Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Microbiology Resource Announcements
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Latest Articles
    • Archive
  • Types of Resources
    • Amplicon Sequence Collections
    • Culture Collections/Mutant Libraries
    • Databases and Software
    • Omics Data Sets
    • Other Genetic Resources
    • Genome Sequences
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About MRA
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Microbiology Resource Announcements
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Microbiology Resource Announcements
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Latest Articles
    • Archive
  • Types of Resources
    • Amplicon Sequence Collections
    • Culture Collections/Mutant Libraries
    • Databases and Software
    • Omics Data Sets
    • Other Genetic Resources
    • Genome Sequences
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About MRA
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
Genome Sequences

Complete Genome Assemblies for Three Variants of the Wolbachia Endosymbiont of Drosophila melanogaster

Preston J. Basting, Casey M. Bergman
Julie C. Dunning Hotopp, Editor
Preston J. Basting
aInstitute of Bioinformatics, University of Georgia, Athens, Georgia, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Preston J. Basting
Casey M. Bergman
aInstitute of Bioinformatics, University of Georgia, Athens, Georgia, USA
bDepartment of Genetics, University of Georgia, Athens, Georgia, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Casey M. Bergman
Julie C. Dunning Hotopp
University of Maryland School of Medicine
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/MRA.00956-19
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Here, we report genome assemblies for three strains of Wolbachia pipientis, assembled from unenriched, unfiltered long-read shotgun sequencing data of geographically distinct strains of Drosophila melanogaster. Our simple methodology can be applied to long-read data sets of other Wolbachia-infected species with limited Wolbachia-host lateral gene transfers to produce complete assemblies for this important model symbiont.

ANNOUNCEMENT

Wolbachia pipientis is a widespread bacterial endosymbiont that infects 40% of arthropod species (1) and induces a wide range of effects including cytoplasmic incompatibility, feminization, male killing, and parthenogenesis (2). Currently, our understanding of the impact of Wolbachia on its hosts is limited by the lack of complete reference genomes for different Wolbachia strains, with only 18 of 84 Wolbachia assemblies in the NCBI assembly database as of August 2019 defined as complete.

Recently, Faddeeva-Vakhrusheva et al. (3) showed that a complete assembly of Wolbachia could be generated as a by-product of assembling the genome of a Wolbachia-infected arthropod species using PacBio long-read sequences. Based on this observation, we attempted to generate complete Wolbachia assemblies using long-read shotgun sequencing data for three geographically distinct Drosophila melanogaster lines (I23 from Ithaca, NY; N25 from the Netherlands; and ZH26 from Zimbabwe) (4) that were previously identified by Early and Clark (5) as being infected with variants of the Wolbachia strain wMel. These flies were reared on a diet of 10% yeast, 10% glucose, and 1% agar at 25°C (J. Chaston, personal communication). As described by Long et al. (4), DNA was extracted by grinding ∼200 adult flies in liquid nitrogen, transferring to a solution of buffer G2 with 38 μl of RNase A (100 mg/ml) and 500 μl of proteinase K (catalog number 158920; Qiagen), incubating the solution overnight at 50°C, and then extracting DNA using the Qiagen Genomic-tip kit (catalog number 10243). DNA was then sequenced on a PacBio Sequel instrument (Pacific Biosciences, Inc.) using 2 or 3 single-molecule real-time (SMRT) cells per sample. Additionally, public short-read Illumina sequencing was used for the same D. melanogaster lines (5). As described by Early and Clark (5), DNA for these samples was extracted from 50 adult female flies using the Qiagen DNeasy blood and tissue kit. Sequencing was performed on an Illumina HiSeq 2000 instrument to produce 100-bp paired-end reads with a 450- to 500-bp insert size. No quality control steps were applied to the PacBio or Illumina sequencing reads prior to assembly and polishing.

All reads from PacBio whole-genome shotgun sequences of each strain were assembled using CANU v1.8 (genomeSize=137.7m, useGrid=False) (6). Assemblies for each strain generated only one contig matching the wMel reference genome (GenBank accession number NC_002978) by BLASTN v2.9.0 search with default parameters (7), which in each case corresponded to the entire Wolbachia genome. Repetitive regions from the ends of uncircularized Wolbachia contigs were trimmed using minimus2 from AMOS v3.1.0 with default parameters (8). The trimmed Wolbachia contigs were then adjusted using BLASTN v2.9.0 (7) and faFrag (9) with default parameters so that the start of each contig matched the wMel reference start. The contigs were then polished using Arrow (SMRTlink v6.0.0.47841; Pacific Biosciences) and Pilon v1.23 (10) using Illumina reads from Early and Clark (5) (Table 1) mapped to the contigs using BWA-MEM v0.7.17 with default parameters (11).

View this table:
  • View inline
  • View popup
  • Download powerpoint
TABLE 1

Accession numbers and statistics for assemblies produced and raw sequencing data used in this study

After polishing, we identified 54, 13, and 18 single-nucleotide polymorphism (SNP)/indel variants for the I23, N25, and ZH26 Wolbachia strains, respectively, relative to the wMel reference genome. The higher similarity of the N25 and ZH26 Wolbachia strains and increased divergence of the I23 Wolbachia strain relative to the wMel reference genome are consistent with previous work showing that Wolbachia genomes from lines N25 and ZH26 are both in clade III of the wMel phylogeny (which also contains the wMel reference genome), while the Wolbachia genome from line I23 is in clade I of the wMel phylogeny (which is more divergent from the wMel reference genome) (5, 12).

Our work extends that of Faddeeva-Vakhrusheva et al. (3) by showing that high-quality, complete genome assemblies of Wolbachia strains can be generated without experimental enrichment of symbiont DNA (e.g., references 13 and 14). Successful de novo assembly of complete Wolbachia genomes directly from unenriched long-read sequences also demonstrates that it is unnecessary to computationally filter symbiont reads from host reads based on similarity to Wolbachia reference genomes prior to assembly (15, 16). We expect this process to be particularly useful for Wolbachia-infected hosts with few host-symbiont lateral gene transfer events, such as D. melanogaster (17), in which there will be few hybrid reads between host and symbiont to confound the assembly process. As the cost of long-read sequencing decreases, we argue that direct sequencing and assembly of unenriched, unfiltered long-read data sets could be applied easily to other Wolbachia-infected arthropod and nematode species to expand the number of complete Wolbachia reference genomes.

Data availability.The assemblies produced in this study were deposited at NCBI under accession number PRJNA557362. PacBio data used to generate these assemblies were published by Long et al. (4) and are available under SRA accession number SRP142531. Illumina data used to polish the assemblies were published by Early and Clark (5) and are available under SRA accession SRP050151. Accession numbers for assemblies produced and raw read data used in this study are given in Table 1.

ACKNOWLEDGMENTS

We thank the Georgia Advanced Computing Resource Center for computational resources, Joshua Udall (Iowa State University) for providing access to raw PacBio Sequel data, and John Chaston (Brigham Young University) for information about PacBio samples used in this project.

This work was supported by a University of Georgia Research Education Award Traineeship (P.J.B.) and by the University of Georgia Research Foundation (C.M.B.).

FOOTNOTES

    • Received 12 August 2019.
    • Accepted 15 October 2019.
    • Published 7 November 2019.
  • Copyright © 2019 Basting and Bergman.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

REFERENCES

  1. 1.↵
    1. Zug R,
    2. Hammerstein P
    . 2012. Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS One 7:e38544. doi:10.1371/journal.pone.0038544.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Werren JH,
    2. Baldo L,
    3. Clark ME
    . 2008. Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 6:741–751. doi:10.1038/nrmicro1969.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    1. Faddeeva-Vakhrusheva A,
    2. Kraaijeveld K,
    3. Derks MFL,
    4. Anvar SY,
    5. Agamennone V,
    6. Suring W,
    7. Kampfraath AA,
    8. Ellers J,
    9. Le Ngoc G,
    10. van Gestel CAM,
    11. Mariën J,
    12. Smit S,
    13. van Straalen NM,
    14. Roelofs D
    . 2017. Coping with living in the soil: the genome of the parthenogenetic springtail Folsomia candida. BMC Genomics 18:493. doi:10.1186/s12864-017-3852-x.
    OpenUrlCrossRef
  4. 4.↵
    1. Long E,
    2. Evans C,
    3. Chaston J,
    4. Udall JA
    . 2018. Genomic structural variations within five continental populations of Drosophila melanogaster. G3 (Bethesda) 8:3247–3253. doi:10.1534/g3.118.200631.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. Early AM,
    2. Clark AG
    . 2013. Monophyly of Wolbachia pipientis genomes within Drosophila melanogaster: geographic structuring, titre variation and host effects across five populations. Mol Ecol 22:5765–5778. doi:10.1111/mec.12530.
    OpenUrlCrossRefWeb of Science
  6. 6.↵
    1. Koren S,
    2. Walenz BP,
    3. Berlin K,
    4. Miller JR,
    5. Bergman NH,
    6. Phillippy AM
    . 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736. doi:10.1101/gr.215087.116.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Camacho C,
    2. Coulouris G,
    3. Avagyan V,
    4. Ma N,
    5. Papadopoulos J,
    6. Bealer K,
    7. Madden TL
    . 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421. doi:10.1186/1471-2105-10-421.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Treangen TJ,
    2. Sommer DD,
    3. Angly FE,
    4. Koren S,
    5. Pop M
    . 2011. Next generation sequence assembly with AMOS. Curr Protoc Bioinformatics Chapter 11:Unit 11.8. doi:10.1002/0471250953.bi1108s33.
    OpenUrlCrossRef
  9. 9.↵
    1. Kuhn RM,
    2. Haussler D,
    3. Kent WJ
    . 2013. The UCSC genome browser and associated tools. Brief Bioinform 14:144–161. doi:10.1093/bib/bbs038.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Walker BJ,
    2. Abeel T,
    3. Shea T,
    4. Priest M,
    5. Abouelliel A,
    6. Sakthikumar S,
    7. Cuomo CA,
    8. Zeng Q,
    9. Wortman J,
    10. Young SK,
    11. Earl AM
    . 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. doi:10.1371/journal.pone.0112963.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Li H,
    2. Durbin R
    . 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. doi:10.1093/bioinformatics/btp324.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    1. Richardson MF,
    2. Weinert LA,
    3. Welch JJ,
    4. Linheiro RS,
    5. Magwire MM,
    6. Jiggins FM,
    7. Bergman CM
    . 2012. Population genomics of the Wolbachia endosymbiont in Drosophila melanogaster. PLoS Genet 8:e1003129. doi:10.1371/journal.pgen.1003129.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Iturbe-Ormaetxe I,
    2. Woolfit M,
    3. Rancès E,
    4. Duplouy A,
    5. O'Neill SL
    . 2011. A simple protocol to obtain highly pure Wolbachia endosymbiont DNA for genome sequencing. J Microbiol Methods 84:134–136. doi:10.1016/j.mimet.2010.10.019.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    1. Lefoulon E,
    2. Vaisman N,
    3. Frydman HM,
    4. Sun L,
    5. Foster JM,
    6. Slatko BE
    . 2019. Large enriched fragment targeted sequencing (LEFT-SEQ) applied to capture of Wolbachia genomes. Sci Rep 9:5939. doi:10.1038/s41598-019-42454-w.
    OpenUrlCrossRef
  15. 15.↵
    1. Salzberg SL,
    2. Dunning Hotopp JC,
    3. Delcher AL,
    4. Pop M,
    5. Smith DR,
    6. Eisen MB,
    7. Nelson WC
    . 2005. Serendipitous discovery of Wolbachia genomes in multiple Drosophila species. Genome Biol 6:R23. doi:10.1186/gb-2005-6-3-r23.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Pascar J,
    2. Chandler CH
    . 2018. A bioinformatics approach to identifying Wolbachia infections in arthropods. PeerJ 6:e5486. doi:10.7717/peerj.5486.
    OpenUrlCrossRef
  17. 17.↵
    1. Huang W,
    2. Massouras A,
    3. Inoue Y,
    4. Peiffer J,
    5. Ramia M,
    6. Tarone AM,
    7. Turlapati L,
    8. Zichner T,
    9. Zhu D,
    10. Lyman RF,
    11. Magwire MM,
    12. Blanken- Burg K,
    13. Carbone MA,
    14. Chang K,
    15. Ellis LL,
    16. Fernandez S,
    17. Han Y,
    18. High- Nam G,
    19. Hjelmen CE,
    20. Jack JR,
    21. Javaid M,
    22. Jayaseelan J,
    23. Kalra D,
    24. Lee S,
    25. Lewis L,
    26. Munidasa M,
    27. Ongeri F,
    28. Patel S,
    29. Perales L,
    30. Perez A,
    31. Pu L,
    32. Rollmann SM,
    33. Ruth R,
    34. Saada N,
    35. Warner C,
    36. Williams A,
    37. Wu YQ,
    38. Yamamoto A,
    39. Zhang Y,
    40. Zhu Y,
    41. Anholt RRH,
    42. Korbel JO,
    43. Mittel- Man D,
    44. Muzny DM,
    45. Gibbs RA,
    46. Barbadilla A,
    47. Johnston JS,
    48. Stone EA,
    49. Richards S,
    50. Deplancke B,
    51. Mackay T
    . 2014. Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines. Genome Res 24:1193–1208. doi:10.1101/gr.171546.113.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Complete Genome Assemblies for Three Variants of the Wolbachia Endosymbiont of Drosophila melanogaster
Preston J. Basting, Casey M. Bergman
Microbiology Resource Announcements Nov 2019, 8 (45) e00956-19; DOI: 10.1128/MRA.00956-19

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print
Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Microbiology Resource Announcements article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Complete Genome Assemblies for Three Variants of the Wolbachia Endosymbiont of Drosophila melanogaster
(Your Name) has forwarded a page to you from Microbiology Resource Announcements
(Your Name) thought you would be interested in this article in Microbiology Resource Announcements.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Complete Genome Assemblies for Three Variants of the Wolbachia Endosymbiont of Drosophila melanogaster
Preston J. Basting, Casey M. Bergman
Microbiology Resource Announcements Nov 2019, 8 (45) e00956-19; DOI: 10.1128/MRA.00956-19
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • ANNOUNCEMENT
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About MRA
  • Editor in Chief
  • Board of Editors
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • Getting Started
  • Submit a Manuscript
  • Author Warranty
  • Ethics
  • Contact Us
  • ASM Author Center

Follow #MRAJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Online ISSN: 2576-098X