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Scrutinizing key steps for reliable metabarcoding of environmental samples [Methods in Ecology and Evolution]

Keywords : biodiversity assessment, environmental DNA, faecal samples, high throughput sequencing, metabarcoding primers, molecular diet analyses, operational taxonomic unit, PCR replicates, primer bias, taxonomic assignment

par Frédéric Magné - publié le , mis à jour le

1. 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.(...)

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