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DNA Barcoding and Metabarcoding Protocols for Species Identification

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DNA Barcoding

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2744))

Abstract

The article presents the several steps to be performed on a plant, fungal, insect, or soil sample to obtain DNA sequences for DNA barcode analysis. The chapter begins with a description of sample preparation including procedures for cleaning and proceeds to DNA extraction with methods adapted for the specific type of sample. Next, DNA quantification is described so the proper amount is used for the amplification of the selected barcode regions. Information is provided for reaction mixes and amplification conditions for several referenced barcode primer pairs tuned for the individual sample of interest. This is followed by a description of procedures to access the success of amplification, cleanup, and quantification of the product ready for either Sanger sequencing or library preparation for massive parallel sequencing (MPS). Finally, procedures are provided for Sanger sequencing, library preparation, and MPS sequencing. The chapter provides several references of barcode regions for different sample types.

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References

  1. Cui X, Li W, Wei J, Qi Y, Li R, Yang Y et al (2020) Assessing the identity of commercial herbs from a Cambodian market using DNA barcoding. Front Pharmacol 11:244. https://doi.org/10.3389/fphar.2020.00244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Robertson JM, Damaso N, Meiklejohn KA (2021) DNA-based analysis of plant material in forensic investigations. In: Dash HR et al (eds) Handbook of DNA profiling. Springer Nature Singapore. https://doi.org/10.1007/978-981-15-9364-2_59-1

    Chapter  Google Scholar 

  3. Pikitch EK (2018) A tool for finding rare marine species. Science 360:1180–1182. https://doi.org/10.1126/science.aa03787

    Article  CAS  PubMed  Google Scholar 

  4. Bandelt H-J, Lahermo P, Richards M, Macaulay (2001) Detecting errors in mtDNA data by phylogenetic analysis. Int J Legal Med 115:64–69. https://doi.org/10.1007/s004140100228

    Article  CAS  PubMed  Google Scholar 

  5. Kress W, Erickson D (2012) DNA barcodes: methods and protocols. Methods Mol Biol 858:3–8. https://doi.org/10.1007/978-1-61779-591-6_1

    Article  CAS  PubMed  Google Scholar 

  6. EMP500 Sample Submission Guide. https://earthmicrobiome.org/protocols-and-standards/emp500-sample-submission-guide/

  7. OSAC 2022-N-0039 collecting and preserving entomological evidence from a terrestrial environment. https://www.nist.gov/system/files/documents/2022/08/31/OSAC%202022-N-0039_Collecting%20and%20Preserving%20Entomological%20Evidence%20from%20a%20Terrestrial%20Environment.OPEN%20CO.pdf

  8. ISO 11063.2020-Direct extraction of soil DNA. Iso.org/standard/75810.html

  9. ISO 23611 Part 1–6 Extraction of live specimens of various groups of soil fauna. ISO.org

  10. ISO 23611-6:2012 (en) Soil quality-sampling of soil invertrebates-Part6: Guidance for the design of sampling programmes with soil invertebrates. ISO.org/standard/50778.html

  11. Thompson LR, Sanders JG, Mc Donald D, Amir A, Jansson JK, Gilbert JA, Knight R, The Earth Microbiome Project Consortium (2017) A communal catalogue reveals earth’s multiscale microbial diversity. Nature 551:457–463. https://doi.org/10.1038/nature24621. https://earthmicrobiome.org

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Harris SE, Bellino M (2013) DNA barcoding from NYC to Belize. Science 342:1462–1463. https://doi.org/10.1126/science.1230006

    Article  CAS  PubMed  Google Scholar 

  13. Handy SM, Deeds JR, Ivanova NV, Herbert PDN, Hanner R, Ormes A et al (2011) Single laboratory validated method for DNA-barcoding for the species identification of fish. https://www.fda.gov/food/dna-based-seafood-identification/single-laboratory-validated-method-dna-barcoding-species-identification-fish

  14. Meiklejohn KA, Jackson ML, Stern LA, Robertson JM (2018) A protocol for obtaining DNA barcodes from plant and insect fragments isolated from forensic-type soils. Intl J Legal Med 132:1515–1526. https://doi.org/10.1007/s00414-018-1772-1

    Article  Google Scholar 

  15. Meiklejohn KA, Damaso N, Robertson JM (2019) Assessment of BOLD and GenBank-their accuracy and reliability for the identification of biological materials. PLoS One 14:e0217084. https://doi.org/10.1371/journal.pone.0217084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Boggs LM, Scheible MKR, Machado G, Meiklejohn KA (2019) Single fragment or bulk soil DNA metabarcoding: which is better for characterizing biological taxa found in surface soils for sample separation? Genes 10(6):431. https://doi.org/10.3390/genes10060431

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nelson LA, Wallman JF, Dowton M (2007) Using COI barcodes to identify forensically and medically important blowflies. Med Vet Entomol 21:44–52. https://doi.org/10.1111/j.1365-2915.2007.00664x

    Article  CAS  PubMed  Google Scholar 

  18. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Marine Biol Biotechnol 3:294–299

    CAS  Google Scholar 

  19. Hebert PD, Penton EH, Burns JM, Janzen DH, Hallwachs W (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci U S A 101:14812–14817. https://doi.org/10.1073/pnas/0406166101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hajibabaei M, Janzen DH, Burns JM, Hallwachs W, Hebert PD (2006) DNA barcodes distinguish species of tropical Lepidoptera. Proc Natl Acad Sci U S A 103:968–971. https://doi.org/10.1073/pnas.0510466103

    Article  PubMed  PubMed Central  Google Scholar 

  21. Meusnier I, Singer GAC, Landry J-F, Hickey DA, Hebert PDN, Hajibabaei M (2008) A universal DNA mini-barcode for biodiversity analysis. BMC Genomics 9(1):214. https://doi.org/10.1186/1471-2164-9-214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, Cambridge, pp 315–322

    Google Scholar 

  23. Levin RA, Wagner WL, Hoch PC, Nepokroeff M, Pires JC, Zimmer EA (2003) Family-level relationships of onagraceae based on chloroplast rbcl and ndhf data. Am J Bot 90:107–115. https://doi.org/10.3732/ajb.90.1.107

    Article  CAS  PubMed  Google Scholar 

  24. Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O et al (2009) Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proc Natl Acad Sci U S A 106:18621–18626. https://doi.org/10.1073/pnas.0909820106

    Article  PubMed  PubMed Central  Google Scholar 

  25. Fazekas AJ, Burgess KS, Kesanakurti PR, Graham SW, Newmaster SG, Husband BC et al (2008) Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS One 3:e2802. https://doi.org/10.1371/journal.pone.0002802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Little DP (2014) A DNA mini-barcode for land plants. Mol Ecol Res 14:437–446. https://doi.org/10.1111/1755-0998.12194

    Article  CAS  Google Scholar 

  27. Fazekas AJ, Kuzmina ML, Newmaster SG, Hollingsworth PM (2012) Methods in molecular biology. In: Erickson D, Kress W (eds) DNA barcodes methods and protocols. Humana Press, New York, pp 223–252. https://doi.org/10.1007/978-1-61779-591-5-1

    Chapter  Google Scholar 

  28. Costion C, Ford A, Cross H, Crayn D, Harrington M, Lowe A (2011) Plant DNA barcodes can accurately estimate species richness in poorly known floras. PLoS One 6:e26841. https://doi.org/10.1371/journal.pone.0026841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ford CS, Ayres KL, Toomey N, Haider N, Stahl JVA, Kelly LJ et al (2009) Selection of candidate coding DNA barcoding regions for use on land plants. Bot J Linn Soc 159:1–11. https://doi.org/10.1111/j.1095-8339.2008.00938.x

    Article  Google Scholar 

  30. Dunning LT, Savolainen V (2010) Broad-scale amplification of matK for DNA barcoding plants, a technical note. Bot J Linn Soc 164:1–9. https://doi.org/10.1111/j.1095-8339.2010.01071.x

    Article  Google Scholar 

  31. Wojciechowski MF, Lavin M, Sanderson MJ (2004) A phylogeny of legumes (Leguminosae) based on the analysis of the plastid matK gene resolves many well-supported subclades within the family. Am J Bot 91(11):1846–1862. https://doi.org/10.3732/ajb.91.11.1846

    Article  CAS  PubMed  Google Scholar 

  32. Cuénoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase MW (2002) Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am J Bot 89:132–144. https://doi.org/10.3732/ajb.89.1.132

    Article  PubMed  Google Scholar 

  33. Li Y, Gao LM, Poudel RC, Li DZ, Forrest A (2011) High universality of matK primers for barcoding gymnosperms. J Syst Evol 49:169–175. https://doi.org/10.1111/j.1759-6831.2011.00128.x

    Article  Google Scholar 

  34. Forrest A (2012) DNA barcoding of plants: matK primers for mosses. Royal Botanic Garden Edinburgh, Edinburgh

    Google Scholar 

  35. Forrest A (2012) DNA barcoding of plants: matK primers for ferns and allies. Royal Botanic Garden Edinburgh, Edinburgh

    Google Scholar 

  36. Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am J Bot 84:1120–1136. https://doi.org/10.2307/2446155

    Article  CAS  PubMed  Google Scholar 

  37. Tate JA, Simpson BB (2003) Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Syst Bot 28:723–737. https://doi.org/10.1043/02-64.1

    Article  Google Scholar 

  38. Hamilton M (1999) Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific variation. Mol Ecol 8:521–523

    CAS  PubMed  Google Scholar 

  39. Li X, Yang Y, Henry RJ, Rossetto M, Wang Y, Chen S (2015) Plant DNA barcoding: from gene to genome. Biol Rev 90:157–166. https://doi.org/10.1111/brv.12104

    Article  PubMed  Google Scholar 

  40. Garrido-Sanz L, Senar MÀ, Piñol J (2019) Estimation of the relative abundance of species in artificial mixtures of insects using low-coverage shotgun metagenomics. Metabarcoding and Metagenomics 4:e48281. https://doi.org/10.3897/mbmg.4.48281

    Article  Google Scholar 

  41. Nevill PG, Zhong X, Tonti-Filippini J, Byrne M, Hislop M, Thiele K, van Leeuwen S, Boykin LM, Small I (2020) Large-scale skimming from herbarium material for accurate plant identification and phylogenetics. Plant Methods 16:1. Doi: https://doi.org/10.1186/s13007-019-0534-5

  42. Wagemaker CAM, Mommer L, Visser EJW, Weigelt A, van Gurp TP, Postuma M, Smit-Tiekstra AE, de Kroon H (2021) msGBS: a new high-throughput approach to quantify the relative species abundance in root samples of multispecies plant communities. Mol Ecol Resour 21:1021–1036. https://doi.org/10.1111/1755-0998.13278

    Article  CAS  PubMed  Google Scholar 

  43. Scientific Working Group on DNA Analysis Methods (SWGDAM). Contamination Prevention and Detection Guidelines for Forensic DNA Laboratories. January 12th, 2017. https://www.swgdam.org/_files/ugd/4344b0_c4d4dbba84f1400a98eaa2e48f2bf291.pdf

  44. Miller DN, Bryant JE, Madsen EL, Ghiorse WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 65(11):4715–4724. https://doi.org/10.1128/AEM.65.11.4715-4724.1999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This is publication number 23.07 of the Laboratory Division of the Federal Bureau of Investigation. Names of commercial products and manufacturers are provided for information only and inclusion does not imply endorsement by the FBI or the US Government. The views expressed are those of the authors and do not necessarily reflect the official policy or position of the FBI or the US Government.

Natalie Damaso is currently an MIT Lincoln Laboratory employee. No MIT Laboratory funding or resources were used to produce the result/findings reported in this publication.

Author information

Authors and Affiliations

  1. Research & Support Unit, Laboratory Division, Federal Bureau of Investigation, Quantico, VA, USA

    Kyleen E. Elwick & James M. Robertson

  2. Counter WMD Systems, Massachusetts Institute of Technology, Lincoln Laboratory, Lexington, MA, USA

    Natalie Damaso

Authors
  1. Kyleen E. Elwick
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  2. Natalie Damaso
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  3. James M. Robertson
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Corresponding author

Correspondence to James M. Robertson .

Editor information

Editors and Affiliations

  1. Division of Invertebrate Zoology, American Museum of Natural History, New York, NY, USA

    Robert DeSalle

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Elwick, K.E., Damaso, N., Robertson, J.M. (2024). DNA Barcoding and Metabarcoding Protocols for Species Identification. In: DeSalle, R. (eds) DNA Barcoding. Methods in Molecular Biology, vol 2744. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3581-0_9

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  • DOI: https://doi.org/10.1007/978-1-0716-3581-0_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3580-3

  • Online ISBN: 978-1-0716-3581-0

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