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DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

  • G.-J. Brandon-Mong (a1) (a2), H.-M. Gan (a3) (a4), K.-W. Sing (a1) (a2), P.-S. Lee (a1) (a2), P.-E. Lim (a5) and J.-J. Wilson (a1) (a2)...
Abstract
Abstract

Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

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Corresponding author
* Author for correspondence Fax: +603-7967-4178 E-mail: johnwilson@um.edu.my
References
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Altschul S.F., Gish W., Miller W., Myers E.W. & Lipman D.J. (1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403410.
Bartram A.K., Lynch M.D., Stearns J.C., Moreno-Hagelsieb G. & Neufeld J.D. (2011) Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end Illumina reads. Applied and Environmental Microbiology 77, 38463852.
Bohmann K., Monadjem A., Lehmkuhl-Noer C., Rasmussen M., Zeale M.R., Clare E., Jones G., Willerslev E. & Gilbert M.T. (2011) Molecular diet analysis of two African free-tailed bats (molossidae) using high throughput sequencing. PLoS ONE 6, e21441.
Boyer F., Mercier C., Bonin A., Taberlet P. & Coissac E. (2014) OBITools: a Unix-inspired software package for DNA metabarcoding. Available online at http://metabarcoding.org/obitools/doc/index.html (accessed 28 February 2015).
Boykin L.M., Armstrong K.F., Kubatko L. & De Barro P. (2012). Species delimitation and global biosecurity. Evolutionary Bioinformatics Online 8, 137.
Bucklin A., Steinke D. & Blanco-Bercial L. (2011) DNA barcoding of marine metazoa. Annual Review of Marine Science 3, 471508.
Burgar J.M., Murray D.C., Craig M.D., Haile J., Houston J., Stokes V. & Bunce M. (2014) Who's for dinner? High-throughput sequencing reveals bat dietary differentiation in a biodiversity hotspot where prey taxonomy is largely undescribed. Molecular Ecology 23, 36053617.
Clarke L.J., Soubrier J., Weyrich L.S. & Cooper A. (2014) Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias. Molecular Ecology Resources 14, 11601170.
Coissac E., Riaz T. & Puillandre N. (2012) Bioinformatic challenges for DNA metabarcoding of plants and animals. Molecular Ecology 21, 18341847.
Cristescu M.E. (2014) From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity. Trends in Ecology and Evolution 29, 566571.
Deagle B.E., Jarman S.N., Coissac E., Pompanon F. & Taberlet P. (2014) DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match. Biology Letters 10, 20140562.
Dereeper A., Guignon V., Blanc G., Audic S. & Buffet S. (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research 36, W465W469.
Edgar R.C. (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods 10, 996998.
Edgar R.C., Haas B.J., Clemente J.C., Quince C. & Knight R. (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 21942200.
Fu L., Niu B., Zhu Z., Wu S. & Li W. (2012) CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 31503152.
Fišer-Pečnikar Ž. & Buzan E.V. (2014) 20 years since the introduction of DNA barcoding: from theory to application. Journal of Applied Genetics 55, 4352.
Floyd R.M., Wilson J.J. & Hebert P.D.N. (2009) DNA barcodes and insect biodiversity. pp. 417431 in Foottit R.G. & Aler P.H. (Eds) Insect Biodiversity: Science and Society. Oxford, Blackwell Publishing Ltd.
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. Molecular Marine Biology and Biotechnology 3, 294299.
Gibson J., Shokralla S., Porter T.M., King I., van-Konynenburg S., Janzen D.H., Hallwachs W. & Hajibabaei M. (2014) Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics. Proceedings of the National Academy of Sciences of the United States of America 111, 80078012.
Gillison A.N., Bignell D.E., Brewer K.R.W., Fernandes E.C.M. & Jones D.T. (2013) Plant functional types and traits as biodiversity indicators for tropical forests: two biogeographically separated case studies including birds, mammals and termites. Biodiversity and Conservation 22, 19091930.
Glenn T.C. (2014) 2014 NGS Field Guide: Overview (The Molecular Ecologist). Available online at http://www.molecularecologist.com/next-gen-fieldguide-2014/ (accessed 28 February 2015).
Hall T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 9598.
Haas B.J., Gevers D., Earl A.M., Feldgarden M., Ward D.V., Giannoukos G., Ciulla D., Tabbaa D., Highlander S.K., Sodergren E., Methé B., DeSantis T.Z., Petrosino J.F., Knight R. & Birren B.W. (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Research 21, 494504.
Hart L.A., Bowker M.B., Tarboton W. & Downs C.T. (2014) Species composition, distribution and habitat types of Odonata in the iSimangaliso Wetland Park, KwaZulu-Natal, South Africa and the associated conservation implications. PLoS ONE 9, e92588.
Hajibabaei M., Smith M.A., Janzen D.H., Rodriguez J.J. & Whitefield J.B. (2006) A minimalist barcode can identify a specimen whose DNA is degraded. Molecular Ecology 6, 959964.
Hajibabaei M., Shokralla S., Zhou X., Singer G.A.C. & Baird D.J. (2011) Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos. PLoS ONE 6, e17497.
Hannon Lab. (2014) FASTX-Toolkit: FASTQ/A short-reads pre-processing tools. Available online at http://hannonlab.cshl.edu/fastx_toolkit/index.html (accessed 5 May 2015).
Hebert P.D.N., Cywinska A., Ball S.L. & deWaard J.R. (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B, Biological Sciences 270, 313321.
Hebert P.D.N., Penton E.H., Burns J.M., Janzen D.H. & Hallwachs W. (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator . Proceedings of the National Academy of Sciences of the United States of America 101, 1481214817.
Hope P.R., Bohmann K., Gilbert M.T., Zepeda-Mendoza M.L., Razgour O. & Jones G. (2014) Second generation sequencing and morphological faecal analysis reveal unexpected foraging behaviour by Myotis nattereri (Chiroptera, Vespertilionidae) in winter. Frontiers in Zoology 11, 39.
Ishii K. & Fukui M. (2001) Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR. Applied and Environmental Microbiology 67, 37533755.
Ji Y., Ashton L., Pedley S.M., Edwards D.P., Tang Y., Nakamura A., Kitching R., Dolman P.M., Woodcock P., Edwards F.A., Larsen T.H., Hsu W.W., Benedick S., Hamer K.C., Wilcove D.S., Bruce C., Wang X., Levi T., Lott M., Emerson B.C. & Yu D.W. (2013) Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding. Ecology Letters 16, 12451257.
Kress W.J., García-Robledo C., Uriarte M. & Erickson D.L. (2015) DNA barcodes for ecology, evolution, and conservation. Trends in Ecology and Evolution 30, 2535.
Kircher M., Heyn P. & Kelso J. (2011) Addressing challenges in the production and analysis of Illumina sequencing data. BMC Genomics 12, 382.
Korasaki V., Lopes J., Gardner-Brown G. & Louzada J. (2013) Using dung beetles to evaluate the effects of urbanization on Atlantic Forest biodiversity. Insect Science 20, 393406.
Lahr D.J. & Katz L.A. (2009) Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase. BioTechniques 47, 857866.
Lange A. (2015) Statistical analysis of amplicon data of the same sample to identify artefacts. Available online at http://cran.r-project.org/web/packages/AmpliconDuo/AmpliconDuo.pdf (accessed 5 May 2015).
Lee P.S., Sing K.W. & Wilson J.J. (2015) Reading mammal diversity from flies: the persistence period of amplifiable mammal mtDNA in blowfly guts (Chrysomya megacephala) and a new DNA mini-barcode target. PLoS ONE 10, e0123871.
Leray M. & Knowlton N. (2015) DNA barcoding and metabarcoding of standardized samples reveal patterns of marine benthic diversity. Proceedings of the National Academy of Sciences of the United States of America 112, 20762081.
Leray M., Yang J.Y., Meyer C.P., Mills S.C., Agudelo N., Ranwez V., Boehm J.T. & Machida R.J. (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. Frontiers in Zoology 10, 34.
Liu S., Li Y., Lu J., Su X., Tang M., Zhang R., Zhou L., Zhou C., Yang Q., Ji Y., Yu D.W. & Zhou X. (2013) SOAPBarcode: revealing arthropod biodiversity through assembly of Illumina shotgun sequences of PCR amplicons. Methods in Ecology and Evolution 4, 11421150.
Magnacca K.N. & Brown M.J. (2010) Mitochondrial heteroplasmy and DNA barcoding in Hawaiian Hylaeus (Nesoprosopis) bees (Hymenoptera: Colletidae). BMC Evolutionary Biology 10, 174.
Martin D.P., Lemey P., Lott M., Moulton V., Posada D. & Lefeuvre P. (2010) RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26, 24622463.
Meusnier I., Singer G.A., Landry J.F., Hickey D.A., Hebert P.D.N. & Hajibabaei M. (2008) A universal DNA mini-barcode for biodiversity analysis. BMC Genomics 9, 214.
Mora C., Tittensor D.P., Adl S., Simpson A.G. & Worm B. (2011) How many species are there on Earth and in the ocean? PLOS Biology 9, e1001127.
Moulton M.J., Song H. & Whiting M.F. (2010) Assessing the effects of primer specificity on eliminating numt coamplification in DNA barcoding: a case study from Orthoptera (Arthropoda: Insecta). Molecular Ecology Resources 10, 615627.
Nelson M.C., Morrison H.G., Benjamino J., Grim S.L. & Graf J. (2014) Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys. PLoS ONE 9, e94249.
Piñol J., San-Andrés V., Clare E.L., Mir G. & Symondson W.O. (2014 a) A pragmatic approach to the analysis of diets of generalist predators: the use of next-generation sequencing with no blocking probes. Molecular Ecology Resources 14, 1826.
Piñol J., Mir G., Gomez-Polo P. & Agustí N. (2014 b) Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods. Molecular Ecology Resources 15, 819830.
Quail M.A., Smith M., Coupland P., Otto T.D., Harris S.R., Connor T.R., Bertoni A., Swerdlow H.P. & Gu Y. (2012) A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13, 341.
Ratnasingham S. & Hebert P.D.N. (2007) BOLD: the Barcode of Life Data System (www.barcodinglife.org). Molecular Ecology Notes 7, 355364.
Razgour O., Clare E.L., Zeale M.R., Hanmer J., Schnell I.B., Rasmussen M., Gilbert T.P. & Jones G. (2011) High-throughput sequencing offers insight into mechanisms of resource partitioning in cryptic bat species. Ecology and Evolution 1, 556570.
Rozen S. & Skaletsky H.J. (2000) Primer3 on the www for general users and for biologist programmers. pp. 365386 in Krawetz S. & Misener S. (Eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. New Jersey, Humana Press.
Rubinoff D., Cameron S. & Will K. (2006) A genomic perspective on the shortcomings of mitochondrial DNA for ‘barcoding’ identification. Journal of Heredity 97, 581594.
Russo L., Stehouwer R., Heberling J.M. & Shea K. (2011) The composite insect trap: an innovative combination trap for biologically diverse sampling. PLoS ONE 6, e21079.
Shokralla S., Gibson J.F., Nikbakht H., Janzen D.H., Hallwachs W. & Hajibabaei M. (2014) Next-generation barcoding: using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens. Molecular Ecology Resources 14, 892901.
Sipos R., Székely A.J., Palatinszky M., Révész S., Márialigeti K. & Nikolausz M. (2007) Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis. FEMS Microbiology Ecology 60, 341350.
Smith M.A., Fisher B.L. & Hebert P.D.N. (2005) DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 360, 18251834.
Smith M.A., Poyarkov N.A. Jr. & Hebert P.D.N. (2008 a) CO1 DNA barcoding amphibians: take the chance, meet the challenge. Molecular Ecology Resources 8, 235246.
Smith M.A. & Rodriguez J.J., Whitfield J.B., Deans A.R., Janzen D.H., Hallwachs W. & Hebert P.D.N. (2008 b) Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections. Proceedings of the National Academy of Sciences of the United States of America 105, 1235912364.
Song H., Buhay J.E., Whiting M.F. & Crandall K.A. (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proceedings of the National Academy of Sciences of the United States of America 105, 1348613491.
Stevens J.L., Jackson R.L. & Olson J.B. (2013) Slowing PCR ramp speed reduces chimera formation from environmental samples. Journal of Microbiological Methods 93, 203205.
Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30, 27252729.
Tang M., Tan M., Meng G., Yang S., Su X., Liu S., Song W., Li Y., Wu Q., Zhang A. & Zhou X. (2014) Multiplex sequencing of pooled mitochondrial genomes-a crucial step toward biodiversity analysis using mito-metagenomics. Nucleic Acids Research 42, e166.
Townsend J.P. & Rand D.M. (2004) Mitochondrial genome size variation in New World and Old World populations of Drosophila melanogaster . Heredity 93, 98103.
Triplehorn C.A. & Johnson N.F. (2005) Borror and DeLong's Introduction to the Study of Insects. 7th edn. California, Thomson Brooks/Cole.
Vesterinen E.J., Lilley T., Laine V.N. & Wahlberg N. (2013) Next generation sequencing of fecal DNA reveals the dietary diversity of the widespread insectivorous predator Daubenton's Bat (Myotis daubentonii) in Southwestern Finland. PLoS ONE 8, e82168.
Wilson J.J. (2012) DNA barcodes for insects. pp. 1745 in Kress W.J. & Erikson D.L. (Eds) DNA Barcodes: Methods and Protocols. New York, Humana Press.
Wilson J.J. & Sing K.W. (2013) DNA barcoding can successfully identify Penaeus monodon, associate life cycle stages, and generate hypotheses of unrecognized diversity. Sains Malaysiana 42, 18271829.
Wilson J.J., Rougerie R., Schonfeld J., Janzen D.H., Hallwachs W., Hajibabaei M., Kitching I.J., Haxaire J. & Hebert P.D.N. (2011) When species matches are unavailable are DNA barcodes correctly assigned to higher taxa? An assessment using sphingid moths. BMC Ecology 11, 18.
Wong M.M., Lim C.L. & Wilson J.J. (2015) DNA barcoding implicates 23 species and four orders as potential pollinators of Chinese knotweed (Persicaria chinensis) in peninsular Malaysia. Bulletin of Entomological Research 105, 515520.
Yang C.X., Wang X.Y., Miller J.A., Marleen-de-Blécourt , Ji Y.Q., Yang C.Y., Harrison R.D. & Yu D.W. (2014) Using metabarcoding to ask if easily collected soil and leaf-litter samples can be used as a general biodiversity indicator. Ecological Indicators 46, 379389.
Yu D.W., Ji Y.Q., Emerson B.C., Wang X.Y., Ye C.X., Yang C.Y. & Ding Z.L. (2012) Biodiversity soup: metabarcoding of arthropods for rapid biodiversity assessment and biomonitoring. Methods in Ecology and Evolution 4, 613623.
Zeale M.R.K., Butlin R.K., Barker G.L.A., Lees D.C. & Jones G. (2011) Taxon-specific PCR for DNA barcoding arthropod prey in bat faeces. Molecular Ecology Resources 11, 236244.
Zhang J., Kobert K., Flouri T. & Stamatakis A. (2014) PEAR: a fast and accurate Illumina paired-end reAD mergeR. Bioinformatics 30, 614620.
Zhou X., Li Y., Liu S., Yang Q., Su X., Zhou L., Tang M., Fu R., Li J. & Huang Q. (2013) Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification. GigaScience 2, 4.
Zografou K., Kati V., Grill A., Wilson R.J., Tzirkalli E., Pamperis L.N., Halley J.M. (2014) Signals of climate change in butterfly communities in a Mediterranean protected area. PLoS ONE 9, e87245.
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