Can non-destructive DNA extraction of bulk invertebrate samples be used for metabarcoding?

Carew, Melissa E.; Coleman, Rhys A.; Hoffmann, Ary A.

doi:10.7717/peerj.4980

Cited by 66 publications

(88 citation statements)

References 56 publications

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“…Also, studies are needed on the differential recovery of DNA from different taxa due to variation in body characteristics (e.g. soft vs. hard bodied arthropods), which can affect metabarcoding results (Carew, Coleman, & Hoffmann, 2018;Zizka et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

Martins

Galhardo

Filipe

et al. 2019

Molecular Ecology Resources

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DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.

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Section: Discussionmentioning

confidence: 99%

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

Martins

Galhardo

Filipe

et al. 2019

Molecular Ecology Resources

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“…DNA may even be extracted directly from the collection medium (e.g., ethanol), because specimens leave trace amounts of DNA in the medium (Hajibabaei et al 2012;Martins et al 2019). However, the DNA recovered from nondestructive extraction of community samples may be biased toward those taxonomic groups that release DNA more readily than others, e.g., soft-bodied animals (Carew et al 2018;Marquina et al 2019).…”

Section: Community Metabarcodingmentioning

confidence: 99%

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

et al. 2020

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Large-scale studies on community ecology are highly desirable but often difficult to accomplish due to the considerable investment of time, labor and, money required to characterize richness, abundance, relatedness, and interactions. Nonetheless, such large-scale perspectives are necessary for understanding the composition, dynamics, and resilience of biological communities. Small invertebrates play a central role in ecosystems, occupying critical positions in the food web and performing a broad variety of ecological functions. However, it has been particularly difficult to adequately characterize communities of these animals because of their exceptionally high diversity and abundance. Spiders in particular fulfill key roles as both predator and prey in terrestrial food webs and are hence an important focus of ecological studies. In recent years, large-scale community analyses have benefitted tremendously from advances in DNA barcoding technology. High-throughput sequencing (HTS), particularly DNA metabarcoding, enables community-wide analyses of diversity and interactions at unprecedented scales and at a fraction of the cost that was previously possible. Here, we review the current state of the application of these technologies to the analysis of spider communities. We discuss amplicon-based DNA barcoding and metabarcoding for the analysis of community diversity and molecular gut content analysis for assessing predator-prey relationships. We also highlight applications of the third generation sequencing technology for long read and portable DNA barcoding. We then address the development of theoretical frameworks for community-level studies, and finally highlight critical gaps and future directions for DNA analysis of spider communities.

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“…While Comprehensive sample homogenisation is a method that has demonstrated promising results, other techniques using fixatives from bulk samples are being developed (Blackman et al, 2019; Carew, Coleman, & Hoffmann, 2018; Erdozain et al, 2019; Martins et al, 2019; Zizka, Leese, Peinert, & Geiger, 2019) and would be advantageous in that the sample remains intact for morphological identification if needed. The strength of eDNA methods for whole catchment assessments is promising (Blackman et al, 2019) and has the potential to benefit biomonitoring protocols by detecting different organisms and offering a safer alternative for assessing dangerous rivers.…”

Section: Resultsmentioning

confidence: 99%

Metabarcoding unsorted kick-samples facilitates macroinvertebrate-based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA

Pereira-da-Conceicoa

Elbrecht

Hall

et al. 2019

Preprint

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Many studies have highlighted the potential of DNA-based methods for the biomonitoring of freshwater macroinvertebrates, however only a few studies have investigated homogenisation of bulk samples that include debris to reduce sample-processing time. In order to explore the use of DNA-based methods in water quality assessment in South Africa, this study compares morphological and molecular-based identification of freshwater macroinvertebrates at the mixed higher taxon and mOTU level while investigating abundance and comparing mOTU recovery with historical species records. From seven sites across three rivers in South Africa, we collected a biomonitoring sample, an intensive-search comprehensive sample and an eDNA sample per site. The biomonitoring sample was picked and scored according to standard protocols and the leftover debris and comprehensive samples were homogenised including all debris. DNA-based methods recovered higher diversity than morphology, but did not always recover the same taxa, even at the family level. Regardless of the differences in taxon scores, most DNA-based methods except some eDNA samples, returned the same water quality assessment category as the standard morphology-based assessment. Homogenised comprehensive samples recovered more freshwater invertebrate diversity than all other methods. The eDNA samples recovered 2 to 10 times more mOTUs than any other method, however 90% of reads were non-target and as a result eDNA recovered the lowest target diversity. However, eDNA did find some target taxa that the other methods failed to detect. This study shows that unsorted samples recover the same water quality scores as a morphology-based assessment and much higher diversity scores than both picked and eDNA samples. As a result, there is potential to integrate DNA-based approaches into existing metrics quickly while providing much more information for the development of more refined metrics at the species or mOTU level with distributional data which can be used for conservation and biodiversity management.

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Can non-destructive DNA extraction of bulk invertebrate samples be used for metabarcoding?

Cited by 66 publications

References 56 publications

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

Metabarcoding unsorted kick-samples facilitates macroinvertebrate-based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA

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