Comparison of commonly used software pipelines for analyzing fungal metabarcoding data

Area of Science:

• Fungal Ecology
• Bioinformatics
• Environmental Microbiology

Background:

• Metabarcoding of the internal transcribed spacer (ITS) region is standard for fungal community analysis.
• Bioinformatic pipelines for ITS sequence processing are not standardized and can yield variable results.
• The use of amplicon sequencing variants (ASVs) for fungal ITS data is debated, unlike its established use for prokaryotic 16S rRNA.

Purpose of the Study:

• To compare the performance of two common bioinformatic pipelines, DADA2 (ASVs) and mothur (OTUs), for fungal metabarcoding.
• To evaluate pipeline-associated biases in fungal community analysis.
• To determine the most appropriate method for processing fungal ITS metabarcoding data.

Main Methods:

• Comparison of DADA2 and mothur pipelines using fungal metabarcoding sequences.
• Analysis of sequences from environmental samples: bovine feces and pasture soil.
• Evaluation of fungal richness and relative abundance homogeneity across technical replicates.

Main Results:

• Mothur, using operational taxonomic unit (OTU) clustering at 99% similarity, identified higher fungal richness than DADA2.
• Mothur produced homogenous relative abundances across 18 technical replicates.
• DADA2 generated highly heterogeneous relative abundances across the same replicates.

Conclusions:

• Bioinformatic pipeline choice introduces bias in fungal metabarcoding data analysis.
• OTU clustering at 97% similarity is suggested as the most appropriate method for processing fungal metabarcoding data.
• This recommendation is based on observed homogeneity of relative abundances and better detection of fungal diversity.