Computational protocol: Surfactant-associated bacteria in the near-surface layer of the ocean

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[…] From a total of 88,198 raw sequences of 8 samples collected on 10 July, 2010 and 10 September, 2011, we obtained 23,778 quality-filtered, non-chimeric sequences. Of those filtered sequences, 10, 593 were used in .Sequences were filtered out during the demultiplexing step, if they had >6 ambiguous base calls, minimum average quality score <25, minimum sequence length <200nt, maximum length of homopolymer >6, and contain any primer mismatches. The demultiplexed sequences were denoised using QIIME denoiser. Operational Taxonomic Units (OTUs) were picked according to their sequence similarity to 97% with UCLUST algorithm. PyNAST was then used to align the OTUs with an alignment template obtained from the Greengenes 16S rRNA gene database. Chimeric sequences were removed using ChimeraSlayer before taxonomy was assigned with the Ribosomal Database Project (RDP) classifier. OTU tables were generated and rarefied to 382 reads in order to match the lowest sequence number obtained for the slick sea surface microlayer (Slick SML) 2010 sample ( and ). Alpha diversity rarefaction plots were created in QIIME using Observed Species metrics (). The rarefied OTU table was used to determine the relative abundance of surfactant-associated genera (). The raw sequence reads have been deposited in the NCBI Sequence Read Archive database under accession number PRJNA280411.In a separate project, the samples taken on July 10, 2010 have also been analyzed using Real time PCR for genus Bacillus and shown consistent results with our sequencing described above (). […]

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