Whole-Genome Enrichment Using RNA Probes and Sequencing of Chlamydia trachomatis Directly from Clinical Samples

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1616)

Abstract

Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow-growing, or obligate intracellular pathogens, such as Chlamydia trachomatis. However clinical samples typically contain too low levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a non-mutagenic approach, we can capture all known variations found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

Key words

Whole-genome enrichment Whole-genome sequencing Chlamydia trachomatis Clinical samples SureSelectXT 

References

  1. 1.
    Köser CU, Ellington MJ, Cartwright EJP et al (2012) Routine use of microbial whole genome sequencing in diagnostic and public health microbiology. PLoS Pathog 8:e1002824CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Köser CU, Bryant JM, Becq J et al (2013) Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med 369:290–292CrossRefPubMedGoogle Scholar
  3. 3.
    Olsen RJ, Long SW, Musser JM (2012) Bacterial genomics in infectious disease and the clinical pathology laboratory. Arch Pathol Lab Med 136:1414–1422CrossRefPubMedGoogle Scholar
  4. 4.
    WHO (2012) | Global incidence and prevalence of selected curable sexually transmitted infections - 2008. ISBN: 978 92 4 150383 9Google Scholar
  5. 5.
    WHO (2011) | Prevalence and incidence of selected sexually transmitted infections. Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis and Trichomonas vaginalis. Methods and results used by WHO to generate 2005 estimates. ISBN: 978 92 4 150245 0Google Scholar
  6. 6.
    Mylonas I (2012) Female genital Chlamydia trachomatis infection: where are we heading? Arch Gynecol Obstet 285:1271–1285CrossRefPubMedGoogle Scholar
  7. 7.
    Mariotti SP, Pascolini D, Rose-Nussbaumer J (2009) Trachoma: global magnitude of a preventable cause of blindness. Br J Ophthalmol 93:563–568CrossRefPubMedGoogle Scholar
  8. 8.
    Blandford JM, Gift TL (2006) Productivity losses attributable to untreated chlamydial infection and associated pelvic inflammatory disease in reproductive-aged women. Sex Transm Dis 33:S117–S121CrossRefPubMedGoogle Scholar
  9. 9.
    Burton MJ, Mabey DCW (2009) The global burden of trachoma: a review. PLoS Negl Trop Dis 3:e460CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Pedersen LN, Herrmann B, Møller JK (2009) Typing Chlamydia trachomatis: from egg yolk to nanotechnology. FEMS Immunol Med Microbiol 55:120–130CrossRefPubMedGoogle Scholar
  11. 11.
    Millman KL, Tavaré S, Dean D (2001) Recombination in the ompA gene but not the omcB gene of Chlamydia contributes to serovar-specific differences in tissue tropism, immune surveillance, and persistence of the organism. J Bacteriol 183:5997–6008CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Psarrakos P, Papadogeorgakis E, Sachse K et al (2011) Chlamydia trachomatis ompA genotypes in male patients with urethritis in Greece: conservation of the serovar distribution and evidence for mixed infections with Chlamydophila abortus. Mol Cell Probes 25:168–173CrossRefPubMedGoogle Scholar
  13. 13.
    Stothard DR, Boguslawski G, Jones RB (1998) Phylogenetic analysis of the Chlamydia trachomatis major outer membrane protein and examination of potential pathogenic determinants. Infect Immun 66:3618–3625PubMedPubMedCentralGoogle Scholar
  14. 14.
    Harris SR, Clarke IN, Seth-Smith HMB et al (2012) Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing. Nat Genet 44:413–419. S1CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    O’Neill CE, Seth-Smith HMB, Van Der Pol B et al (2013) Chlamydia trachomatis clinical isolates identified as tetracycline resistant do not exhibit resistance in vitro: whole-genome sequencing reveals a mutation in porB but no evidence for tetracycline resistance genes. Microbiology 159:748–756CrossRefPubMedGoogle Scholar
  16. 16.
    Seth-Smith HMB, Harris SR, Scott P et al (2013) Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA. Nat Protoc 8:2404–2412CrossRefPubMedGoogle Scholar
  17. 17.
    Seth-Smith HMB, Harris SR, Skilton RJ et al (2013) Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture. Genome Res 23:855–866CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Christiansen MT, Brown AC, Kundu S et al (2014) Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples. BMC Infect Dis 14:591CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Depledge DP, Palser AL, Watson SJ et al (2011) Specific capture and whole-genome sequencing of viruses from clinical samples. PLoS One 6:e27805CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Depledge DP, Kundu S, Jensen NJ et al (2014) Deep sequencing of viral genomes provides insight into the evolution and pathogenesis of varicella zoster virus and its vaccine in humans. Mol Biol Evol 31:397–409CrossRefPubMedGoogle Scholar
  21. 21.
    Brown AC, Bryant JM, Einer-Jensen K et al (2015) Rapid whole genome sequencing of M. tuberculosisdirectly from clinical samples. J Clin Microbiol 53(7):2230–2237Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Oxford Gene TechnologyOxfordUK
  2. 2.Department of Microbiology and ImmunologyCornell UniversityIthacaUSA
  3. 3.University College LondonLondonUK

Personalised recommendations