A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy

Weigand, Alexander; Macher, Jan‐Niklas

doi:10.3897/mbmg.2.26869

Cited by 45 publications

(38 citation statements)

References 76 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The remainder of the metazoans were mainly branchiopods and copepods. Similar results with non-target taxa were reported in other papers using degenerate COI primers for freshwater community metabarcording (Weigand & Macher, 2018). There has been some debate about the usability of the standard COI barcode region defined by Hebert et al (2003) within DNA- and eDNA-based analyses, but thus far the benefit of an extensive COI database seems to outweigh the drawbacks (Andújar et al, 2018), as also witnessed by the many primer sets that have been designed for macroinvertebrate metabarcoding studies (Leray et al, 2013; Bista et al, 2017; Elbrecht & Leese, 2017).…”

Section: Discussionsupporting

confidence: 90%

The effects of spatial and temporal replicate sampling on eDNA metabarcoding

Beentjes

Speksnijder

Schilthuizen

et al. 2019

PeerJ

View full text Add to dashboard Cite

Background The heterogeneous nature of environmental DNA (eDNA) and its effects on species detection and community composition estimates has been highlighted in several studies in the past decades. Mostly in the context of spatial distribution over large areas, in fewer occasions looking at spatial distribution within a single body of water. Temporal variation of eDNA, similarly, has mostly been studied as seasonality, observing changes over large periods of time, and often only for small groups of organisms such as fish and amphibians. Methods We analyzed and compared small-scale spatial and temporal variation by sampling eDNA from two small, isolated dune lakes for 20 consecutive weeks. Metabarcoding was performed on the samples using generic COI primers. Molecular operational taxonomic unit (MOTUs) were used to assess dissimilarities between spatial and temporal replicates. Results Our results show large differences between samples taken within one lake at one point in time, but also expose the large differences between temporal replicates, even those taken only 1 week apart. Furthermore, between-site dissimilarities showed a linear correlation with time frame, indicating that between-site differences will be inflated when samples are taken over a period of time. We also assessed the effects of PCR replicates and processing strategies on general patterns of dissimilarity between samples. While more inclusive PCR replicate strategies lead to higher richness estimations, dissimilarity patterns between samples did not significantly change. Conclusions We conclude that the dissimilarity of temporal replicates at a 1 week interval is comparable to that of spatial replicate samples. It increases, however, for larger time intervals, which suggests that population turnover effects can be stronger than community heterogeneity. Spatial replicates alone may not be enough for optimal recovery of taxonomic diversity, and cross-comparisons of different locations are susceptible to inflated dissimilarities when performed over larger time intervals. Many of the observed MOTUs could be classified as either phyto- or zooplankton, two groups that have gained traction in recent years as potential novel bio-indicator species. Our results, however, indicate that these groups might be susceptible to large community shifts in relatively short periods of time, highlighting the need to take temporal variations into consideration when assessing their usability as water quality indicators.

show abstract

Section: Discussionsupporting

confidence: 90%

The effects of spatial and temporal replicate sampling on eDNA metabarcoding

Beentjes

Speksnijder

Schilthuizen

et al. 2019

PeerJ

View full text Add to dashboard Cite

show abstract

“…sample, sub‐sample, or PCR. This result is consistent with Weigand and Macher () who found that taxon detection among PCR replicates mostly overlapped. However, we did find that detection of taxa increased slightly as levels of replication were combined.…”

Section: Discussionsupporting

confidence: 92%

Towards routine DNA metabarcoding of macroinvertebrates using bulk samples for freshwater bioassessment: Effects of debris and storage conditions on the recovery of target taxa

et al. 2019

View full text Add to dashboard Cite

Macroinvertebrates are commonly sampled for bioassessment of freshwater ecosystems. However, current bioassessment protocols involve laborious sorting of the animals from the debris (sample matrix) and morphological identification, where species level identifications are often difficult. DNA metabarcoding has the potential to improve bioassessment by reducing the time taken to process samples and improve the accuracy and speed of macroinvertebrate species identification. In this study, we evaluated DNA metabarcoding of macroinvertebrate samples, which include macroinvertebrates and the debris collected in the sample nets, to test if bulk, unsorted samples can be used to assess macroinvertebrate diversity. First, we tested if the sample matrix prevented the detection of six target macroinvertebrate taxa when DNA metabarcoding. Second, we tested if sample storage influenced the detection of the same six target macroinvertebrates. We also explored different levels of replication at the sample, sub‐sample, and polymerase chain reaction levels and compared the overall macroinvertebrate families detected using DNA metabarcoding to those identified morphologically. We found that the presence of the sample matrix did not interfere with or inhibit the detection of the six target macroinvertebrate taxa. Furthermore, we found that the various sample storage methods did not affect target macroinvertebrate detection. The reliability of detection of the target macroinvertebrates improved as hierarchical levels of replication were combined. We found strong overlap between the detection of overall macroinvertebrate family diversity when comparing DNA metabarcoding to morphological identification. Extracting DNA from the bulk macroinvertebrate samples that included the sample matrix and using this for DNA metabarcoding could improve bioassessment by removing the need for laborious sorting of samples. Furthermore, DNA metabarcoding detection of the six target taxa was not dependent on sample storage of up to 1 year in 95% ethanol, at room temperature or after heating. DNA metabarcoding had the advantage of identifying macroinvertebrate species, but good DNA barcode libraries are needed for widespread species identifications. Further investigation should focus on including multiple samples with different macroinvertebrate composition and densities to refine and standardise bulk sample processing protocols, and on building comprehensive DNA barcode libraries for aquatic macroinvertebrates.

show abstract

“…Trombidiformes, Dugesia , see Table S3 in Elbrecht and Leese), where detection rate clearly decreased with increasing filtering thresholds. Furthermore, taxa which are known to amplify less successfully with the universal BF2/BR2 primers (Nematoda, Gastropoda, see Elbrecht and Leese (), Weigand & Macher, ) were less frequently detected. The highest number of taxa was constantly detected in samples processed with the TruSeq Nano DNA Library Preparation kit (threshold 0.004–0.01: on average 48.9 [±1] taxa, threshold 0.02–0.09:46.4 [±1.2 taxa]).…”

Section: Resultsmentioning

confidence: 99%

Assessing the influence of sample tagging and library preparation on DNA metabarcoding

Zizka

Elbrecht

Macher

et al. 2019

Molecular Ecology Resources

Self Cite

View full text Add to dashboard Cite

Metabarcoding is increasingly used to assess species diversity by high‐throughput sequencing where millions of sequences can be generated in parallel and multiple samples can be analysed in one sequencing run. Generating amplified fragments with a unique sequence identifier ('tag') for each sample is crucial, as it allows assigning sequences to the original samples. The tagging through so‐called fusion primers is a fast and cheap alternative to commercially produced ligation‐based kits. However, little is known about potential bias and inconsistencies introduced by the long nucleotide tail attached to those primers, which could lead to deficient reports of community composition in metabarcoding studies. We therefore tested the consistency and taxa detection efficiency of fusion primers in (1) a one‐step and (2) two‐step PCR protocol as well as (3) a commercially manufactured Illumina kit using mock communities of known composition. The Illumina kit delivered the most consistent results and detected the highest number of taxa. However, success of the two‐step PCR approach was only marginally lower compared to the kit with the additional advantage of a much more competitive price per library. While most taxa were also detected with the one‐step PCR approach, the consistency between replicates including read abundance was substantially lower. Our results highlight that method choice depends on the precision needed for analysis as well as on economic considerations and recommend the Illumina kit to obtain most accurate results and the two‐step PCR approach as a much cheaper yet very robust alternative.

show abstract

A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy

Cited by 45 publications

References 76 publications

The effects of spatial and temporal replicate sampling on eDNA metabarcoding

The effects of spatial and temporal replicate sampling on eDNA metabarcoding

Towards routine DNA metabarcoding of macroinvertebrates using bulk samples for freshwater bioassessment: Effects of debris and storage conditions on the recovery of target taxa

Assessing the influence of sample tagging and library preparation on DNA metabarcoding

Contact Info

Product

Resources

About