Towards reproducible metabarcoding data: Lessons from an international cross‐laboratory experiment

Abstract

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (

Fish community surveys in eelgrass beds using both eDNA metabarcoding and seining: implications for biodiversity monitoring in the coastal zone

“…Although eDNA metabarcoding has been widely used to survey fish biodiversity, there is currently no consensus for a standardized eDNA metabarcoding protocol and bioinformatic pipelines (Spens et al 2017;Prodan et al 2020;Zaiko et al 2021). Results can be affected by changes at one or more of the many steps in the protocol: sample collection, water filtration, DNA extraction, genetic marker selection, library preparation, sequence quality control, feature (e.g., ASV vs. OTU) table construction, and taxonomy assignment.…”

Temporal and Spatial Variation of the Skin-Associated Bacteria from Healthy Participants and Atopic Dermatitis Patients

“…Consequently, a mean of 12.7% of reads per sample was removed during these final processing steps. Read counts were normalized with a fourth-root transformation to counter PCR biases ( 44 ), which were then converted to relative abundances for downstream statistical analyses.…”

Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding

“…PCR-generated artifacts have also been found to increase as species diversity increases (Qiu et al 2001); thus, amplicon-sequence data of 16S rRNA or 18S rRNA from environmental DNAs may contain several artifacts. Furthermore, in a crosslaboratory experiment, the choice of the polymerase had a consistently significant effect on the metabarcoding data variability explained by the differential performance of the polymerases used (Zaiko et al 2021).…”

“…The resulting HTS data is influenced by various factors from DNA extraction to bioinformatics data analysis (Oliver et al 2015;Deiner et al 2018;Nichols et al 2018;Jeunen et al 2019;Santoferrara 2019;van der Loos and Nijland 2020;Zaiko et al 2021). Amongst those factors, PCR amplification can influence the diversity detected and relative sequence abundances obtained by the HTS data (Haas et al 2011;Brandarriz-Fontes et al 2015;Kelly et al 2019).…”