# Draft Genome Sequences of Four Pseudomonas aeruginosa Clinical Strains with Various Biofilm Phenotypes

Amine Boukerb, Marjolaine Simon, Erwan Pernet, Albane Jouault, Emilie Portier, Elise Persyn, Emeline Bouffartigues, Alexis Bazire, Sylvie Chevalier, Marc G J Feuilloley, et al.

<figure><img src="image_1.png" /></figure>

To cite this version:

Amine Boukerb, Marjolaine Simon, Erwan Pernet, Albane Jouault, Emilie Portier, et al. Draft Genome Sequences of Four Pseudomonas aeruginosa Clinical Strains with Various Biofilm Phenotypes. Microbiology Resource Announcements, 2020, 9 (1), 10.1128/MRA.01286-19. hal-02428484

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Distributed under a Creative Commons Attribution 4.0 International License
# Draft Genome Sequences of Four Pseudomonas aeruginosa Clinical Strains with Various Biofilm Phenotypes

Amine Boukerb, Marjolaine Simon, Erwan Pernet, Albane Jouault, Emilie Portier, Elise Persyn, Emeline Bouffartigues, Alexis Bazire, Sylvie Chevalier, Marc G J Feuilloley, et al.

<figure><img src="image_1.png" /></figure>

To cite this version:

Amine Boukerb, Marjolaine Simon, Erwan Pernet, Albane Jouault, Emilie Portier, et al. Draft Genome Sequences of Four Pseudomonas aeruginosa Clinical Strains with Various Biofilm Phenotypes. Microbiology Resource Announcements, 2020, 9 (1), 10.1128/MRA.01286-19. hal-02428484

HAL Id: hal-02428484

https://hal.science/hal-02428484v1

Submitted on 22 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Draft Genome Sequences of Four *Pseudomonas aeruginosa* Clinical Strains with Various Biofilm Phenotypes

Amine M. Boukerb,ª Marjolaine Simon,ᵇ Erwan Pernet,ᵇ* Albane Jouault,ᵇ Emilie Portier,ᵇ Elise Persyn,ᶜ Emeline Bouffartigues,ª Alexis Bazire,ᵇ Sylvie Chevalier,ª Marc G. J. Feuilloley,ª Olivier Lesouhaitier,ª Jocelyne Caillon,ᶜ Alain Dufourᵇ

**ABSTRACT** Biofilms produced by *Pseudomonas aeruginosa* present a serious threat to cystic fibrosis patients. Here, we report the draft genome sequences of four cystic fibrosis isolates displaying various mucoid and biofilm phenotypes. The estimated average genome size was about 6,255,986 ± 50,202 bp with a mean G+C content of 66.52 ± 0.06%.

The lungs of cystic fibrosis (CF) patients are prone to infections by *Pseudomonas aeruginosa* (1). CF lungs are generally colonized by nonmucoid *P. aeruginosa* strains forming biofilms, and chronic infections occur upon the emergence of mucoid strains overproducing alginate (2). Their biofilms are highly resistant to antibiotics and immune mediators and lead to pulmonary decline (2, 3).

*P. aeruginosa* strains were isolated from sputum samples of adult CF patients suffering from chronic infection and followed at the Centre Hospitalier Universitaire, Nantes, France. Since these sputum samples were used only to isolate bacteria but not to work on human cells or on human DNA, French law (Décret no 2016-1537, 16 November 2016) does not require that the study be reviewed and approved by an institutional ethics committee or that the participants provide their written or verbal informed consent. Bacteria from sputum samples were grown on blood agar medium at 37°C and identified as *P. aeruginosa* using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS [Vitek; bioMérieux, Marcy-l'Étoile, France]). A single isolate from each patient was used. Based mainly on their biofilm structure and mucoid phenotype, the isolates MUC-N1, MUC-N2, MUC-P4, and MUC-P5 were selected to constitute a strain panel for testing antibiofilm compounds (M. Simon, E. Pernet, A. Jouault, E. Portier, A. M. Boukerb, S. Pineau, J. Vieillard, E. Bouffartigues, C. Poc-Duclairoir, M. G. J. Feuilloley, O. Lesouhaitier, J. Caillon, S. Chevalier, A. Bazire, and A. Dufour, submitted for publication), which prompted us to sequence their genomes.

Each strain was grown at 37°C in liquid LB medium inoculated with a single colony picked from an LB agar plate, and its genomic DNA was extracted using a GeneJet genomic DNA purification kit (Thermo Fisher Scientific, France) following the manufacturer's recommendations and assessed using the double-stranded DNA (dsDNA) high-sensitivity kit on a Qubit fluorometer (Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis. Sequencing libraries were prepared using the Illumina Nextera XT DNA library prep kit following the manufacturer's protocol. Pooled libraries were sequenced on a MiSeq instrument (LMSM Genomics platform, Rouen Normandy University, Evreux, France) with dual-index paired-end reads using the MiSeq reagent kit v.3 (2 × 250 bp). Default parameters were used for all software unless otherwise noted. The reads were trimmed using Trimmomatic v.0.36 (4), and their quality was checked with MultiQC

**Citation** Boukerb AM, Simon M, Pernet E, Jouault A, Portier E, Persyn E, Bouffartigues E, Bazire A, Chevalier S, Feuilloley MGJ, Lesouhaitier O, Caillon J, Dufour A. 2020. Draft genome sequences of four *Pseudomonas aeruginosa* clinical strains with various biofilm phenotypes. Microbiol Resour Announc 9:e01286-19. https://doi.org/10.1128/MRA.01286-19.

**Editor** Vincent Bruno, University of Maryland School of Medicine

**Copyright** © 2020 Boukerb et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Alain Dufour, alain.dufour@univ-ubs.fr.

* Present address: Erwan Pernet, Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Montreal, Canada.

Received 11 October 2019

Accepted 20 November 2019

Published 2 January 2020

<figure><img src="image_3.png" /></figure>

<footnote>ªLaboratoire de Microbiologie Signaux et Micro-Environnement, LMSM EA4312, Université de Rouen-Normandie, Normandie Université, Evreux, France</footnote>

<footnote>ᵇUniversité de Bretagne-Sud, EA 3884, LBCM, IUEM, Lorient, France</footnote>

<footnote>ᶜUniversité de Nantes, Faculté de Médecine, EA3826 Thérapeutiques Cliniques et Expérimentales des Infections, Nantes, France</footnote>
<figure><img src="image_3.png" /></figure>

# Draft Genome Sequences of Four *Pseudomonas aeruginosa* Clinical Strains with Various Biofilm Phenotypes

Amine M. Boukerb,ª Marjolaine Simon,ᵇ Erwan Pernet,ᵇ* Albane Jouault,ᵇ Emilie Portier,ᵇ Elise Persyn,ᶜ

Emeline Bouffartigues,ª Alexis Bazire,ᵇ Sylvie Chevalier,ª Marc G. J. Feuilloley,ª Olivier Lesouhaitier,ª Jocelyne Caillon,ᶜ

Alain Dufourᵇ

**ABSTRACT** Biofilms produced by *Pseudomonas aeruginosa* present a serious threat to cystic fibrosis patients. Here, we report the draft genome sequences of four cystic fibrosis isolates displaying various mucoid and biofilm phenotypes. The estimated average genome size was about 6,255,986 ± 50,202 bp with a mean G+C content of 66.52 ± 0.06%.

The lungs of cystic fibrosis (CF) patients are prone to infections by *Pseudomonas aeruginosa* (1). CF lungs are generally colonized by nonmucoid *P. aeruginosa* strains forming biofilms, and chronic infections occur upon the emergence of mucoid strains overproducing alginate (2). Their biofilms are highly resistant to antibiotics and immune mediators and lead to pulmonary decline (2, 3).

*P. aeruginosa* strains were isolated from sputum samples of adult CF patients suffering from chronic infection and followed at the Centre Hospitalier Universitaire, Nantes, France. Since these sputum samples were used only to isolate bacteria but not to work on human cells or on human DNA, French law (Décret no 2016-1537, 16 November 2016) does not require that the study be reviewed and approved by an institutional ethics committee or that the participants provide their written or verbal informed consent. Bacteria from sputum samples were grown on blood agar medium at 37°C and identified as *P. aeruginosa* using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS [Vitek; bioMérieux, Marcy-l'Étoile, France]). A single isolate from each patient was used. Based mainly on their biofilm structure and mucoid phenotype, the isolates MUC-N1, MUC-N2, MUC-P4, and MUC-P5 were selected to constitute a strain panel for testing antibiofilm compounds (M. Simon, E. Pernet, A. Jouault, E. Portier, A. M. Boukerb, S. Pineau, J. Vieillard, E. Bouffartigues, C. Poc-Duclairoir, M. G. J. Feuilloley, O. Lesouhaitier, J. Caillon, S. Chevalier, A. Bazire, and A. Dufour, submitted for publication), which prompted us to sequence their genomes.

Each strain was grown at 37°C in liquid LB medium inoculated with a single colony picked from an LB agar plate, and its genomic DNA was extracted using a GeneJet genomic DNA purification kit (Thermo Fisher Scientific, France) following the manufacturer's recommendations and assessed using the double-stranded DNA (dsDNA) high-sensitivity kit on a Qubit fluorometer (Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis. Sequencing libraries were prepared using the Illumina Nextera XT DNA library prep kit following the manufacturer's protocol. Pooled libraries were sequenced on a MiSeq instrument (LMSM Genomics platform, Rouen Normandy University, Evreux, France) with dual-index paired-end reads using the MiSeq reagent kit v.3 (2 × 250 bp). Default parameters were used for all software unless otherwise noted. The reads were trimmed using Trimmomatic v.0.36 (4), and their quality was checked with MultiQC

**Citation** Boukerb AM, Simon M, Pernet E, Jouault A, Portier E, Persyn E, Bouffartigues E, Bazire A, Chevalier S, Feuilloley MGJ, Lesouhaitier O, Caillon J, Dufour A. 2020. Draft genome sequences of four *Pseudomonas aeruginosa* clinical strains with various biofilm phenotypes. Microbiol Resour Announc 9:e01286-19. https://doi.org/10.1128/MRA.01286-19.

**Editor** Vincent Bruno, University of Maryland School of Medicine

**Copyright** © 2020 Boukerb et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Alain Dufour, alain.dufour@univ-ubs.fr.

* Present address: Erwan Pernet, Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Montreal, Canada.

Received 11 October 2019

Accepted 20 November 2019

Published 2 January 2020

<footnote>ªLaboratoire de Microbiologie Signaux et Micro-Environnement, LMSM EA4312, Université de Rouen-Normandie, Normandie Université, Evreux, France</footnote>

<footnote>ᵇUniversité de Bretagne-Sud, EA 3884, LBCM, IUEM, Lorient, France</footnote>

<footnote>ᶜUniversité de Nantes, Faculté de Médecine, EA3826 Thérapeutiques Cliniques et Expérimentales des Infections, Nantes, France</footnote>
<figure><img src="image_4.png" /><figcaption>TABLE 1 Overview of the draft genome assemblies of the four *P. aeruginosa* strains</figcaption></figure>

<table><thead><tr><th rowspan="2">Parameter</th><th colspan="4">Data for strain:</th></tr><tr><th>MUC-N1</th><th>MUC-N2</th><th>MUC-P4</th><th>MUC-P5</th></tr></thead><tbody><tr><td>GenBank accession no.</td><td>WBZH00000000</td><td>WBZG00000000</td><td>WBZF00000000</td><td>WBZE00000000</td></tr><tr><td>SRA accession no.</td><td>SRR10230517</td><td>SRR10230516</td><td>SRR10230515</td><td>SRR10230514</td></tr><tr><td>No. of total reads</td><td>1,617,598</td><td>1,508,174</td><td>1,658,332</td><td>1,669,232</td></tr><tr><td>Mean coverage (×)</td><td>65</td><td>61</td><td>67</td><td>66</td></tr><tr><td>G+C content (%)</td><td>66.58</td><td>66.58</td><td>66.50</td><td>66.46</td></tr><tr><td>N<sub>50</sub> (bp)</td><td>185,163</td><td>162,332</td><td>160,065</td><td>209,989</td></tr><tr><td>No. of contigs</td><td>105</td><td>98</td><td>108</td><td>92</td></tr><tr><td>Genome size (bp)</td><td>6,206,374</td><td>6,205,784</td><td>6,209,920</td><td>6,306,189</td></tr><tr><td>No. of CDS</td><td>5,642</td><td>5,634</td><td>5,616</td><td>5,795</td></tr><tr><td>No. of tRNAs</td><td>67</td><td>67</td><td>68</td><td>62</td></tr><tr><td>No. of rRNAs</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>Prophage loci<sup>a</sup></td><td>Pseudo YMC11/02/<br/>R656 (NC028657)</td><td>Pseudo YMC11/02/<br/>R656 (NC028657)</td><td>Pseudo YMC11/02/<br/>R656* (NC028657);<br/>Pseudo phiCTX*<br/>(NC003278)</td><td>Pseudo B3 (NC006548); Escher vB EcoM<br/>ECO1230 10 (NC027995); Vibrio<br/>VP882 (NC009016); Shigel SfIV*<br/>(NC022749); Burkho BcepMu* (NC005882);<br/>Pseudo Pf1* (NC001331)</td></tr></tbody></table>

v.1.6 (5). De novo assembly was performed with Unicycer v.0.4.7 (6), and assembly metrics were calculated using QUAST v.5.0.0 (7). Prokka v.1.14.0 (8) was used for structural gene prediction and functional annotation. Multilocus sequence typing (MLST), the resistome, and the virulome were assessed using the "reads to report" Nullarbor pipeline v.2.0.20181010 (9), with sequence identity and coverage thresholds fixed at 70% and 90%, respectively. Prophage regions were identified through the integrated search and annotation tool PHASTER (10).

The average total size of the draft genomes was 6,255,986 ± 50,202 bp, arranged into 100 ± 8 contigs (Table 1). The genome sequence data were at an average coverage of 64 ± 3× with a mean $N_{50}$ value of 185,027 ± 24,962 bp and a mean G+C content of 66.52 ± 0.06%. These genomes contain an average of 5,706 ± 90 coding sequences (CDS), genes for 3 rRNAs, 65 ± 3 tRNAs, and 1 transfer-messenger RNA (tmRNA). MLST typing identified MUC-N1 and MUC-N2 with sequence type 549 (ST549), MUC-P4 with ST675, and MUC-P5 with ST649, all of which clustered within the PAO1 clade. Major virulence factors involved in human pathogenicity were identified, including the *exoS*, *exoT*, and *exoY* genes. The four strains shared the following antibiotic resistance genes: *aph(3')-IIb* (aminoglycosides), *bla*<sub>PDC-55</sub> (beta-lactam antibiotics), *catB7* (phenicols), and *fosA* (phosphonic acids). Three additional beta-lactam resistance genes were found, *bla*<sub>OXA-50</sub> in MUC-N1 and MUC-N2, *bla*<sub>OXA-395</sub> in MUC-P4, and *bla*<sub>OXA-396</sub> in MUC-P5. Ten prophage loci were predicted (eight different prophages), five of which are complete (Table 1). MUC-P5 contains six prophages (three are complete), whereas the other strains contain one or two prophages. Altogether, the genetic features of these four strains are of interest, especially in the perspective of using them as a panel to investigate antibiofilm molecules.

**Data availability.** The draft sequences have been deposited at DDBJ/ENA/GenBank under the accession numbers cited in Table 1.

## ACKNOWLEDGMENTS

We acknowledge the contribution of the Normandy Network of Microbial Metagenomics (RNAmb).

This work was supported by grant RF20120600634 from Vaincre La Mucoviscidose. M.S. and A.J. were the recipients of doctoral fellowships from the Université de Bretagne-Sud and the Région Bretagne. The LBCM is supported by the Région Bretagne and European Union (FEDER). The LMSM is supported by the Région Normandie and European Union (FEDER). A.M.B. is the recipient of a postdoctoral grant from Région Normandie and European Union (FEDER).

<footnote><sup>a</sup> Asterisks (*) indicate incomplete prophage loci.</footnote>
<figure><img src="image_4.png" /><figcaption>TABLE 1 Overview of the draft genome assemblies of the four *P. aeruginosa* strains</figcaption></figure>

<table><thead><tr><th rowspan="2">Parameter</th><th colspan="4">Data for strain:</th></tr><tr><th>MUC-N1</th><th>MUC-N2</th><th>MUC-P4</th><th>MUC-P5</th></tr></thead><tbody><tr><td>GenBank accession no.</td><td>WBZH00000000</td><td>WBZG00000000</td><td>WBZF00000000</td><td>WBZE00000000</td></tr><tr><td>SRA accession no.</td><td>SRR10230517</td><td>SRR10230516</td><td>SRR10230515</td><td>SRR10230514</td></tr><tr><td>No. of total reads</td><td>1,617,598</td><td>1,508,174</td><td>1,658,332</td><td>1,669,232</td></tr><tr><td>Mean coverage (×)</td><td>65</td><td>61</td><td>67</td><td>66</td></tr><tr><td>G+C content (%)</td><td>66.58</td><td>66.58</td><td>66.50</td><td>66.46</td></tr><tr><td>N<sub>50</sub> (bp)</td><td>185,163</td><td>162,332</td><td>160,065</td><td>209,989</td></tr><tr><td>No. of contigs</td><td>105</td><td>98</td><td>108</td><td>92</td></tr><tr><td>Genome size (bp)</td><td>6,206,374</td><td>6,205,784</td><td>6,209,920</td><td>6,306,189</td></tr><tr><td>No. of CDS</td><td>5,642</td><td>5,634</td><td>5,616</td><td>5,795</td></tr><tr><td>No. of tRNAs</td><td>67</td><td>67</td><td>68</td><td>62</td></tr><tr><td>No. of rRNAs</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>Prophage loci<sup>a</sup></td><td>Pseudo YMC11/02/<br/>R656 (NC028657)</td><td>Pseudo YMC11/02/<br/>R656 (NC028657)</td><td>Pseudo YMC11/02/<br/>R656* (NC028657);<br/>Pseudo phiCTX*<br/>(NC003278)</td><td>Pseudo B3 (NC006548); Escher vB EcoM<br/>ECO1230 10 (NC027995); Vibrio<br/>VP882 (NC009016); Shigel SflV*<br/>(NC022749); Burkho BcepMu* (NC005882);<br/>Pseudo Pf1* (NC001331)</td></tr></tbody></table>

v.1.6 (5). De novo assembly was performed with Unicycer v.0.4.7 (6), and assembly metrics were calculated using QUAST v.5.0.0 (7). Prokka v.1.14.0 (8) was used for structural gene prediction and functional annotation. Multilocus sequence typing (MLST), the resistome, and the virulome were assessed using the "reads to report" Nullarbor pipeline v.2.0.20181010 (9), with sequence identity and coverage thresholds fixed at 70% and 90%, respectively. Prophage regions were identified through the integrated search and annotation tool PHASTER (10).

The average total size of the draft genomes was 6,255,986 ± 50,202 bp, arranged into 100 ± 8 contigs (Table 1). The genome sequence data were at an average coverage of 64 ± 3× with a mean $N_{50}$ value of 185,027 ± 24,962 bp and a mean G+C content of 66.52 ± 0.06%. These genomes contain an average of 5,706 ± 90 coding sequences (CDS), genes for 3 rRNAs, 65 ± 3 tRNAs, and 1 transfer-messenger RNA (tmRNA). MLST typing identified MUC-N1 and MUC-N2 with sequence type 549 (ST549), MUC-P4 with ST675, and MUC-P5 with ST649, all of which clustered within the PAO1 clade. Major virulence factors involved in human pathogenicity were identified, including the *exoS*, *exoT*, and *exoY* genes. The four strains shared the following antibiotic resistance genes: *aph(3')-llb* (aminoglycosides), *bla*<sub>PDC-55</sub> (beta-lactam antibiotics), *catB7* (phenicols), and *fosA* (phosphonic acids). Three additional beta-lactam resistance genes were found, *bla*<sub>OXA-50</sub> in MUC-N1 and MUC-N2, *bla*<sub>OXA-395</sub> in MUC-P4, and *bla*<sub>OXA-396</sub> in MUC-P5. Ten prophage loci were predicted (eight different prophages), five of which are complete (Table 1). MUC-P5 contains six prophages (three are complete), whereas the other strains contain one or two prophages. Altogether, the genetic features of these four strains are of interest, especially in the perspective of using them as a panel to investigate antibiofilm molecules.

**Data availability.** The draft sequences have been deposited at DDBJ/ENA/GenBank under the accession numbers cited in Table 1.

## ACKNOWLEDGMENTS

We acknowledge the contribution of the Normandy Network of Microbial Metagenomics (RNAmb).

This work was supported by grant RF20120600634 from Vaincre La Mucoviscidose. M.S. and A.J. were the recipients of doctoral fellowships from the Université de Bretagne-Sud and the Région Bretagne. The LBCM is supported by the Région Bretagne and European Union (FEDER). The LMSM is supported by the Région Normandie and European Union (FEDER). A.M.B. is the recipient of a postdoctoral grant from Région Normandie and European Union (FEDER).

<footnote>a Asterisks (*) indicate incomplete prophage loci.</footnote>
<figure><img src="image_5.png" /></figure>

REFERENCES

1. Lyczak JB, Cannon CL, Pier GB. 2002. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15:194-222. https://doi.org/10.1128/ cmr.15.2.194-222.2002.

2. Ramsey DM, Wozniak DJ. 2005. Understanding the control of *Pseudomonas aeruginosa* alginate synthesis and the prospects for management of chronic infections in cystic fibrosis. Mol Microbiol 56:309-922. https:// doi.org/10.1111/j.1365-2958.2005.04552.x.

3. Mann EE, Wozniak DJ. 2012. *Pseudomonas* biofilm matrix composition and niche biology. FEMS Microbiol Rev 36:893-916. https://doi.org/10. 1111/j.1574-6976.2011.00322.x.

4. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120. https://doi.org/10.1093/bioinformatics/btu170.

5. Ewels P, Magnusson M, Lundin S, Kaller M. 2016. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinforma- matics 32:3047-3048. https://doi.org/10.1093/bioinformatics/btw354.

6. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycer: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. https://doi.org/10.1371/journal.pcbi .1005595.

7. Mikheenko A, Prjibelski A, Saveliev V, Antipov D, Gurevich A. 2018. Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 34:i142-i150. https://doi.org/10.1093/bioinformatics/bty266.

8. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinforma- matics 30:2068-2069. https://doi.org/10.1093/bioinformatics/btu153.

9. Seemann T, Goncalves da Silva A, Bulach DM, Schultz MB, Kwong JC, Howden BP. Nullarbor. https://github.com/tseemann/nullarbor.

10. Arndt D, Marcu A, Liang Y, Wishart DS. 2017. PHAST, PHASTER and PHASTEST: tools for finding prophage in bacterial genomes. Brief Bioin- form 20:1560-1567. https://doi.org/10.1093/bib/bbx121.
<figure><img src="image_5.png" /></figure>

REFERENCES

1. Lyczak JB, Cannon CL, Pier GB. 2002. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15:194-222. https://doi.org/10.1128/ cmr.15.2.194-222.2002.

2. Ramsey DM, Wozniak DJ. 2005. Understanding the control of *Pseudomonas aeruginosa* alginate synthesis and the prospects for management of chronic infections in cystic fibrosis. Mol Microbiol 56:309-922. https:// doi.org/10.1111/j.1365-2958.2005.04552.x.

3. Mann EE, Wozniak DJ. 2012. *Pseudomonas* biofilm matrix composition and niche biology. FEMS Microbiol Rev 36:893-916. https://doi.org/10. 1111/j.1574-6976.2011.00322.x.

4. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120. https://doi.org/10. 1093/bioinformatics/btu170.

5. Ewels P, Magnusson M, Lundin S, Kaller M. 2016. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinfor- matics 32:3047-3048. https://doi.org/10.1093/bioinformatics/btw354.

6. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. https://doi.org/10.1371/journal.pcbi .1005595.

7. Mikheenko A, Prjibelski A, Saveliev V, Antipov D, Gurevich A. 2018. Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 34:i142-i150. https://doi.org/10.1093/bioinformatics/bty266.

8. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinfor- matics 30:2068-2069. https://doi.org/10.1093/bioinformatics/btu153.

9. Seemann T, Goncalves da Silva A, Bulach DM, Schultz MB, Kwong JC, Howden BP. Nullarbor. https://github.com/tseemann/nullarbor.

10. Arndt D, Marcu A, Liang Y, Wishart DS. 2017. PHAST, PHASTER and PHASTEST: tools for finding prophage in bacterial genomes. Brief Bioin- form 20:1560-1567. https://doi.org/10.1093/bib/bbx121.