Computational protocol: Comparative Molecular Microbial Ecology of the Spring Haptophyte Bloom in a Greenland Arctic Oligosaline Lake

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Protocol publication

[…] We performed genomic DNA amplifications using eukaryotic and bacterial-specific primers targeting the V9 (Amaral-Zettler et al., ) or V6–V4 (Morrison and Sogin, in preparation) regions, respectively. Eukaryotic sequences were generated using a Genome Sequencer FLX (Roche, Switzerland) with the GS-LR70 long-read sequencer kit at the Marine Biological Laboratory Keck Sequencing Facility. Amplifications and sequencing for eukaryotic sequences were after Amaral-Zettler et al. (). We sequenced the V6–V4 hypervariable region of the bacterial 16S rRNA gene using bacterial primers 515F and 1046R on a Roche GS FLX pyrosequencer using GS FLX Titanium Series reagents (Roche Diagnostics, Basel, Switzerland) following manufacturer’s protocols. Sequences were trimmed and screened for quality after Huse et al. (). To assign taxonomy to the remaining quality-controlled tags, we used the Global Alignment for Sequence Taxonomy (GAST) algorithm (Huse et al., ). Tag sequences were grouped into Operational Taxonomic Units using SLP-PWAL (refer to Huse et al., ), with bacterial sequences clustered at 3% and eukaryotic sequences clustered at 6%. Venn diagrams were constructed using BioVenn (Hulsen et al., ). Bacterial diversity estimates were calculated using EstimateS v8.2.0 (Colwell, ) and CatchAll (Bunge, ). The open source Investigation/Study/Assay (ISA) (Sansone et a., ) metadata-tracking framework was used to curate the datasets and format them for submission to the NCBI SRA database. All sequences have been deposited in the NCBI Sequence Read Archive (SRA) under the SRA number SRA059384, and are MIMARKS compliant (Yilmaz et al., ). […]

Pipeline specifications

Software tools BioVenn, MIxS
Application Miscellaneous
Organisms Hemisus marmoratus