Background: The ability to compare samples or studies easily using metabarcodingso as to better interpret microbial ecology results is an upcoming challenge. A grow‑ing number of metabarcoding pipelines are available, each with its own benefts andlimitations. However, very few have been developed to ofer the opportunity to charac‑terize various microbial communities (e.g., archaea, bacteria, fungi, photosyntheticmicroeukaryotes) with the same tool.Results: BIOCOM-PIPE is a fexible and independent suite of tools for processing datafrom high-throughput sequencing technologies, Roche 454 and Illumina platforms,and focused on the diversity of archaeal, bacterial, fungal, and photosynthetic microeu‑karyote amplicons. Various original methods were implemented in BIOCOM-PIPE to (1)remove chimeras based on read abundance, (2) align sequences with structure-basedalignments of RNA homologs using covariance models, and (3) a post-clustering tool(ReClustOR) to improve OTUs consistency based on a reference OTU database. Thecomparison with two other pipelines (FROGS and mothur) and Amplicon SequenceVariant defnition highlighted that BIOCOM-PIPE was better at discriminating land usegroups.Conclusions: The BIOCOM-PIPE pipeline makes it possible to analyze 16S, 18S and23S rRNA genes in the same packaged tool. The new post-clustering approach defnesa biological database from previously analyzed samples and performs post-clusteringof reads with this reference database by using open-reference clustering. This makes iteasier to compare projects from various sequencing runs, and increased the congru‑ence among results. For all users, the pipeline was developed to allow for adding ormodifying the components, the databases and the bioinformatics tools easily, givinghigh modularity for each analysis.