Computational protocol: High planktonic diversity in mountain lakes contains similar contributions of autotrophic, heterotrophic and parasitic eukaryotic life forms

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Protocol publication

[…] High-speed multiplexed 18S rRNA gene sequencing with the Illumina MiSeq System (2 × 150 bp) was carried out with the “universal” 1391 f and EukBr eukaryotic primers (V9 region). These primers are known to recover the known eukaryotic diversity without major qualitative or quantitative biases,. Guidelines from the Earth microbiome project (EMP) protocol, and the genomics core facilities and methods of the RTSF-MSU (Michigan State University, USA) were followed. Raw sequences were analysed with UPARSE. Overall quality of sequences was high, with 77.6% reaching Q30. The total number of sequences before quality filtering was 12,288,923. After merging of read pairs and filtering by read length (above or equal to 161 pb), quality-score distribution (ASCII ‘B’) and an expected error of 0.25, we kept 6,384,407 sequences. Out of them 921,749 were unique and 205,694 not singletons. The latter were clustered at 97% identity, after chimera removal, resulting in 6398 OTUs. Sequences were mapped back into OTUs and classified with the SILVA 119 database. Overall, we retrieved a mean of 24,428 sequences per lake (range 10677–45940). [...] Sequences were classified using the RDP classifier by default as implemented in Qiime and the Silva-NGS pipeline with the Silva 119 database. Relevant discrepancies were double-checked through local BLAST (search September 2015), and if not resolved left as “Unclassified”. Around 7% of the OTUs could not be classified beyond “domain”. Mean number of OTUs per lake (Richness) and average occurrence of OTUs were determined. Shannon diversity per lake (alpha diversity), Shannon diversity in the whole region (gamma diversity) and Berger-Parker dominance indexes were calculated.For classification ad hoc taxonomical levels reflecting the main ecological life-styles were used. Furthermore, preferences for carbon source, nutrition type and habitat were labelled. Thus, groups corresponded to distinct taxonomic hierarchical levels but shared the common characteristic that most of the species within the group showed a similar life form and functional role within the planktonic food web. For the first ten most relevant groups, we evaluated in detail more different diversity patterns (Table ). […]

Pipeline specifications

Software tools UPARSE, RDP Classifier, QIIME
Databases EMP
Application 16S rRNA-seq analysis
Organisms Toxoplasma gondii
Diseases Pulmonary Fibrosis