Computational protocol: Inter-annual variability of transparent exopolymer particles in the Arctic Ocean reveals high sensitivity to ecosystem changes

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Protocol publication

[…] Raw sequence reads were processed using the analysis pipeline Quantitative Insights Into Microbial Ecology Version 1.8.0 (QIIME)1. The primer set used in this study amplifies a PCR product of ~500 bp including the V4-region of the 18 S rRNA gene. The forward primer 528 F, used for the sequencing, attaches approximately 25 bp upstream of the V4 region, which has an approximate length of 230 bp. Thus, sequence reads with a length under 250 bp were excluded from further analysis to ensure including the complete V4 region in the analysis and to omit short reads. The quality score was set to 25 and eight homopolymers and two primer mismatches were allowed. Chimeric sequences in the remaining data set were eliminated from further analyses based on an assessment using the software UCHIME within QIIME. The resulting high quality reads were subsampled to allow comparison of sequence abundance in the different samples. Subsequently these high quality sequences were grouped into operational taxonomic units (OTUs) at the 97% similarity level using UCLUST. The 97% similarity level has shown to be the most suitable to reproduce original eukaryotic diversity and also has the effect of bracing most sequencing errors. Furthermore, known intragenomic SSU polymorphism levels can vary by 2.9% in dinoflagellate species. OTUs composed of less than 4 sequence reads were removed from the analysis. The remaining sequences were aligned using the SILVA reference database (SSU Ref 119). Unassigned OTUs and those assigned to Ophistakonta were excluded from further analysis in a final cleaning step. […]

Pipeline specifications

Software tools QIIME, UCHIME, UCLUST
Application 16S rRNA-seq analysis
Chemicals Carbon Dioxide