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Protocol publication

[…] Amplification of the V4 region of the 18S rRNA genes (rDNA) and 18S rRNA was performed using the eukaryotic primers Ek-NSF573 (CGCGGTAATTCCAGCTCCA) and Ek-NSR951 (TTGGYRAATGCTTTCGC) (Mangot et al., ). Pyrosequencing was achieved by the GINA Platform (Clermont-Ferrand, France), using a Roche 454 GS-FLX system with titanium chemistry. Pyrosequencing data for both 18S rDNA and 18S rRNA datasets represented 304,315 raw sequences (total for 2 and 80 m depths). The cleaning procedure of the reads is summarized in Supplementary Figure . Briefly, cleaning procedures consisted in the elimination of sequences presenting ambiguous bases “N,” a quality score < 25, length shorter 200 bp, with a mismatch in the forward primer and chimeras (Edgar, ). The remaining sequences were clustered at a 95% similarity threshold as suggested by Debroas et al. () with UCLUST (Edgar, ) and representative sequence for each OTU were aligned (Eddy, ) and inserted in phylogenetic trees with Fasttree (Price et al., ) for taxonomic annotation. After the cleaning of 454 raw data and discarding the singletons and OTUs without rDNA reads, we defined 7737 OTUs for the monimolimnion and 6883 for the mixolimnion. This process was automated by PANAM that also computed richness and diversity indexes, Chao1 and Shannon respectively (http://code.google.com/p/panam-phylogenetic-annotation/downloads/list) (Taib et al., ). We also performed a hierarchical cluster analysis based on Bray Curtis distance in order to compare the structure of eukaryotic communities for all DNA samples between 2 and 80 m (XLSTAT 2015 4.01). The monimolimnion (80 m depth) dataset contained a total of 44,000 sequences for the 18S rDNA dataset and 10,324 sequences for the 18S rRNA dataset (the latter was obtained for one date, 05/07/2011). The data were normalized to have an equal number of rRNA and rDNA reads per sample in order to infer on the activity of eukaryotic taxa (rRNA:rDNA ratio). The data have been deposited in the Dryad Digital Repository. […]

Pipeline specifications

Software tools UCLUST, FastTree, PANAM
Application Phylogenetics
Organisms Hemisus marmoratus
Chemicals Oxygen