Skip to main content
PMC home page
Genome Announcements logoLink to Genome Announcements
. 2016 Apr 21;4(2):e00231-16. doi: 10.1128/genomeA.00231-16

Draft Genome Sequences of 63 Pseudomonas aeruginosa Isolates Recovered from Cystic Fibrosis Sputum

Theodore Spilker 1, John J LiPuma 1,✉
PMCID: PMC4841125  PMID: 27103710

Abstract

Here, we report the draft genome sequences of 63 Pseudomonas aeruginosa isolates, recovered in culture of sputum from 15 individuals with cystic fibrosis (CF) receiving care in a single CF care center over a 13-year period. These sequences add value to studies of within-host evolution of bacterial pathogens during chronic infection.

GENOME ANNOUNCEMENT

Individuals with cystic fibrosis (CF) are susceptible to infection of the respiratory tract with Pseudomonas aeruginosa, which undergoes well-characterized phenotypic adaptation during chronic infection of CF airways (1). Recent studies have also noted considerable genetic variation among isolates serially recovered from CF patients during the course of chronic infection (2–4). This genetic variation may confound genotyping of P. aeruginosa isolates, which is important for monitoring potential interpatient spread of specific strains in this patient population. We determined the whole-genome sequences of 63 P. aeruginosa isolates cultured from 15 adult CF patients during a period of 13 years. The patients ranged in age from 19 years to 55 years (mean, 33 years). Between two and seven isolates were available from each patient; isolates were cultured from each patient during periods of time ranging from 4 to 13 years (mean, 6 years).

Bacteria were grown in Mueller-Hinton broth overnight at 37°C in an orbital shaker. Five ml of bacterial culture was pelleted, resuspended in 1 ml of 1X Tris-EDTA buffer and adjusted to an optical density of 0.55, corresponding to approximately 108 CFU. Genomic DNA was extracted from 350 µl of the adjusted suspension using the MagNA Pure Compact nucleic acid isolation kit (Roche) following the manufacturer’s instructions. Genomic DNA libraries were prepared using an Illumina TruSEQ DNA library kit and sequenced on an Illumina HiSEQ 2500 paired-end flow cell (2 × 125-bp read length, V4 chemistry) at the University of Michigan Medical School DNA Sequencing Core. Output files containing the fastq reads were checked and edited using Trimmomatic version 0.33 (5). Read correction and assembly of draft genomes was carried out using SPAdes version 3.5.0 (6). Genomic alignments, phylogenetics, and single nucleotide polymorphism (SNP) visualization was performed using Gingr and Parsnp programs of the Harvest tools version 1.2 suite (7). SNP analysis was carried out using the ISG pipeline (8), employing MUMMER version 3.23 (9), BWA version 0.7.12 (10), and GATK version 3.4 (11). Genomes were annotated using NCBI’s whole-genome shotgun submission portal containing the automated Prokaryotic Genomic Annotation Pipeline (PGAP) option.

Draft genomes ranged in size from 6,118,548 bp to 6,884,695 bp, and contained 5,617 to 6,333 coding sequences (Table 1). The N50 of the draft genomes ranged from 111,223 to 588,702 bp, with 33 to 124 contiguous pieces. Pairwise comparison of draft genomes of isolates from any one patient revealed that SNPs ranged from a low of 43 and a high of 3,160 with a mean range of 771 and a median range of 719.

TABLE 1 .

Global statistics of draft genome sequences of 63 Pseudomonas aeruginosa isolates

Patient Isolatea Yr isolated BioSample no. Accession no. Genome size (bp) CDSsb No. of contigs N50
1 AU6462 2003 SAMN04436460 LRYR00000000 6,678,504 6,119 81 178,306
1 AU9381 2005 SAMN04436461 LRYD00000000 6,712,367 6,193 84 160,605
1 AU10014 2005 SAMN04436462 LRYE00000000 6,515,569 5,989 76 168,780
1 AU10015 2005 SAMN04436463 LRYF00000000 6,697,715 6,192 105 130,232
1 AU10714 2006 SAMN04436464 LRYG00000000 6,518,682 6,031 89 138,224
1 AU11866 2006 SAMN04436465 LRYH00000000 6,441,026 5,909 87 158,236
2 AU2342 2000 SAMN04436466 LRYI00000000 6,644,537 6,137 59 247,442
2 AU5471 2003 SAMN04436467 LRYJ00000000 6,576,117 6,061 48 417,292
2 AU10241 2005 SAMN04436468 LRYK00000000 6,517,933 6,000 57 333,007
3 AU6854 2004 SAMN04436469 LRYL00000000 6,746,537 6,202 66 276,987
3 AU9739 2005 SAMN04436470 LRYM00000000 6,861,787 6,327 66 195,665
3 AU20916 2010 SAMN04436471 LRYN00000000 6,837,226 6,300 70 231,549
4 AU6923 2004 SAMN04436472 LRYO00000000 6,490,208 6,056 57 233,457
4 AU12175 2006 SAMN04436473 LRYP00000000 6,529,487 6,105 76 193,222
4 AU13212 2007 SAMN04436474 LRYQ00000000 6,519,696 6,061 38 349,744
4 AU13213 2007 SAMN04436475 LRYS00000000 6,508,051 6,056 44 329,608
4 AU16960 2008 SAMN04436476 LRYT00000000 6,499,229 6,051 33 588,702
5 AU7032 2004 SAMN04436477 LRYU00000000 6,539,801 6,026 33 492,301
5 AU15431 2008 SAMN04436478 LRYV00000000 6,545,379 6,063 39 426,848
6 AU7033 2004 SAMN04436479 LRYW00000000 6,366,246 5,851 95 149,269
6 AU10583 2006 SAMN04436480 LRYX00000000 6,353,322 5,816 55 272,732
6 AU18068 2009 SAMN04436481 LRYY00000000 6,355,037 5,813 60 254,892
6 AU24156 2012 SAMN04436482 LRYZ00000000 6,356,852 5,808 59 232,432
7 AU7198 2004 SAMN04436483 LRZA00000000 6,548,147 6,081 91 130,397
7 AU10272 2005 SAMN04436484 LRZB00000000 6,588,605 6,093 53 202,738
7 AU10409 2005 SAMN04436485 LRZC00000000 6,475,849 5,985 56 184,471
7 AU10410 2005 SAMN04436486 LRZD00000000 6,580,210 6,096 56 202,738
7 AU10836 2006 SAMN04436487 LRZE00000000 6,536,502 6,110 124 111,223
7 AU16821 2008 SAMN04436488 LRZF00000000 6,572,055 6,095 54 253,358
8 AU8251 2004 SAMN04436489 LRZG00000000 6,293,365 5,797 56 209,586
8 AU12528 2006 SAMN04436490 LRZH00000000 6,327,593 5,810 49 233,151
8 AU17907 2009 SAMN04436491 LRZI00000000 6,336,865 5,820 54 211,550
9 AU9017 2005 SAMN04436492 LRZJ00000000 6,328,267 5,757 39 370,552
9 AU10502 2006 SAMN04436493 LRZK00000000 6,321,192 5,754 49 370,708
9 AU11990 2006 SAMN04436494 LRZL00000000 6,321,329 5,752 39 428,683
9 AU11991 2006 SAMN04436495 LRZM00000000 6,321,358 5,758 47 345,115
9 AU12424 2006 SAMN04436496 LRZN00000000 6,320,793 5,749 38 424,798
9 AU18274 2009 SAMN04436497 LRZO00000000 6,319,222 5,764 61 226,542
9 AU25116 2012 SAMN04436498 LRZP00000000 6,317,546 5,772 80 215,654
10 AU9899 2005 SAMN04436499 LRZQ00000000 6,240,563 5,706 64 213,685
10 AU13210 2007 SAMN04436500 LRZR00000000 6,239,781 5,688 49 318,484
10 AU19803 2010 SAMN04436501 LRZS00000000 6,158,966 5,617 52 232,854
10 AU19804 2010 SAMN04436502 LRZT00000000 6,161,159 5,626 66 179,368
11 AU10658 2006 SAMN04436503 LRZU00000000 6,341,036 5,836 77 176,289
11 AU17091 2008 SAMN04436504 LRZV00000000 6,264,463 5,733 35 355,061
11 AU21076 2010 SAMN04436505 LRZW00000000 6,230,429 5,700 64 193,145
11 AU25210 2012 SAMN04436506 LRZX00000000 6,268,083 5,733 38 335,848
12 AU10713 2006 SAMN04436507 LRZY00000000 6,262,596 5,719 51 238,345
12 AU17550 2009 SAMN04436508 LRZZ00000000 6,255,564 5,701 35 418,726
12 AU18132 2009 SAMN04436509 LSAA00000000 6,260,665 5,740 89 150,226
12 AU19319 2009 SAMN04436510 LSAB00000000 6,257,108 5,707 43 268,203
12 AU24526 2012 SAMN04436511 LSAC00000000 6,253,538 5,706 46 220,311
13 AU10756 2006 SAMN04436512 LSAD00000000 6,825,418 6,267 53 373,936
13 AU20671 2010 SAMN04436513 LSAE00000000 6,884,695 6,333 70 186,989
13 AU24807 2012 SAMN04436514 LSAF00000000 6,740,949 6,211 89 139,380
14 AU14820 2007 SAMN04436515 LSAG00000000 6,667,350 6,123 46 291,575
14 AU17965 2009 SAMN04436516 LSAH00000000 6,692,467 6,139 40 375,961
14 AU23529 2011 SAMN04436517 LSAI00000000 6,692,578 6,147 51 322,358
15 AU1215 1999 SAMN04436518 LRSF00000000 6,487,674 5,986 80 160,063
15 AU7511 2004 SAMN04436519 LRSG00000000 6,430,803 5,932 60 215,080
15 AU13626 2007 SAMN04436520 LRSH00000000 6,423,934 5,912 48 260,857
15 AU22632 2011 SAMN04436521 LRSI00000000 6,118,548 5,711 103 117,443
15 AU24787 2012 SAMN04436522 LRSJ00000000 6,150,557 5,707 61 192,465
Open in a new tab
a

All isolates are included in Bioproject PRJNA309533 and SRA SRP068878.

b

CDSs, coding sequences.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited in GenBank under the accession numbers listed in Table 1. The versions described in this paper are the first versions.

ACKNOWLEDGMENTS

We thank Robert Lyon, Christina McHenry, Katherine Borysko, and the University of Michigan Medical School DNA Sequencing Core Facility for their technical expertise.

This work was supported by the University of Michigan Medical School Host Microbiome Initiative.

Funding Statement

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Footnotes

Citation Spilker T, LiPuma JJ. 2016. Draft genome sequences of 63 Pseudomonas aeruginosa isolates recovered from cystic fibrosis sputum. Genome Announc 4(2):e00231-16. doi:10.1128/genomeA.00231-16.

REFERENCES

  • 1.Hauser AR, Jain M, Bar-Meir M, McColley SA. 2011. Clinical significance of microbial infection and adaptation in cystic fibrosis. Clin Microbiol Rev 24:29–70. doi: 10.1128/CMR.00036-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Hansen SK, Rau MH, Johansen HK, Ciofu O, Jelsbak L, Yang L, Folkesson A, Jarmer HØ, Aanæs K, von Buchwald C, Høiby N, Molin S. 2012. Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection. ISME J 6:31–45. doi: 10.1038/ismej.2011.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Caballero JD, Clark ST, Coburn B, Zhang Y, Wang PW, Donaldson SL, Tullis DE, Yau YCW, Waters VJ, Hwang DM, Guttman DS. 2015. Selective sweeps and parallel pathoadaptation drive Pseudomonas aeruginosa evolution in the cystic fibrosis lung. MBio 6:e00981-15. doi: 10.1128/mBio.00981-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Jorth P, Staudinger BJ, Wu X, Hisert KB, Hayden H, Garudathri J, Harding CL, Radey MC, Rezayat A, Bautista G, Berrington WR, Goddard AF, Zheng C, Angermeyer A, Brittnacher MJ, Kitzman J, Shendure J, Fligner CL, Mittler J, Aitken ML, Manoil C, Bruce JE, Yahr TL, Singh PK. 2015. Regional isolation drives bacterial diversification within cystic fibrosis lungs. Cell Host Microbe 18:307–319. doi: 10.1016/j.chom.2015.07.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.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]
  • 6.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Treangen TJ, Ondov BD, Koren S, Phillippy AM. 2014. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol 15:524. doi: 10.1186/s13059-014-0524-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Sahl JW, Beckstrom-Sternberg SM, Babic-Sternberg J, Gillece JD, Hepp CM, Auerbach RK, Tembe W, Wagner DM, Keim PS, Pearson T. 2015. The In Silico Genotyper (ISG): an open-source pipeline to rapidly identify and annotate nucleotide variants for comparative genomics applications. bioRxiv. doi: 10.1101/015578. [DOI] [Google Scholar]
  • 9.Delcher AL, Phillippy A, Carlton J, Salzberg SL. 2002. Fast algorithms for large-scale genome alignment and comparison. Nucleic Acids Res 30:2478–2483. doi: 10.1093/nar/30.11.2478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. doi: 10.1093/bioinformatics/btp324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. 2010. The Genome. Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303. doi: 10.1101/gr.107524.110. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genome Announcements are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES