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Microbial Genomics logo Microbial Genomics

Volume 11, Issue 11

cultured in MGIT media for whole-genome sequencing application: a systematic literature review and meta-analysis Open Access

Abstract

Whole-genome sequencing (WGS) holds promise for accurate and comprehensive diagnosis of drug resistance in and identification of transmission events. ‘Early positive cultures’ (EPC) are increasingly used when WGS is implemented to guide clinical care to reduce the turnaround time. We performed a systematic literature review to compare methods used for EPC-based WGS and performed an individual sample data meta-analysis to identify variables associated with bioinformatic quality measures. Of 423 studies identified, 15 met eligibility criteria. We analysed 1,065 FASTQ files from 11 studies using Illumina sequencing; 96.1% passed all quality control thresholds. Median genome coverage was 65× (IQR, 63–82), with a pooled mapping percentage of 91.2%. The meta-analysis showed that the number of sequencing cycles was significantly associated with improved sequencing quality, while other laboratory variables had no consistent effect. Based on these findings, we suggest replacing the term EPC with ‘clinical primary culture’ and propose a standardized workflow and reporting checklist for WGS on primary Mycobacteria Growth Indicator Tube (MGIT) cultures.

  • Received:
  • Accepted:
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Keyword(s): DNA extraction , early positive culture , liquid culture , primary culture , tuberculosis and whole-genome sequencing
Funding
This study was supported by the:
  • Key Digital Technologies Joint Undertaking (Award 101103171)
    • Principal Award Recipient: Túliode Oliveira
  • South African Medical Research Council
    • Principal Award Recipient: RobinMark Warren
  • Research Foundation Flanders (FWO) through the TORCH consortium FWO TBM (Applied Biomedical Research with a Primary Social finality) (Award T001018N)
    • Principal Award Recipient: AnneliesVan Rie
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2025-11-24
2025-12-16

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References

  1. Rageade F, Picot N, Blanc-Michaud A, Chatellier S, Mirande C et al. Performance of solid and liquid culture media for the detection of Mycobacterium tuberculosis in clinical materials: meta-analysis of recent studies. Eur J Clin Microbiol Infect Dis 2014; 33:867–870 [View Article] [PubMed]
     [Google Scholar]
  2. Wilson ML, Stone BL, Hildred MV, Reves RR. Comparison of recovery rates for mycobacteria from BACTEC 12B vials, Middlebrook 7H11-selective 7H11 biplates, and lowenstein jensen slants in a public health mycobacteriology laboratory. J Clin Microbiol 1995; 33:2516–2518 [View Article] [PubMed]
     [Google Scholar]
  3. Middlebrook G, Reggiardo Z, Tigertt WD. Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. Am Rev Respir Dis 1977; 115:1066–1069 [View Article] [PubMed]
     [Google Scholar]
  4. Tortoli E, Lavinia F, Simonetti MT. Early detection of Mycobacterium tuberculosis in BACTEC cultures by ligase chain reaction. J Clin Microbiol 1998; 36:2791–2792 [View Article] [PubMed]
     [Google Scholar]
  5. Ellner PD, Kiehn TE, Cammarata R, Hosmer M. Rapid detection and identification of pathogenic mycobacteria by combining radiometric and nucleic acid probe methods. J Clin Microbiol 1988; 26:1349–1352 [View Article] [PubMed]
     [Google Scholar]
  6. Peterson EM, Lu R, Floyd C, Nakasone A, Friedly G et al. Direct identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare from amplified primary cultures in BACTEC media using DNA probes. J Clin Microbiol 1989; 27:1543–1547 [View Article] [PubMed]
     [Google Scholar]
  7. Body BA, Warren NG, Spicer A, Henderson D, Chery M. Use of gen-probe and bactec for rapid isolation and identification of mycobacteria. Correlation of probe results with growth index. Am J Clin Pathol 1990; 93:415–420 [View Article] [PubMed]
     [Google Scholar]
  8. Köser CU, Bryant JM, Becq J, Török ME, Ellington MJ et al. Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med 2013; 369:290–292 [View Article] [PubMed]
     [Google Scholar]
  9. Votintseva AA, Pankhurst LJ, Anson LW, Morgan MR, Gascoyne-Binzi D et al. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures. J Clin Microbiol 2015; 53:1137–1143 [View Article] [PubMed]
     [Google Scholar]
  10. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. Int J Surg 2021; 88:105906 [View Article] [PubMed]
     [Google Scholar]
  11. Heupink TH, Verboven L, Warren RM, Van Rie A. Comprehensive and accurate genetic variant identification from contaminated and low-coverage Mycobacterium tuberculosis whole genome sequencing data. Microbial Genomics 2021; 7: [View Article]
     [Google Scholar]
  12. Heupink TH, Verboven L, Sharma A, Rennie V, de Diego Fuertes M et al. The MAGMA pipeline for comprehensive genomic analyses of clinical Mycobacterium tuberculosis samples. PLoS Comput Biol 2023; 19:e1011648 [View Article] [PubMed]
     [Google Scholar]
  13. Bogaerts B, Delcourt T, Soetaert K, Boarbi S, Ceyssens P-J et al. A bioinformatics whole-genome sequencing workflow for clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches. J Clin Microbiol 2021; 59:e00202-21 [View Article] [PubMed]
     [Google Scholar]
  14. Soetaert K, Ceyssens P-J, Boarbi S, Bogaerts B, Delcourt T et al. Retrospective evaluation of routine whole genome sequencing of Mycobacterium tuberculosis at the Belgian National Reference Center, 2019. Acta Clin Belg 2022; 77:853–860 [View Article] [PubMed]
     [Google Scholar]
  15. Wu X, Tan G, Sha W, Liu H, Yang J et al. Use of whole-genome sequencing to predict Mycobacterium tuberculosis complex drug resistance from early positive liquid cultures. Microbiol Spectr 2022; 10:e0251621 [View Article] [PubMed]
     [Google Scholar]
  16. Pankhurst LJ, Del Ojo Elias C, Votintseva AA, Walker TM, Cole K et al. Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study. Lancet Respir Med 2016; 4:49–58 [View Article] [PubMed]
     [Google Scholar]
  17. Cirillo DM, Cabibbe AM, De Filippo MR, Trovato A, Simonetti T et al. Use of WGS in Mycobacterium tuberculosis routine diagnosis. Int J Mycobacteriol 2016; 5 Suppl 1:S252–S253 [View Article] [PubMed]
     [Google Scholar]
  18. Nimmo C, Shaw LP, Doyle R, Williams R, Brien K et al. Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture. BMC Genomics 2019; 20: [View Article]
     [Google Scholar]
  19. Zakham F, Laurent S, Esteves Carreira AL, Corbaz A, Bertelli C et al. Whole-genome sequencing for rapid, reliable and routine investigation of Mycobacterium tuberculosis transmission in local communities. New Microbes New Infect 2019; 31:100582 [View Article] [PubMed]
     [Google Scholar]
  20. Votintseva AA, Bradley P, Pankhurst L, del Ojo Elias C, Loose M et al. Same-day diagnostic and surveillance data for tuberculosis via whole-genome sequencing of direct respiratory samples. J Clin Microbiol 2017; 55:1285–1298 [View Article]
     [Google Scholar]
  21. Wyllie DH, Robinson E, Peto T, Crook DW, Ajileye A et al. Identifying mixed Mycobacterium tuberculosis infection and laboratory cross-contamination during mycobacterial sequencing programs. J Clin Microbiol 2018; 56:e00923-18 [View Article] [PubMed]
     [Google Scholar]
  22. Doyle RM, Burgess C, Williams R, Gorton R, Booth H et al. Direct whole-genome sequencing of sputum accurately identifies drug-resistant Mycobacterium tuberculosis faster than MGIT culture sequencing. J Clin Microbiol 2018; 56:e00666-18 [View Article] [PubMed]
     [Google Scholar]
  23. Shea J, Halse TA, Lapierre P, Shudt M, Kohlerschmidt D et al. Comprehensive whole-genome sequencing and reporting of drug resistance profiles on clinical cases of Mycobacterium tuberculosis in New York state. J Clin Microbiol 2017; 55:1871–1882 [View Article] [PubMed]
     [Google Scholar]
  24. Shea J, Halse TA, Kohlerschmidt D, Lapierre P, Modestil HA et al. Low-level rifampin resistance and rpoB mutations in Mycobacterium tuberculosis: an analysis of whole-genome sequencing and drug susceptibility test data in New York. J Clin Microbiol 2021; 59:e01885-20 [View Article] [PubMed]
     [Google Scholar]
  25. Smith C, Halse TA, Shea J, Modestil H, Fowler RC et al. Assessing nanopore sequencing for clinical diagnostics: a comparison of next-generation sequencing (NGS) methods for Mycobacterium tuberculosis. J Clin Microbiol 2020; 59:e00583-20 [View Article] [PubMed]
     [Google Scholar]
  26. Metcalfe JZ, Streicher E, Theron G, Colman RE, Penaloza R et al. Mycobacterium tuberculosis subculture results in loss of potentially clinically relevant heteroresistance. Antimicrob Agents Chemother 2017; 61:e00888-17 [View Article] [PubMed]
     [Google Scholar]
  27. Dohál M, Porvazník I, Pršo K, Rasmussen EM, Solovič I et al. Whole-genome sequencing and Mycobacterium tuberculosis: challenges in sample preparation and sequencing data analysis. Tuberculosis (Edinb) 2020; 123:101946 [View Article] [PubMed]
     [Google Scholar]
  28. Tyler AD, Christianson S, Knox NC, Mabon P, Wolfe J et al. Comparison of sample preparation methods used for the next-generation sequencing of Mycobacterium tuberculosis. PLoS One 2016; 11:e0148676 [View Article] [PubMed]
     [Google Scholar]
  29. van Soolingen D, de Haas PE, Hermans PW, Groenen PM, van Embden JD. Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis. J Clin Microbiol 1993; 31:1987–1995 [View Article] [PubMed]
     [Google Scholar]
  30. Motro Y, Moran-Gilad J. Next-generation sequencing applications in clinical bacteriology. Biomol Detect Quantif 2017; 14:1–6 [View Article] [PubMed]
     [Google Scholar]
  31. Illumina Nextera XT DNA Library Prep (Reference Guide) Illumina, Inc; 2019
     [Google Scholar]
  32. Biolabs NE. NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (Instruction Manual) New England Biolabs: Inc; 2022
     [Google Scholar]
  33. Illumina TruSeq® Nano DNA Library Prep (Reference Guide) Illumina, Inc; 2015
     [Google Scholar]
  34. Hess JF, Kohl TA, Kotrová M, Rönsch K, Paprotka T et al. Library preparation for next generation sequencing: a review of automation strategies. Biotechnol Adv 2020; 41:107537 [View Article] [PubMed]
     [Google Scholar]
  35. Illumina Balancing sample coverage for whole-genome sequencing: Index correction strategies for Illumina DNA PCR-Free Prep [Internet]; 2021 https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/illumina-dna-prep-pcr-free-index-correction-tech-note-m-gl-00005/illumina-dna-pcr-free-index-correction-tech-note-m-gl-00005.pdf accessed 11 January 2023
  36. Head SR, Komori HK, LaMere SA, Whisenant T, Van Nieuwerburgh F et al. Library construction for next-generation sequencing: overviews and challenges. Biotechniques 2014; 56:61–64 [View Article] [PubMed]
     [Google Scholar]
  37. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55:611–622 [View Article] [PubMed]
     [Google Scholar]
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Mycobacterium tuberculosis cultured in MGIT media for whole-genome sequencing application: a systematic literature review and meta-analysis
M Gen 11, 001565 (2025); https://doi.org/10.1099/mgen.0.001565
/content/journal/mgen/10.1099/mgen.0.001565
/content/journal/mgen/10.1099/mgen.0.001565
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