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.

Download Full-text

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.

Download Full-text

Validation of COI metabarcoding primers for terrestrial arthropods

10.7287/peerj.preprints.27801v1 ◽

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.

Download Full-text

In silico and empirical evaluation of twelve COI & 16S metabarcoding primer sets for insectivorous diet analyses

10.1101/742874 ◽

2019 ◽

Cited By ~ 2

Author(s):

Orianne Tournayre ◽

Maxime Leuchtmann ◽

Ondine Filippi-Codaccioni ◽

Marine Trillat ◽

Sylvain Piry ◽

...

Keyword(s):

In Silico ◽

Empirical Evaluation ◽

Environmental Dna ◽

Degraded Dna ◽

Taxonomic Identification ◽

Mock Community ◽

Predator Detection ◽

Primer Sets ◽

Diet Analyses

AbstractThis last decade, environmental DNA metabarcoding approaches have been developed and improved to minimize biological and technical biases; some challenges, however, remain, as the design of primers. Here we have performed a comprehensive assessment of ten COI and two 16S primer sets. We have combined in silico, in vivo-mock community of 33 arthropod taxa from 16 orders and guano analyses to identify primer sets that should maximize arthropod detection and taxonomic identification, whilst identifying bat species and minimizing labour time and cost. We have focused on two insectivorous bat species living in mixed-colonies, the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy’s bat (Myotis emarginatus). We have found that the level of primer degeneracy is the main factor influencing arthropod detection for in silico and mock community analyses, while the amplicon length is critical for the detection of arthropods from degraded DNA samples. Our results confirm the importance of performing predator detection and taxonomic identification, simultaneously with arthropod sequencing, as faeces samples can be contaminated by different insectivorous species. Moreover, amplifying bat DNA does not affect the primers’ capacity to detect arthropods. We therefore recommend the systematic simultaneous identification of predator and prey. Finally, we evidenced that one third of the prey occurrences are unreliable and probably not of primary interest in diet studies, which might decrease the relevance of combining several primer sets instead of using one efficient primer set. In conclusion, this study provides general criteria enabling the selection of primers whilst considering different scientific and methodological constraints.

Download Full-text

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.

Download Full-text

The effect of temperature and method of feeding on the diestibility of two milk substitutes and on the performance of lambs

The Journal of Agricultural Science ◽

10.1017/s0021859600037254 ◽

1977 ◽

Vol 88 (3) ◽

pp. 579-589 ◽

Cited By ~ 12

Author(s):

P. D. Penning ◽

Ines M. Penning ◽

T. T. Treacher

Keyword(s):

Dry Matter ◽

Body Weight Gain ◽

Coconut Oil ◽

The Body ◽

Effect Of Temperature ◽

Minor Effect ◽

Feeding Regime ◽

Dry Matter Digestibility ◽

Milk Substitute ◽

Milk Substitutes

SUMMARYThe effects of temperature and method of feeding on the intake characteristics and digestibilities of two milk substitutes were examined.Forty Finnish Landrace x Dorset Horn lambs (20 males and 20 females) were fed one of two milk substitutes from 3 to 25 days of age when they were slaughtered and chemical composition of the empty body was measured.The two milk substitutes contained 30% fat in the dry matter as butter fat (diet B), or tallow and coconut oil (diet TC).The diets were offered at either 34°C (W) or 5 °C (C) and three methods of feeding were used. The diets were given: ad libitumcold (AL1), four feeds to appetite per day either warm or cold (AL2W and AL2C) and four feeds restricted to an intake of 62 g D.M./kg live weight0·5 either warm or cold (RW or RC).Digestibilities of the milk substitutes were not affected by the temperature at which they were offered or the feeding regime used. Diet B had an apparent dry-matter digestibility of 97·6% and diet TC 92·5%. The fat digestibility of diet TC was 13·6 units lower than diet B and this affected the digestibility of all the other dietary components.Lambs tended to consume more of diet B and grow faster.Lambs on treatment AL1 consumed 21% more milk substitute than those on treatment AL2.The composition of the body-weight gain was found to alter with rate of gain.The temperature at which the milk substitutes were offered had only a minor effect on the performance of the lambs. The type of milk substitute and feeding regime used had a greater influence on both lamb intake and growth.

Download Full-text

The Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains*

Journal of Forensic Sciences ◽

10.1111/j.1556-4029.2006.00077.x ◽

2006 ◽

Vol 51 (2) ◽

pp. 351-356 ◽

Cited By ~ 27

Author(s):

Kerry L. Opel ◽

Denise T. Chung ◽

Jiri Drabek ◽

Nancy E. Tatarek ◽

Lee Meadows Jantz ◽

...

Keyword(s):

Skeletal Remains ◽

Degraded Dna ◽

Human Skeletal Remains ◽

Primer Sets

Download Full-text

The development of miniplex primer sets for the analysis of degraded DNA

10.1117/12.603080 ◽

2005 ◽

Author(s):

Bruce McCord ◽

Kerry Opel ◽

Denise Chung ◽

Jiri Drabek ◽

Nancy Tatarek ◽

...

Keyword(s):

Degraded Dna ◽

Primer Sets

Download Full-text

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.

Download Full-text

Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding

10.7287/peerj.preprints.26667 ◽

2018 ◽

Author(s):

Aimee L van der Reis ◽

Olivier Laroche ◽

Andrew G Jeffs ◽

Shane D Lavery

Keyword(s):

New Zealand ◽

Preliminary Analysis ◽

18S Rrna ◽

18S Rrna Gene ◽

Coi Gene ◽

Taxonomic Resolution ◽

Gut Contents ◽

Rrna Gene ◽

Diverse Range ◽

Dna Metabarcoding

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable, but metabarcoding outcomes indicated that the ethanol samples produced better results from the COI gene. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta, however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctate), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitza), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

Download Full-text