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Increased performance of DNA metabarcoding of macroinvertebrates by taxonomic sorting.

ABSTRACT: DNA-based identification through the use of metabarcoding has been proposed as the next step in the monitoring of biological communities, such as those assessed under the Water Framework Directive (WFD). Advances have been made in the field of metabarcoding, but challenges remain when using complex samples. Uneven biomass distributions, preferential amplification and reference database deficiencies can all lead to discrepancies between morphological and DNA-based taxa lists. The effects of different taxonomic groups on these issues remain understudied. By metabarcoding WFD monitoring samples, we analyzed six different taxonomic groups of freshwater organisms, both separately and combined. Identifications based on metabarcoding data were compared directly to morphological assessments performed under the WFD. The diversity of taxa for both morphological and DNA-based assessments was similar, although large differences were observed in some samples. The overlap between the two taxon lists was 56.8% on average across all taxa, and was highest for Crustacea, Heteroptera, and Coleoptera, and lowest for Annelida and Mollusca. Taxonomic sorting in six basic groups before DNA extraction and amplification improved taxon recovery by 46.5%. The impact on ecological quality ratio (EQR) scoring was considerable when replacing morphology with DNA-based identifications, but there was a high correlation when only replacing a single taxonomic group with molecular data. Different taxonomic groups provide their own challenges and benefits. Some groups might benefit from a more consistent and robust method of identification. Others present difficulties in molecular processing, due to uneven biomass distributions, large genetic diversity or shortcomings of the reference database. Sorting samples into basic taxonomic groups that require little taxonomic knowledge greatly improves the recovery of taxa with metabarcoding. Current standards for EQR monitoring may not be easily replaced completely with molecular strategies, but the effectiveness of molecular methods opens up the way for a paradigm shift in biomonitoring.

SUBMITTER: Beentjes KK

PROVIDER: S-EPMC6913968 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Increased performance of DNA metabarcoding of macroinvertebrates by taxonomic sorting.

Beentjes Kevin K KK Speksnijder Arjen G C L AGCL Schilthuizen Menno M Hoogeveen Marten M Pastoor Rob R van der Hoorn Berry B BB

PloS one 20191216 12

DNA-based identification through the use of metabarcoding has been proposed as the next step in the monitoring of biological communities, such as those assessed under the Water Framework Directive (WFD). Advances have been made in the field of metabarcoding, but challenges remain when using complex samples. Uneven biomass distributions, preferential amplification and reference database deficiencies can all lead to discrepancies between morphological and DNA-based taxa lists. The effects of diffe ...[more]

PMID: 31841568

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Project description:BackgroundThe DNA metabarcoding approach has become one of the most used techniques to study the taxa composition of various sample types. To deal with the high amount of data generated by the high-throughput sequencing process, a bioinformatics workflow is required and the QIIME2 platform has emerged as one of the most reliable and commonly used. However, only some pre-formatted reference databases dedicated to a few barcode sequences are available to assign taxonomy. If users want to develop a new custom reference database, several bottlenecks still need to be addressed and a detailed procedure explaining how to develop and format such a database is currently missing. In consequence, this work is aimed at presenting a detailed workflow explaining from start to finish how to develop such a curated reference database for any barcode sequence.ResultsWe developed DB4Q2, a detailed workflow that allowed development of plant reference databases dedicated to ITS2 and rbcL, two commonly used barcode sequences in plant metabarcoding studies. This workflow addresses several of the main bottlenecks connected with the development of a curated reference database. The detailed and commented structure of DB4Q2 offers the possibility of developing reference databases even without extensive bioinformatics skills, and avoids 'black box' systems that are sometimes encountered. Some filtering steps have been included to discard presumably fungal and misidentified sequences. The flexible character of DB4Q2 allows several key sequence processing steps to be included or not, and downloading issues can be avoided. Benchmarking the databases developed using DB4Q2 revealed that they performed well compared to previously published reference datasets.ConclusionThis study presents DB4Q2, a detailed procedure to develop custom reference databases in order to carry out taxonomic analyses with QIIME2, but also with other bioinformatics platforms if desired. This work also provides ready-to-use plant ITS2 and rbcL databases for which the prediction accuracy has been assessed and compared to that of other published databases.

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Project description:Understanding interactions between herbivores and parasitoids is essential for successful biodiversity protection and monitoring and for biological pest control. Morphological identifications employ insect rearing and are complicated by insects' high diversity and crypsis. DNA barcoding has been successfully used in studies of host-parasitoid interactions as it can substantially increase the recovered real host-parasitoid diversity distorted by overlooked species complexes, or by species with slight morphological differences. However, this approach does not allow the simultaneous detection and identification of host(s) and parasitoid(s). Recently, high-throughput sequencing has shown high potential for surveying ecological communities and trophic interactions. Using mock samples comprising insect larvae and their parasitoids, we tested the potential of DNA metabarcoding for identifying individuals involved in host-parasitoid interactions to different taxonomic levels, and compared it to standard DNA barcoding and morphological approaches. For DNA metabarcoding, we targeted the standard barcoding marker cytochrome oxidase subunit I using highly degenerate primers, 2*300 bp sequencing on a MiSeq platform, and RTAX classification using paired-end reads. Additionally, using a large host-parasitoid dataset from a Central European floodplain forest, we assess the completeness and usability of a local reference library by confronting the number of Barcoding Index Numbers obtained by standard barcoding with the number of morphotypes. Overall, metabarcoding recovery was high, identifying 92.8% of the taxa present in mock samples, and identification success within individual taxonomic levels did not significantly differ among metabarcoding, standard barcoding, and morphology. Based on the current local reference library, 39.4% parasitoid and 90.7% host taxa were identified to the species level. DNA barcoding estimated higher parasitoid diversity than morphotyping, especially in groups with high level of crypsis. This study suggests the potential of metabarcoding for effectively recovering host-parasitoid diversity, together with more accurate identifications obtained from building reliable and comprehensive reference libraries, especially for parasitoids.

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Increased performance of DNA metabarcoding of macroinvertebrates by taxonomic sorting.

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Increased performance of DNA metabarcoding of macroinvertebrates by taxonomic sorting.

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