Scrutinizing key steps for reliable metabarcoding of environmental samples

Abstract 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. 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…