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Metabarcoding Marine Sediments: Preparation of Amplicon Libraries

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Marine Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1452))

Abstract

The accurate assessment of community composition and ultimately species identification is of utmost importance in any ecological and evolutionary study. Advances in sequencing technologies have allowed the unraveling of levels of biodiversity never imagined before when applied to large-scale environmental DNA studies (also termed metabarcoding/metagenetics/metasystematics/environmental barcoding). Here, we describe a detailed protocol to assess eukaryotic biodiversity in marine sediments, identifying key steps that should not be neglected when preparing Next-Generation Sequencing (NGS) amplicon libraries: DNA extraction, multiple PCR amplification of DNA barcode markers with index/ tag-primers, and final Illumina MiSeq sequencing library preparation.

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References

  1. Metzker ML (2010) Sequencing technologies – the next generation. Nat Rev Genet 11(1):31–46. doi:10.1038/nrg2626

    Article  CAS  PubMed  Google Scholar 

  2. Taberlet P, Coissac E, Pompanon F, Brochmann C, Willerslev E (2012) Towards next-generation biodiversity assessment using DNA metabarcoding. Mol Ecol 21(8):2045–2050. doi:10.1111/j.1365-294X.2012.05470.x

    Article  CAS  PubMed  Google Scholar 

  3. Bik HM, Porazinska DL, Creer S, Caporaso JG, Knight R, Thomas WK (2012) Sequencing our way towards understanding global eukaryotic biodiversity. Trends Ecol Evol 27(4):233–243. doi:10.1016/j.tree.2011.11.010

    Article  PubMed  PubMed Central  Google Scholar 

  4. Lallias D, Hiddink JG, Fonseca VG, Gaspar JM, Sung W, Neill SP, Barnes N, Ferrero T, Hall N, Lambshead PJ, Packer M, Thomas WK, Creer S (2015) Environmental metabarcoding reveals heterogeneous drivers of microbial eukaryote diversity in contrasting estuarine ecosystems. ISME J 9:1208–1221. doi:10.1038/ismej.2014.213

    Article  PubMed  Google Scholar 

  5. Fonseca V, Carvalho G, Nichols B, Quince C, Johnson H, Neill S, Lambshead P, Thomas W, Power D, Creer S (2014) Metagenetic analysis of patterns of distribution and diversity of marine meiobenthic eukaryotes. Glob Ecol Biogeogr 23(11):1293–1302. doi:10.1111/geb.12223

    Article  Google Scholar 

  6. Fonseca V, Carvalho G, Sung W, Johnson H, Power D, Neill S, Packer M, Blaxter M, Lambshead P, Thomas W, Creer S (2010) Second-generation environmental sequencing unmasks marine metazoan biodiversity. Nat Commun 1(7):98. doi:10.1038/ncomms1095

    Article  PubMed  PubMed Central  Google Scholar 

  7. Gibson J, Shokralla S, Porter TM, King I, van Konynenburg S, Janzen DH, Hallwachs W, Hajibabaei M (2014) Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics. Proc Natl Acad Sci U S A 111(22):8007–8012. doi:10.1073/pnas.1406468111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shokralla S, Porter TM, Gibson JF, Dobosz R, Janzen DH, Hallwachs W, Golding GB, Hajibabaei M (2015) Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform. Sci Rep 5:9687. doi:10.1038/srep09687

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Baird DJ, Hajibabaei M (2012) Biomonitoring 2.0: a new paradigm in ecosystem assessment made possible by next-generation DNA sequencing. Mol Ecol 21(8):2039–2044

    Article  PubMed  Google Scholar 

  10. Leray M, Knowlton N (2015) DNA barcoding and metabarcoding of standardized samples reveal patterns of marine benthic diversity. Proc Natl Acad Sci U S A 112(7):2076–2081. doi:10.1073/pnas.1424997112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored "rare biosphere". Proc Natl Acad Sci U S A 103(32):12115–12120. doi:10.1073/pnas.0605127103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bett BJ, Vanreusel A, Vincx M, Soltwedel T, Pfannkuche O, Lambshead PJD, Gooday AJ, Ferrero T, Dinet A (1994) Sampler bias in the quantitative study of deep-sea meiobenthos. Mar Ecol Prog Ser 104(1-2):197–203

    Article  Google Scholar 

  13. Bohmann K, Evans A, Gilbert MT, Carvalho GR, Creer S, Knapp M, Yu DW, de Bruyn M (2014) Environmental DNA for wildlife biology and biodiversity monitoring. Trends Ecol Evol 29(6):358–367. doi:10.1016/j.tree.2014.04.003

    Article  PubMed  Google Scholar 

  14. Creer S, Fonseca V, Porazinska D, Giblin-Davis R, Sung W, Power D, Packer M, Carvalho G, Blaxter M, Lambshead P, Thomas W (2010) Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises. Mol Ecol 19:4–20. doi:10.1111/j.1365-294X.2009.04473.x

    Article  PubMed  Google Scholar 

  15. Chao A, Colwell RK, Lin CW, Gotelli NJ (2009) Sufficient sampling for asymptotic minimum species richness estimators. Ecology 90(4):1125–1133

    Article  PubMed  Google Scholar 

  16. O'Mahony EM, Tay WT, Paxton RJ (2007) Multiple rRNA variants in a single spore of the microsporidian Nosema bombi. J Eukaryot Microbiol 54(1):103–109, doi:JEU232 [pii] 10.1111/j.1550-7408.2006.00232.x

    Article  PubMed  Google Scholar 

  17. Medinger R, Nolte V, Pandey RV, Jost S, Ottenwalder B, Schlotterer C, Boenigk J (2010) Diversity in a hidden world: potential and limitation of next-generation sequencing for surveys of molecular diversity of eukaryotic microorganisms. Mol Ecol 19(Suppl 1):32–40. doi:10.1111/j.1365-294X.2009.04478.x

    Article  PubMed  PubMed Central  Google Scholar 

  18. Wu JY, Jiang XT, Jiang YX, Lu SY, Zou F, Zhou HW (2010) Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method. BMC Microbiol 10:255. doi:10.1186/1471-2180-10-255

    Article  PubMed  PubMed Central  Google Scholar 

  19. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methe B, Desantis TZ, Petrosino JF, Knight R, Birren BW (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21(3):494–504. doi:10.1101/gr.112730.110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fonseca VG, Nichols B, Lallias D, Quince C, Carvalho GR, Power DM, Creer S (2012) Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses. Nucleic Acids Res 40(9), e66. doi:10.1093/nar/gks002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. von Wintzingerode F, Gobel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21(3):213–229

    Article  Google Scholar 

  22. Creer S (2010) Second-generation sequencing derived insights into the temporal biodiversity dynamics of freshwater protists. Mol Ecol 19(14):2829–2831. doi:10.1111/j.1365-294X.2010.04670.x

    Article  PubMed  Google Scholar 

  23. Wang GC, Wang Y (1996) The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species. Microbiology 142:1107–1114

    Article  CAS  PubMed  Google Scholar 

  24. Wang GC, Wang Y (1997) Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. Appl Environ Microbiol 63(12):4645–4650

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Huse SM, Welch DM, Morrison HG, Sogin ML (2010) Ironing out the wrinkles in the rare biosphere through improved OTU clustering. Environ Microbiol 12:1889–1898. doi:10.1111/j.1462-2920.2010.02193.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kunin V, Engelbrektson A, Ochman H, Hugenholtz P (2009) Wrinkles in the rare biosphere: pyrosequencing errors lead to artificial inflation of diversity estimates. Environ Microbiol. doi:10.1111/j.1462-2920.2009.02051.x

    PubMed  Google Scholar 

  27. Quince C, Lanzen A, Curtis TP, Davenport RJ, Hall N, Head IM, Read LF, Sloan WT (2009) Accurate determination of microbial diversity from 454 pyrosequencing data. Nat Methods 6(9):639–641. doi:10.1038/nmeth.1361

    Article  CAS  PubMed  Google Scholar 

  28. Qiu X, Wu L, Huang H, McDonel PE, Palumbo AV, Tiedje JM, Zhou J (2001) Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Appl Environ Microbiol 67(2):880–887. doi:10.1128/AEM.67.2.880-887.2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lahr DJ, Katz LA (2009) Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase. Biotechniques 47(4):857–866. doi:10.2144/000113219

    CAS  PubMed  Google Scholar 

  30. Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F, Ochman H, Hugenholtz P (2010) Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J 4(5):642–647, doi:ismej2009153 [pii] 10.1038/ismej.2009.153

    Article  CAS  PubMed  Google Scholar 

  31. Schloss PD, Gevers D, Westcott SL (2011) Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One 6(12), e27310. doi:10.1371/journal.pone.0027310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hadziavdic K, Lekang K, Lanzen A, Jonassen I, Thompson EM, Troedsson C (2014) Characterization of the 18S rRNA gene for designing universal eukaryote specific primers. PLoS One 9(2), e87624. doi:10.1371/journal.pone.0087624

    Article  PubMed  PubMed Central  Google Scholar 

  33. Amaral-Zettler LA, McCliment EA, Ducklow HW, Huse SM (2009) A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PLoS One 4(7), e6372. doi:10.1371/journal.pone.0006372

    Article  PubMed  PubMed Central  Google Scholar 

  34. Leray M, Yang JY, Meyer CP, Mills SC, Agudelo N, Ranwez V, Boehm JT, Machida RJ (2013) A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents. Front Zool 10:34. doi:10.1186/1742-9994-10-34

    Article  PubMed  PubMed Central  Google Scholar 

  35. Tang CQ, Leasi F, Obertegger U, Kieneke A, Barraclough TG, Fontaneto D (2012) The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna. Proc Natl Acad Sci U S A 109(40):16208–16212. doi:10.1073/pnas.1209160109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Epp LS, Boessenkool S, Bellemain EP, Haile J, Esposito A, Riaz T, Erseus C, Gusarov VI, Edwards ME, Johnsen A, Stenoien HK, Hassel K, Kauserud H, Yoccoz NG, Brathen KA, Willerslev E, Taberlet P, Coissac E, Brochmann C (2012) New environmental metabarcodes for analysing soil DNA: potential for studying past and present ecosystems. Mol Ecol 21(8):1821–1833. doi:10.1111/j.1365-294X.2012.05537.x

    Article  CAS  PubMed  Google Scholar 

  37. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6(8):1621–1624. doi:10.1038/ismej.2012.8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Stoeck T, Bass D, Nebel M, Christen R, Jones MDM, Breiner H-W, Richards TA (2010) Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol Ecol 19:21–31. doi:10.1111/j.1365-294X.2009.04480.x

    Article  CAS  PubMed  Google Scholar 

  39. Walters WA, Caporaso JG, Lauber CL, Berg-Lyons D, Fierer N, Knight R (2011) PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers. Bioinformatics 27(8):1159–1161. doi:10.1093/bioinformatics/btr087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Deagle BE, Jarman SN, Coissac E, Pompanon F, Taberlet P (2014) DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match. Biol Lett 10(9):pii: 20140562. doi:10.1098/rsbl.2014.0562

    Article  Google Scholar 

  41. Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, Pallen MJ (2012) Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol 30(5):434–439. doi:10.1038/nbt.2198

    Article  CAS  PubMed  Google Scholar 

  42. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23):7537–7541. doi:10.1128/AEM.01541-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7(5):335–336. doi:10.1038/nmeth.f.303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Boyer F, Mercier C, Bonin A, Le Bras Y, Taberlet P, Coissac E (2015) Obitools: a unix-inspired software package for DNA metabarcoding. Mol Ecol Resour. doi:10.1111/1755-0998.12428

    Google Scholar 

  45. Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DG, Nilsson RH (2015) metaxa2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Mol Ecol Resour. doi:10.1111/1755-0998.12399

    Google Scholar 

  46. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20(14):2317–2319

    Article  CAS  PubMed  Google Scholar 

  47. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2006) New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl Environ Microbiol 72(9):5734–5741. doi:10.1128/AEM.00556-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ (2011) Removing noise from pyrosequenced amplicons. BMC Bioinformatics 12(1):38, doi:1471-2105-12-38 [pii] 10.1186/1471-2105-12-38

    Article  PubMed  PubMed Central  Google Scholar 

  49. He Y, Caporaso JG, Jiang XT, Sheng HF, Huse SM, Rideout JR, Edgar RC, Kopylova E, Walters WA, Knight R, Zhou HW (2015) Stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity. Microbiome 3:20. doi:10.1186/s40168-015-0081-x

    Article  PubMed  PubMed Central  Google Scholar 

  50. Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, Vanfleteren JR, Mackey LY, Dorris M, Frisse LM, Vida JT, Thomas WK (1998) A molecular evolutionary framework for the phylum Nematoda. Nature 392(6671):71–75

    Article  CAS  PubMed  Google Scholar 

  51. Fonseca V, Power D, Carvalho G, Lambshead J, Packer M, Creer S (2011) Isolation of marine meiofauna from sandy sediments: from decanting to DNA extraction. Nat Protoc Exchange doi:10.1038/nprot.2010.157

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Authors and Affiliations

  1. Zoological Research Museum Alexander Koenig (ZFMK), Centre for Molecular Biodiversity Research, Adenauerallee 160, 53113, Bonn, Germany

    Vera G. Fonseca

  2. Institut National de la Recherche Agronomique (INRA), Unité Génétique en Aquaculture, Centre de Jouy-en-Josas, Domaine de Vilvert, 78350, Jouy-en-Josas, France

    Delphine Lallias

Authors
  1. Vera G. Fonseca
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  2. Delphine Lallias
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Correspondence to Vera G. Fonseca .

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Editors and Affiliations

  1. Department of Marine Sciences, University of Gothenburg, Gothenburg, Sweden

    Sarah J. Bourlat

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Fonseca, V.G., Lallias, D. (2016). Metabarcoding Marine Sediments: Preparation of Amplicon Libraries. In: Bourlat, S. (eds) Marine Genomics. Methods in Molecular Biology, vol 1452. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3774-5_12

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  • DOI: https://doi.org/10.1007/978-1-4939-3774-5_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3772-1

  • Online ISBN: 978-1-4939-3774-5

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