By using the cBot (Illumina) and the TruSeq SR Cluster Kit v2 – cBot–HS (Illumina) the libraries were hybridized to complementary adapter oligonucleotides of the flow cell and amplified isothermally and clonally to form clusters. Sequencing of 50 bp was performed using the TruSeq SBS Kit – HS chemistry (50 cycles) on an Illumina HiSeq 2000 resulting in 172 million single reads (Supplementary

Table S1). These sequences are available from the ENA with the study accession numbers ERP004166. The sequence associated contextual (meta)data are MIxS compliant (Yilmaz et al., 2011). Extraction of 16S rDNA fragments from metatranscriptome and metagenome data as well as pyrotags, and their subsequent taxonomic assignments were AZD2281 clinical trial done with the SILVA pipeline (Quast et al., 2013), which uses the SINA aligner (Pruesse et al., 2012). Details have been described elsewhere (Klindworth et al., 2013). Messenger RNA reads were mapped with the short read mapper ssaha2 (Ning et al., 2001) onto metagenome sequences obtained from the same samples (Teeling et al., 2012). Pfam (Finn et al., 2010) and CAZy (Cantarel et al., 2009) hits with E-values below E-6 were used for functional analyses. In cases

where a metatranscriptome read mapped to multiple genes, the least common denominator in terms of taxonomy and function was used. The Pfam analysis for the two 454 metatranscriptomes Adenosine resulted in 39,518 hits (31/03/2009) http://www.selleckchem.com/products/VX-765.html and 33,215 hits (14/04/2009).

The CAZy analysis revealed 1,210 hits (31/03/2009) and 1,010 hits (14/04/2009). The Illumina metatranscriptome showed 24,283,085 hits to the Pfam database and 602,359 to the CAZy database. In this study, we used novel data in conjunction with previously published data (Table 1). For taxonomic profiling, we used 16S rDNA reads from three different sources, (a) cDNA reads derived from total RNA (non mRNA-enriched), (b) pyrotag reads, and (c) shotgun metagenome reads. The cDNA and pyrotags datasets were on average 25 times larger than those from metagenomes. We have shown previously that results from larger datasets normally do not constitute artifacts of deep sequencing, and thus do not infringe on the comparability of the resulting taxonomic data (Klindworth et al., 2013). For functional profiling, we used metatranscriptome cDNA reads which were compared to the outcome from the metagenome and metaproteome analyses (Teeling et al., 2012). To facilitate traceability each of these datasets has been assigned a token that is used throughout the text (Table 1). As described by Teeling et al. (2012) in 2009 a spring phytoplankton bloom started with increasing sunlight and temperatures in early March in the German Bight of the North Sea, which was most likely boosted by an influx of nutrient-rich estuaries waters.