Similar protocols

Pipeline publication

[…] ssion number PRJEB22033., To perform a hierarchical taxonomic rank assignment for each sequence, a pipeline developed by the Leibniz-Institut DSMZ (Esposito et al., ) was implemented to pre-process raw sequences. Only sequences that contained intact barcode and primer sequences were included in the analyses. Additionally, sequences containing ambiguous bases were removed from the analyses. A quality filter with a Phred score of 20 was applied. After trimming, quality-filtering and pairing the raw reads, 29,064,287 sequences for Bacteria and Archaea and 8,009,549 sequences were obtained for eukaryotes including Fungi. Bacterial and Archaeal sequences were checked for potential chimeras using UCHIME (Edgar et al., ). A taxonomic-independent analysis was conducted for all four communities. Sequences that passed the pre-processing steps were first binned into operational taxonomic units (OTUs) using the open-reference pipeline for OTU picking (Caporaso et al., ) at 97% similarity using UCLUST (Edgar, ), followed by picking a representative sequence for each OTU. Representative sequences were aligned to the Greengenes Core reference alignment (DeSantis et al., ) using PyNAST (Caporaso et al., ), and the resulting sequences were kept for building the OTU tables. Then, OTUs were taxonomically assigned using the Ribosomal Data Project (RDP) Classifier (Cole et al., ) command line version 2.10.1. Sequences were considered as classified at the deepest taxonomic rank with a minimum confidence value of 0.5. Taxonomic assignment of the reads showed that bacterial primers also amplified archaeal phyla. These archaeal reads were discarded for the bacterial OTU analyses and all the bacterial reads for the archaeal OTU analyses., High-quality fungal ITS sequencing data were processed using the same pipeline. Chimeric sequences were removed using a combination of refe […]

Pipeline specifications

Software tools UCHIME, UCLUST, PyNAST