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Multiplexed Non-barcoded Long-Read Sequencing and Assembling Genomes of Bacillus Strains in Error-Free Simulations

  • Jiating Qian
  • Qiao Meng
  • Yifan Feng
  • Xuanxuan Mao
  • Yayue Ling
  • Jie LiEmail author
Article

Abstract

The generation of genomic data from microorganisms has revolutionized our abilities to understand their biology, but it is still challenging to obtain complete genome sequences of microbes in an automated high-throughput and cost-effective manner. While the advent of second-generation sequencing technologies provided significantly higher throughput, their shorter lengths and more pronounced sequence-context bias led to a shift towards resequencing applications. Recently, single molecule real-time (SMRT) DNA sequencing has been used to generate sequencing reads that are much longer than other sequencing platforms, facilitating de novo genome assembly and genome finishing. Here we introduced a novel multiplex strategy to make full use of the capacity and characteristics of SMRT sequencing in microbe genome assembly. We used error-free simulations to evaluate the practicability of assembling SMRT genomic sequencing data from multiple microbes into finished genomes once at a time. Then we compared the influence of two key factors, including sequencing coverage and read length, on multiplex assembling. Our results showed that long-read genomic sequencing inherently provided the ability to assemble genomic sequencing data from multiple microbes into finished genomes due to its long length. This approach might be helpful for the various groups of microbial genome projects or metagenomics research.

Notes

Acknowledgements

This work is supported by Teachers' Research Start-up Fund from Changshu Institute of Technology (KYZ2018009Q), the Natural Science Foundation of Jiangsu Province (BK20181034). The fundings have no role in the design of the study and collection, analysis, and interpretation of data and writing the manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Fraser CM, Eisen JA, Nelson KE, Paulsen IT, Salzberg SL (2002) The value of complete microbial genome sequencing (you get what you pay for). J Bacteriol 184(23):6403–6405CrossRefGoogle Scholar
  2. 2.
    Medini D, Serruto D, Parkhill J, Relman DA, Donati C, Moxon R, Falkow S, Rappuoli R (2008) Microbiology in the post-genomic era. Nat Rev Microbiol 6:419–430CrossRefGoogle Scholar
  3. 3.
    Gagarinova A, Emili A (2012) Genome-scale genetic manipulation methods for exploring bacterial molecular biology. Mol Biosyst 8:1626–1638CrossRefGoogle Scholar
  4. 4.
    Loman NJ, Constantinidou C, Chan JZ, Halachev M, Sergeant M, Penn CW, Robinson ER, Pallen MJ (2012) High-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity. Nat Rev Microbiol 10:599–606CrossRefGoogle Scholar
  5. 5.
    Korlach J (2014) Returning to more finished genomes. Genome Data 2:46–48CrossRefGoogle Scholar
  6. 6.
    Parkhill J, Wren BW (2011) Bacterial epidemiology and biology—lessons from genome sequencing. Genome Biol 12:230CrossRefGoogle Scholar
  7. 7.
    Nagarajan N, Cook C, Di Bonaventura M, Ge H, Richards A, Bishop-Lilly KA, DeSalle R, Read TD, Pop M (2010) Finishing genomes with limited resources: lessons from an ensemble of microbial genomes. BMC Genomics 11:242CrossRefGoogle Scholar
  8. 8.
    Chain PS, Grafham DV, Fulton RS, Fitzgerald MG, Hostetler J, Muzny D, Ali J, Birren B, Bruce DC, Buhay C, Cole JR, Ding Y, Dugan S, Field D, Garrity GM, Gibbs R, Graves T, Han CS, Harrison SH, Highlander S, Hugenholtz P, Khouri HM, Kodira CD, Kolker E, Kyrpides NC, Lang D, Lapidus A, Malfatti SA, Markowitz V, Metha T, Nelson KE, Parkhill J, Pitluck S, Qin X, Read TD, Schmutz J, Sozhamannan S, Sterk P, Strausberg RL, Sutton G, Thomson NR, Tiedje JM, Weinstock G, Wollam A, Genomic Standards Consortium Human Microbiome Project Jumpstart Consortium, Detter JC (2009) Genome project standards in a new era of sequencing. Science 326(5950):236–237CrossRefGoogle Scholar
  9. 9.
    Ricker N, Qian H, Fulthorpe RR (2012) The limitations of draft assemblies for understanding prokaryotic adaptation and evolution. Genomics 100:167–175CrossRefGoogle Scholar
  10. 10.
    Harrison J, Studholme DJ (2014) Recently published Streptomyces genome sequences. Microb Biotechnol 7(5):373–380CrossRefGoogle Scholar
  11. 11.
    Mardis ER (2013) Next-generation sequencing platforms. Annu Rev Anal Chem 6:287–303CrossRefGoogle Scholar
  12. 12.
    Roberts RJ, Carneiro MO, Schatz MC (2013) The advantages of SMRT sequencing. Genome Biol 14(7):405CrossRefGoogle Scholar
  13. 13.
    Koren S, Phillippy AM (2015) One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly comparisons. Curr Opin Microbiol 23:110–120CrossRefGoogle Scholar
  14. 14.
    Ribeiro FJ, Przybylski D, Yin S, Sharpe T, Gnerre S, Abouelleil A, Berlin AM, Montmayeur A, Shea TP, Walker BJ, Young SK, Russ C, Nusbaum C, MacCallum I, Jaffe DB (2012) Finished bacterial genomes from shotgun sequence data. Genome Res 22:2270–2277CrossRefGoogle Scholar
  15. 15.
    Koren S, Schatz MC, Walenz BP, Martin J, Howard JT, Ganapathy G, Wang Z, Rasko DA, McCombie WR, Jarvis ED, Phillippy AM (2012) Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat Biotechnol 30:693–700CrossRefGoogle Scholar
  16. 16.
    Bashir A, Klammer AA, Robins WP, Chin CS, Webster D, Paxinos E, Hsu D, Ashby M, Wang S, Peluso P, Sebra R, Sorenson J, Bullard J, Yen J, Valdovino M, Mollova E, Luong K, Lin S, LaMay B, Joshi A, Rowe L, Frace M, Tarr CL, Turnsek M, Davis BM, Kasarskis A, Mekalanos JJ, Waldor MK, Schadt EE (2012) A hybrid approach for the automated finishing of bacterial genomes. Nat Biotechnol 30:701–707CrossRefGoogle Scholar
  17. 17.
    Prjibelski AD, Vasilinetc I, Bankevich A, Gurevich A, Krivosheeva T, Nurk S, Pham S, Korobeynikov A, Lapidus A, Pevzner PA (2014) ExSPAnder: a universal repeat resolver for DNA fragment assembly. Bioinformatics 30(12):i293–301CrossRefGoogle Scholar
  18. 18.
    Boetzer M, Pirovano W (2014) SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information. BMC Bioinform 15:211CrossRefGoogle Scholar
  19. 19.
    Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J (2013) Nonhybrid, finished microbial genome assemblies from long-read smrtsequencing data. Nat Methods 10(6):563–569CrossRefGoogle Scholar
  20. 20.
    Koren S, Harhay GP, Smith TP, Bono JL, Harhay DM, Mcvey SD, Radune D, Bergman NH, Phillippy AM (2013) Reducing assembly complexity of microbial genomes with single-molecule sequencing. Genome Biol 14(9):R101CrossRefGoogle Scholar
  21. 21.
    Lin HH, Liao YC (2015) Evaluation and validation of assembling corrected PacBio long reads for microbial genome completion via hybrid approaches. PLoS ONE 10(12):e0144305CrossRefGoogle Scholar
  22. 22.
    Sohn JI, Nam JW (2016) The present and future of de novo whole-genome assembly. Brief Bioinform 19:23–40Google Scholar
  23. 23.
    Liao YC, Lin SH, Lin HH (2015) Completing bacterial genome assemblies: strategy and performance. Sci Rep 5:8747CrossRefGoogle Scholar
  24. 24.
    Wong KH, Jin Y, Moqtaderi Z (2013) Multiplex illumina sequencing using DNA barcoding. Curr Protoc Mol Biol 101(7):11Google Scholar
  25. 25.
    Lam KK, Khalak A, Tse D (2014) Near-optimal assembly for shotgun sequencing with noisy reads. BMC Bioinform 15(9):S4CrossRefGoogle Scholar
  26. 26.
    Salmela L, Walve R, Rivals E, Ukkonen E (2016) Accurate self-correction of errors in long reads using de Bruijn graphs. Bioinformatics 33:799–806PubMedCentralGoogle Scholar
  27. 27.
    Wenger AM, Peluso P, Rowell WJ, Chang PC, Hall RJ, Concepcion GT, Ebler J, Fungtammasan A, Kolesnikov A, Olson ND, Töpfer A, Alonge M, Mahmoud M, Qian Y, Chin CS, Phillippy AM, Schatz MC, Myers G, DePristo MA, Ruan J, Marschall T, Sedlazeck FJ, Zook JM, Li H, Koren S, Carroll A, Rank DR, Hunkapiller MW. 2019. Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nat Biotechnol. 1–8Google Scholar
  28. 28.
    Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G, Sekowska A, Vallenet D, Wang T, Moszer I, Médigue C, Danchin A (2009) From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology 155(Pt 6):1758–1775CrossRefGoogle Scholar
  29. 29.
    Rhee MS, Moritz BE, Xie G, Glavina Del Rio T, Dalin E, Tice H, Bruce D, Goodwin L, Chertkov O, Brettin T, Han C, Detter C, Pitluck S, Land ML, Patel M, Ou M, Harbrucker R, Ingram LO, Shanmugam KT (2011) Complete genome sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1. Stand Genomic Sci 5(3):331–340CrossRefGoogle Scholar
  30. 30.
    Djukic M, Poehlein A, Thürmer A, Daniel R (2011) Genome sequence of Brevibacillus laterosporus LMG 15441, a pathogen of invertebrates. J Bacteriol 193(19):5535–5536CrossRefGoogle Scholar
  31. 31.
    Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36:W5–9CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Biology and Food EngineeringChangshu Institute of TechnologySuzhouChina

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