Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

Elbrecht, Vasco; Taberlet, Pierre; Déjean, Tony; Valentini, Alice; Usseglio‐Polatera, Philippe; Beisel, Jean‐Nicolas; Coissac, Éric; Boyer, Frédéric; Leese, Florian

doi:10.7287/peerj.preprints.1855

Cited by 57 publications

(86 citation statements)

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“…Our results demonstrate, however, that the performance of a primer set on real eDNA extracts can radically differ from that predicted from such studies. The Clarke primers have been reported useful for DNA metabarcoding of insects based on in silico and in vitro mock eDNA studies (Clarke et al, 2014;Elbrecht et al, 2016), but in the present study we observe that when they are used on real eDNA extracts, non-target DNA is PCR amplified to such extent that it prevents detection of the taxonomic group that the primers were designed to amplify, namely insects (Figure 2a, b). Hence, instead of relying solely on the results of in silico and in vitro analyses, we encourage researchers to perform pilot studies using a subset of their environmental samples to test the performance of the selected primers in a real setting and to make a decision on whether more than one primer set is needed to detect the desired diversity.…”

Section: Should I Use Multiple Primer Sets?mentioning

confidence: 58%

“…Each of the four extraction rounds included one extraction blank. DNA was PCR amplified from the extracts using four primer sets as detailed in Table 1 and Figure S2, hereafter referred to as Clarke (Clarke et al, 2014;Elbrecht et al, 2016), Epp (Epp et al, 2012), Leray (Geller, Meyer, Parker, & Hawk, 2013;Leray et al, 2013) and Zeale (Zeale, Butlin, & Barker, 2011). After optimizing PCR conditions through qPCR screening (Murray et al, 2015) ( Figure S3), each of the 54 bat faecal extracts, four extraction blanks and two PCR blanks were amplified in three PCR replicates.…”

Section: Laboratory Workmentioning

confidence: 99%

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Scrutinizing key steps for reliable metabarcoding of environmental samples

et al. 2017

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Abstract1. Metabarcoding of environmental samples has many challenges and limitations that require carefully considered laboratory and analysis workflows to ensure reliable results. We explore how decisions regarding study design, laboratory set-up, and bioinformatic processing affect the final results, and provide guidelines for reliable study of environmental samples.2. We evaluate the performance of four primer sets targeting COI and 16S regions characterizing arthropod diversity in bat faecal samples, and investigate how metabarcoding results are affected by parameters including: (1) number of PCR replicates per sample, (2) sequencing depth, (3) PCR replicate processing strategy (i.e. either additively, by combining the sequences obtained from the PCR replicates, or restrictively, by only retaining sequences that occur in multiple PCR replicates for each sample), (4) minimum copy number for sequences to be retained, (5) chimera removal, and (6) similarity thresholds for Operational Taxonomic Unit (OTU) clustering. Lastly, we measure within-and between-taxa dissimilarities when using sequences from public databases to determine the most appropriate thresholds for OTU clustering and taxonomy assignment.3. Our results show that the use of multiple primer sets reduces taxonomic biases and increases taxonomic coverage. Taxonomic profiles resulting from each primer set are principally affected by how many PCR replicates are carried out per sample and how sequences are filtered across them, the sequence copy number threshold and the OTU clustering threshold. We also report considerable diversity differences between PCR replicates from each sample. Sequencing depth increases the dissimilarity between PCR replicates unless the bioinformatic strategies to remove allegedly artefactual sequences are adjusted according to the number of analysed sequences. Finally, we show that the appropriate identity thresholds for OTU clustering and taxonomy assignment differ between markers.4. Metabarcoding of complex environmental samples ideally requires (1) investigation of whether more than one primer sets targeting the same taxonomic group is needed to offset primer biases, (2) more than one PCR replicate per sample, (3) bioinformatic processing of sequences that balance diversity detection with removal of artefactual sequences, and (4) empirical selection of OTU clustering and taxonomy assignment thresholds tailored to each marker and the obtained taxa.

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Section: Should I Use Multiple Primer Sets?mentioning

confidence: 58%

Section: Laboratory Workmentioning

confidence: 99%

Scrutinizing key steps for reliable metabarcoding of environmental samples

et al. 2017

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“…Elbrecht and Leese (2017a) proposed an approach to consider taxa of a region or only specific taxon groups degenerate primers, depending on the research or applied question. It should be noted, however, that primer degeneracy is no main concern when metabarcoding is used for bulk tissue samples, as this 'biodiversity soup' (Yu et al 2012) mostly contains DNA of the target organisms (Elbrecht et al 2016. It becomes a big challenge when trying to detect trace amounts of target DNA in pools of non-target DNA and this is the case with eDNA extracted from water.…”

Section: Introductionmentioning

confidence: 99%

Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

Leese

Sander

Buchner

et al. 2020

Preprint

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Acknowledgments:We would like to thank Marlen Mährlein, Nathalie Kaffenberger, Cristina Hartmann-Fatu and Arne Beermann for help with sample collection or processing. Furthermore, we thank Jan-Niklas Macher for helpful discussions, Beatrice Kulawig for compiling the taxonomic data from the Rhine-Main-Observatory, and Martina Weiss for valuable comments on the structure of the manuscript. This work has been funded by a grant of the Bode Foundation to FL. This work has been conducted as part of COST (European Cooperation in Science and Technology) Action DNAqua-Net (CA15219). AbstractDNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database.However, when universal primers are used on environmental DNA (eDNA) isolated from stream water, macroinvertebrate read and OTU numbers are typically "watered down", i.e. diluted, compared to whole specimen 'bulk samples' due to greater co-amplification of abundant nontarget taxa such as algae and bacteria. Because stream macroinvertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3'-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.

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“…95 bp fragment excluding primers (Taylor 1996) , ii) two pools of tagged amplicons from vampire bat gut contents, PCR amplified with metazoan COI primers (mlCOIintF/jgHCO2198, from here on referred to as 'COI'), amplifying 313 bp fragment of the COI barcode region excluding primers (Geller et al 2013;Leray et al 2013;Bohmann et al 2018) , and iii) two pools of tagged amplicons from insect-eating bat faecal droppings, PCR amplified with insect mitochondrial 16sRNA primers (Ins16s_1shortF /Ins16s_1shortR, from here on referred to as '16sIns'), amplifying a ca. 190 bp fragment (excluding primers) (Clarke et al 2014;Elbrecht et al 2016) .…”

Section: Pools Of Tagged Ampliconsmentioning

confidence: 99%

Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

Carøe

Bohmann

2020

Preprint

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Metabarcoding of environmental DNA (eDNA) and DNA extracted from bulk specimen samples is a powerful tool in studies of biodiversity, diet and ecological interactions as its inherent labelling of amplicons allows sequencing of taxonomically informative genetic markers from many samples in parallel. However, the occurrence of so-called 'tag-jumps' can cause incorrect assignment of sequences to samples and artificially inflate diversity. Two steps during library preparation of pools of 5' nucleotide-tagged amplicons have been suggested to cause tag-jumps; i) T4 DNA polymerase blunt-ending in the end-repair step and ii) post-ligation PCR amplification of amplicon libraries.The discovery of tag-jumps has led to recommendations to only carry out metabarcoding PCR amplifications with primers carrying twin-tags to ensure that tag-jumps cannot result in false assignments of sequences to samples. As this increases both cost and workload, a metabarcoding library preparation protocol which circumvents the two steps that causes tag-jumps is needed. Here, we demonstrate Tagsteady, a metabarcoding Illumina library preparation protocol for pools of nucleotide-tagged amplicons that enables efficient and cost-effective generation of metabarcoding data with virtually no tag-jumps. We use pools of twin-tagged amplicons to investigate the effect of T4 DNA polymerase blunt-ending and post-ligation PCR on the occurrence of tag-jumps. We demonstrate that both blunt-ending and post-ligation PCR, alone or together, can result in detrimental amounts of tag-jumps (here, 1/20 up to ca. 49% of total sequences), while leaving both steps out (the Tagsteady protocol) results in amounts of sequences carrying new combinations of used tags (tag-jumps) comparable to background contamination.

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Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

Cited by 57 publications

References 0 publications

Scrutinizing key steps for reliable metabarcoding of environmental samples

Scrutinizing key steps for reliable metabarcoding of environmental samples

Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

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