Validation of COI metabarcoding primers for terrestrial arthropods

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids ecosystem assessment. However , it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. Thus this study tests the performance of 36 primer sets on a mock community containing 374 species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets where also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from the 21 amplicon pools of the mock community and Malaise trap sample were used to select four primer sets for metabarcoding evaluation at different annealing temperatures (40-60 Co) using the mock community. Temperature did only have a minor effect on taxa recovery that varied with the specific primer pair. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrated that certain primer sets can recover most taxa in a diverse species assemblage. Thus there is no need to employ several primer sets targeting the same amplicon. While we identified several suited primer sets for arthropod metabarcoding, the primer selection depends on the targeted taxonomic groups, as well as DNA quality, desired taxonomic resolution, and sequencing platform employed for analysis.

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Validation of COI metabarcoding primers for terrestrial arthropods

10.7287/peerj.preprints.27801 ◽

2019 ◽

Author(s):

Vasco Elbrecht ◽

Thomas WA Braukmann ◽

Natalia V Ivanova ◽

Sean WJ Prosser ◽

Mehrdad Hajibabaei ◽

...

Keyword(s):

Taxonomic Resolution ◽

Minor Effect ◽

Malaise Trap ◽

Mock Community ◽

Sequencing Platform ◽

Amplification Bias ◽

Terrestrial Arthropods ◽

Primer Sets ◽

Taxonomic Groups ◽

Trap Sample

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids ecosystem assessment. However , it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. Thus this study tests the performance of 36 primer sets on a mock community containing 374 species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets where also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from the 21 amplicon pools of the mock community and Malaise trap sample were used to select four primer sets for metabarcoding evaluation at different annealing temperatures (40-60 Co) using the mock community. Temperature did only have a minor effect on taxa recovery that varied with the specific primer pair. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrated that certain primer sets can recover most taxa in a diverse species assemblage. Thus there is no need to employ several primer sets targeting the same amplicon. While we identified several suited primer sets for arthropod metabarcoding, the primer selection depends on the targeted taxonomic groups, as well as DNA quality, desired taxonomic resolution, and sequencing platform employed for analysis.

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Validation of COI metabarcoding primers for terrestrial arthropods

PeerJ ◽

10.7717/peerj.7745 ◽

2019 ◽

Vol 7 ◽

pp. e7745 ◽

Cited By ~ 22

Author(s):

Vasco Elbrecht ◽

Thomas W.A. Braukmann ◽

Natalia V. Ivanova ◽

Sean W.J. Prosser ◽

Mehrdad Hajibabaei ◽

...

Keyword(s):

Degraded Dna ◽

Taxonomic Resolution ◽

Effect Of Temperature ◽

Gut Contents ◽

Minor Effect ◽

Malaise Trap ◽

Mock Community ◽

Terrestrial Arthropods ◽

Primer Sets ◽

Trap Sample

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

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Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

10.1101/433607 ◽

2018 ◽

Cited By ~ 1

Author(s):

Thomas W. A. Braukmann ◽

Natalia V. Ivanova ◽

Sean W. J. Prosser ◽

Vasco Elbrecht ◽

Dirk Steinke ◽

...

Keyword(s):

Illumina Miseq ◽

Ion Torrent ◽

Mock Community ◽

Species Recovery ◽

Sequencing Platform ◽

Ion Torrent Pgm ◽

Terrestrial Arthropods ◽

Dna Metabarcoding ◽

Powerful Approach ◽

Read Abundance

AbstractDNA metabarcoding is an attractive approach for monitoring biodiversity. However, it is subject to biases that often impede detection of all species in a sample. In particular, the proportion of sequences recovered from each species depends on its biomass, mitome copy number, and primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 BINs, a species proxy. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body partitions (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicon generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, other variables did. As expected, the best recovery was obtained from the Single Leg treatment, but the Bulk Abdomen produced a more uniform read abundance than the Bulk Leg or Composite Leg samples. Primer choice also influenced species recovery. Our results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to obtain reproducible results and increase its cost-effectiveness.

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Effects of Malaise trap spacing on species richness and composition of terrestrial arthropod bulk samples

10.1101/2020.09.30.321430 ◽

2020 ◽

Author(s):

D Steinke ◽

TWA Braukmann ◽

L Manerus ◽

A Woodhouse ◽

V Elbrecht

Keyword(s):

Species Richness ◽

Field Experiments ◽

Collection Efficiency ◽

Malaise Trap ◽

Number Of Factors ◽

Large Numbers ◽

Set Up ◽

Taxonomic Groups ◽

A Site ◽

Malaise Traps

AbstractThe Malaise trap has gained popularity for assessing diverse terrestrial arthropod communities because it collects large samples with modest effort. A number of factors that influence collection efficiency, placement being one of them. For instance, when designing larger biotic surveys using arrays of Malaise traps we need to know the optimal distance between individual traps that maximises observable species richness and community composition. We examined the influence of spacing between Malaise traps by metabarcoding samples from two field experiments at a site in Waterloo, Ontario, Canada. For one experiment, we used two trap pairs deployed at weekly increasing distance (3m increments from 3 to 27 m). The second experiment involved a total of 10 traps set up in a row at 3m distance intervals for three consecutive weeks.Results show that community similarity of samples decreases over distance between traps. The amount of species shared between trap pairs shows drops considerably at about 15m trap-to-trap distance. This change can be observed across all major taxonomic groups and for two different habitat types (grassland and forest). Large numbers of OTUs found only once within samples cause rather large dissimilarity between distance pairs even at close proximity. This could be caused by a large number of transient species from adjacent habitat which arrive at the trap through passive transport, as well as capture of rare taxa, which end up in different traps by chance.

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

Bulletin of Entomological Research ◽

10.1017/s0007485315000681 ◽

2015 ◽

Vol 105 (6) ◽

pp. 717-727 ◽

Cited By ~ 83

Author(s):

G.-J. Brandon-Mong ◽

H.-M. Gan ◽

K.-W. Sing ◽

P.-S. Lee ◽

P.-E. Lim ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Illumina Miseq ◽

Ease Of Use ◽

Malaise Trap ◽

Detection Rates ◽

Operational Taxonomic Units ◽

Arthropod Species ◽

Primer Sets ◽

Arthropod Biodiversity

AbstractMetabarcoding, 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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Estimating intraspecific genetic diversity from community DNA metabarcoding data

10.7287/peerj.preprints.3269v4 ◽

2018 ◽

Author(s):

Vasco Elbrecht ◽

Ecaterina Edith Vamos ◽

Dirk Steinke ◽

Florian Leese

Keyword(s):

Genetic Diversity ◽

Biological Diversity ◽

Intraspecific Diversity ◽

Great Promise ◽

Data Sets ◽

Mock Community ◽

Data Set ◽

Dna Metabarcoding ◽

Intraspecific Genetic Diversity ◽

Primer Sets

Background. DNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTU), losing information on intraspecific diversity in the process. While COI haplotype information is limited in resolution, it is nevertheless useful in a phylogeographic context, helping to formulate hypothesis on taxon dispersal. Methods. This study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotypes from freshwater macroinvertebrate metabarcoding data sets. This novel approach was added to the R package "JAMP" and can be applied to Cytochrome c oxidase subunit I (COI) amplicon datasets. We tested our haplotyping method by sequencing i) a single-species mock community composed of 31 individuals with different haplotypes spanning three orders of magnitude in biomass and ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates. Results. We detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177 - 200 OTUs, each containing an average of 2.40 to 3.30 haplotypes per OTU. Population structures were consistent between replicates, and similar between primer pairs, depending on the primer length. A closer look at abundant taxa in the data set revealed various population genetic patterns, e.g. Taeniopteryx nebulosa and Hydropsyche pellucidula with a difference in north-south haplotype distribution, while Oulimnius tuberculatus and Asellus aquaticus display no clear population pattern but differ in genetic diversity. Discussion. We developed a strategy to infer intraspecific genetic diversity from bulk invertebrate monitoring samples using metabarcoding data. It needs to be stressed that at this point metabarcoding-informed haplotyping is not capable of capture the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and potential taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding data sets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about biological diversity but also underlying genetic diversity.

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Slippage of degenerate primers can cause variation in amplicon length

10.7287/peerj.preprints.26763 ◽

2018 ◽

Author(s):

Vasco Elbrecht ◽

Paul D.N. Hebert ◽

Dirk Steinke

Keyword(s):

Clustering Algorithms ◽

Length Variation ◽

Degenerate Primers ◽

Amplification Bias ◽

Binding Behavior ◽

Operational Taxonomic Units ◽

Amplicon Length ◽

Slippage Effect ◽

Primer Sets ◽

Mock Communities

Metabarcoding studies often employ degenerate primers to reduce amplification bias and increase the number of detected taxa. However, degeneracy has the disadvantage of lowering binding specificity although the exact mechanisms and potential biases introduced by such off-target amplification are not fully understood. We examined sequences recovered from the ten most abundant operational taxonomic units (OTUs) in two mock communities to investigate the specificity and binding behavior of five degenerate primer sets. Our results indicate that primers frequently bound 1-2 bp upstream in taxa where a homopolymer region was present in the amplification direction. As well, although less frequent, degeneracy occasionally led to primer binding 1 bp downstream. Some widely used primer sets were severely affected by this slippage effect, while others were not. Our study shows that primer slippage can produce taxon-specific length variation in amplicons and subsequent length variation in recovered sequences. While this variation will only have small impacts on OTU designation by clustering algorithms that ignore terminal gaps, primer sets employed in metabarcoding projects should be evaluated for their sensitivity to slippage. Moreover, steps should be taken to reduce slippage by improving protocols for primer design. For example, the flanking region adjacent to the 3' end of the primer is not considered by current primer development software although GC clamps in this position could mitigate slippage. While degeneracy is important to ensure the universality of a primer, binding in homopolymer regions should be avoided.

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Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

10.1101/759746 ◽

2019 ◽

Author(s):

Elena Valsecchi ◽

Jonas Bylemans ◽

Simon J. Goodman ◽

Roberto Lombardi ◽

Ian Carr ◽

...

Keyword(s):

Marine Mammals ◽

High Throughput Sequencing ◽

Environmental Dna ◽

16S Rrna Genes ◽

Rrna Genes ◽

Taxonomic Resolution ◽

Universal Primers ◽

Marine Biodiversity ◽

Primer Sets ◽

Marine Megafauna

ABSTRACTMetabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

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Estimating intraspecific genetic diversity from community DNA metabarcoding data

10.7287/peerj.preprints.3269v3 ◽

2018 ◽

Author(s):

Vasco Elbrecht ◽

Ecaterina Edith Vamos ◽

Dirk Steinke ◽

Florian Leese

Keyword(s):

Genetic Diversity ◽

Biological Diversity ◽

Intraspecific Diversity ◽

Great Promise ◽

Data Sets ◽

Mock Community ◽

Data Set ◽

Dna Metabarcoding ◽

Intraspecific Genetic Diversity ◽

Primer Sets

Background. DNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTU), losing information on intraspecific diversity in the process. While COI haplotype information is limited in resolution, it is nevertheless useful in a phylogeographic context, helping to formulate hypothesis on taxon dispersal. Methods. This study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotypes from freshwater macroinvertebrate metabarcoding data sets. This novel approach was added to the R package "JAMP" and can be applied to Cytochrome c oxidase subunit I (COI) amplicon datasets. We tested our haplotyping method by sequencing i) a single-species mock community composed of 31 individuals with different haplotypes spanning three orders of magnitude in biomass and ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates. Results. We detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177 - 200 OTUs, each containing an average of 2.40 to 3.30 haplotypes per OTU. Population structures were consistent between replicates, and similar between primer pairs, depending on the primer length. A closer look at abundant taxa in the data set revealed various population genetic patterns, e.g. Taeniopteryx nebulosa and Hydropsyche pellucidula with a difference in north-south haplotype distribution, while Oulimnius tuberculatus and Asellus aquaticus display no clear population pattern but differ in genetic diversity. Discussion. We developed a strategy to infer intraspecific genetic diversity from bulk invertebrate monitoring samples using metabarcoding data. It needs to be stressed that at this point metabarcoding-informed haplotyping is not capable of capture the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and potential taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding data sets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about biological diversity but also underlying genetic diversity.

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