 18.Wong T., Ly-Trong N., Ren H., Baños H., Roger A., Susko E., Bielow C., De Maio N., Goldman N., Hahn M., Huttley G., Lanfear R., Minh B.Q. IQ-TREE 3: phylogenomic inference software using complex evolutionary models. EcoEvoRxiv. 2025 doi: 10.32942/X2P62N. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Yu G., Lam T.T.-Y., Zhu H., Guan Y. Two methods for mapping and visualizing associated data on phylogeny using Ggtree. Mol. Biol. Evol. 2018;35:3041–3043. doi: 10.1093/molbev/msy194. [DOI] [PMC free article] [PubMed] [Google Scholar]