Thus, dRNA-seq is a powerful method for the selection of freshly initiated transcripts based on the differently phosphorylated 5′ ends. Pretreatment of bacterial RNA with TerminatorTM 5′ phosphate-dependent
exonuclease specifically degraded transcripts with a 5′ mono-phosphate. Subsequently, these samples were treated with tobacco acid pyrophosphatase to produce the RNA 5′ monophosphates necessary for RNA linker ligation, followed by reverse transcription, resulting in a cDNA pool enriched in primary transcripts. For preparation of the RNA-seq library from the 45 m sample, total RNA was reverse-transcribed using random hexamers. For all libraries, fragmented cDNA of 200–500 nt size was paired-end sequenced on an Illumina HiSeq 2000 platform Dinaciclib chemical structure with a read-length of 100 nt. With dRNA-seq, after quality filtering we obtained 77,676,351 paired reads for the 2.5 m sample, 71,291,764 paired reads for the 45 m, and 80,859,071 paired reads for the 440 m sample. Random RNA-seq resulted in 74,260,285 paired reads for the 45 m sample. Ribosomal CDK activity RNA reads were filtered out using SortMeRNA (Kopylova et al., 2012). The remaining non-ribosomal reads were then assembled
de novo with Velvet (Zerbino and Birney, 2008) using the approach of merging multiple Velvet outputs (contiguous sequences, contigs) produced with different kmer lengths. Merging of contigs was done as described in the Rnnotator pipeline (Martin et al., 2010) with Minimus2 (Sommer et al., 2007). To check the validity of the assembly and get the abundance of each contig, the raw reads were mapped back onto the merged contigs plus singleton contigs (those not merged in the Minimus2 step) using
Bowtie2 (Langmead unless and Salzberg, 2012). All steps and corresponding read numbers are presented in Fig. 2. All raw reads can be downloaded from the NCBI Sequence Read Archive under the BioProject accession number PRJNA248420. This work was supported by the Assemble (Association of European Marine Biological Laboratories) Infrastructure Access Call 5 to the Interuniversity Institute for Marine Sciences, Eilat, (IUI) Israel, by a BMBF-MOST JOINT GERMAN-ISRAELI RESEARCH PROJECT, project number GR2378/03F0640A to WRH and IBF and by the EU project MaCuMBA (Marine Microorganisms: Cultivation Methods for Improving their Biotechnological Applications; grant agreement no: 311975) to WRH. For support during the sampling we thank Martin Hagemann, University of Rostock, and especially the captain of the research ship “Sam Rothberg”, Sefi Baruch, Assaf Rivlin and the IUI logistic support teams. “
“Hydrocarbons can be major contaminants of the marine and coastal ecosystems and can have significant socio-ecological impacts. Although microbial consortia indigenous to areas with constitutively increased concentrations of hydrocarbons are well known for their ability to degrade these contaminants (Vila et al.