Fecal metagenomics for the simultaneous assessment of diet, parasites, and population genetics of an understudied primate

Srivathsan, Amrita; Ang, Andie; Vogler, Alfried P.; Meier, Rudolf

doi:10.1186/s12983-016-0150-4

Cited by 110 publications

(125 citation statements)

References 43 publications

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“…This is consistent with the levels found in other faecal samples in other studies (e.g. Srivathsan, Ang, Vogler, & Meier, ). Low levels of template DNA is a cause of PCR stochasticity in metabarcoding studies (Murray et al., ).…”

Section: Discussionsupporting

confidence: 93%

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: Discussionsupporting

confidence: 93%

Scrutinizing key steps for reliable metabarcoding of environmental samples

et al. 2017

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“…Fortunately, we are starting to move towards a point where markers used in different applications are better understood and alternative less‐biased approaches are being explored. This includes the use of multiple target markers (Stat et al., ) and PCR‐free approaches (Srivathsan, Ang, Vogler, & Meier, ) that can be combined with prey DNA enrichment (Krehenwinkel, Kennedy, Pekár, & Gillespie, ). Inclusion of control materials in sequencing runs can also ensure consistency between experiments (Hardwick, Deveson, & Mercer, ).…”

Section: A View Of the Way Forward In Interpreting Sequence Countsmentioning

confidence: 99%

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

et al. 2018

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Advances in DNA sequencing technology have revolutionized the field of molecular analysis of trophic interactions, and it is now possible to recover counts of food DNA sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of a consumer's diet should we work strictly with data sets summarizing frequency of occurrence of different food taxa, or is it possible to use relative number of sequences? Both approaches are applied to obtain semi-quantitative diet summaries, but occurrence data are often promoted as a more conservative and reliable option due to taxa-specific biases in recovery of sequences. We explore representative dietary metabarcoding data sets and point out that diet summaries based on occurrence data often overestimate the importance of food consumed in small quantities (potentially including low-level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that using relative read abundance (RRA) information often provides a more accurate view of population-level diet even with moderate recovery biases incorporated; however, RRA summaries are sensitive to recovery biases impacting common diet taxa. Both approaches are more accurate when the mean number of food taxa in samples is small. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue addressing methodological challenges and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research.

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“…Third, it is unclear how one can obtain reliable abundance and biomass information from metabarcoding data although such data are often needed for ecological analyses (Gibb et al., ). Lastly, metabarcoding studies of poorly known faunas tend to generate large numbers of barcodes that cannot be identified to species (Lim et al., ; Srivathsan, Ang, Vogler, & Meier, ; Srivathsan, Sha, Vogler, & Meier, ), because the available barcoding databases typically have poor species coverage, especially for invertebrates (Kwong, Srivathsan, & Meier, ). This interferes with biological interpretation of the data because unidentified sequences remain singular observations, while sequences that can be identified to species yield scientific names that allow for accessing the relevant scientific literature.…”

Section: Introductionmentioning

confidence: 99%

Sorting specimen‐rich invertebrate samples with cost‐effective NGS barcodes: Validating a reverse workflow for specimen processing

Wang

Srivathsan

Foo

et al. 2018

Molecular Ecology Resources

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Biologists frequently sort specimen-rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a "reverse workflow" is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next-generation sequencing (NGS) barcoding pipeline that allows for cost-effective high-throughput generation of short specimen-specific barcodes (313 bp of COI; laboratory cost <$0.50 per specimen) through next-generation sequencing of tagged amplicons. We applied our approach to a large sample of tropical ants, obtaining barcodes for 3,290 of 4,032 specimens (82%). NGS barcodes and their corresponding specimens were then sorted into molecular operational taxonomic units (mOTUs) based on objective clustering and Automated Barcode Gap Discovery (ABGD). High diversity of 88-90 mOTUs (4% clustering) was found and morphologically validated based on preserved vouchers. The mOTUs were overwhelmingly in agreement with morphospecies (match ratio 0.95 at 4% clustering). Because of lack of coverage in existing barcode databases, only 18 could be accurately identified to named species, but our study yielded new barcodes for 48 species, including 28 that are potentially new to science. With its low cost and technical simplicity, the NGS barcoding pipeline can be implemented by a large range of laboratories. It accelerates invertebrate species discovery, facilitates downstream taxonomic work, helps with building comprehensive barcode databases and yields precise abundance information.

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Fecal metagenomics for the simultaneous assessment of diet, parasites, and population genetics of an understudied primate

Cited by 110 publications

References 43 publications

Scrutinizing key steps for reliable metabarcoding of environmental samples

Scrutinizing key steps for reliable metabarcoding of environmental samples

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

Sorting specimen‐rich invertebrate samples with cost‐effective NGS barcodes: Validating a reverse workflow for specimen processing

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