Microbial methods matter: Identifying discrepancies between microbiome denoising pipelines using a leaf biofilm...

Area of Science:

• Microbial Ecology
• Bioinformatics
• Paleontology

Background:

• Microorganisms on aquatic macrophyte fossils suggest biofilms aid preservation.
• Understanding microbial impact on leaf preservation requires studies on living plants and microbial amplicon sequencing.
• Accurate data interpretation hinges on selecting the optimal bioinformatic pipeline for microbial community composition.

Purpose of the Study:

• To compare the performance of three bioinformatic pipelines (DADA2, Deblur, UNOISE) for analyzing microbial communities in aquatic leaf biofilms.
• To evaluate the impact of different denoising algorithms on microbial community composition estimates.
• To determine the most suitable pipeline for analyzing 16S rRNA, 18S rRNA, and ITS amplicon regions.

Main Methods:

• Analysis of biofilms from floating and submerged leaves of *Nymphaea alba* and *Nuphar lutea*.
• Use of mock communities to assess pipeline accuracy.
• Application of primers for 16S ribosomal RNA (rRNA), 18S rRNA, and ITS amplicon regions.
• Comparison of microbial community compositions derived from DADA2, Deblur, and UNOISE pipelines.

Main Results:

• The choice of denoising pipeline alters the number of identified sequences and specific taxa.
• Microbial communities differed between leaf depths (floating vs. submerged).
• Environmental effects on microbial communities varied depending on the amplicon region analyzed.

Conclusions:

• DADA2 is recommended for analyzing 16S rRNA and 18S rRNA amplicon data due to its performance in identifying amplicon sequence variants (ASVs).
• For the ITS region, Deblur identified the most ASVs and showed compositional similarity to DADA2, making it a viable option.
• Pipeline selection is critical for accurate microbial community profiling in aquatic plant biofilms.