Similarity percentage analysis (SIMPER) and principal component analysis (PCA), overlain with Bray-Curtis similarity using PRIMER 6 (PRIMER Ltd., Plymouth, UK, Plymouth Marine Laboratory, UK) ( Clarke 1993), were used to identify the TRFs that contributed most to the dissimilarity between stations. One microlitre of DNA extract from sample E54 was the template for the PCR reaction, using universal bacterial primers GM3 (5′-AGA GTT TGA

TCC TGG C-3′) and 1507R Pexidartinib (5′-TAC CTT GTT ACG ACT T-3′) for the 16S rRNA gene (Muyzer et al. 1995). The PCR reaction contained 25 μl PCR Master Mix (Promega GmbH, Mannheim, Germany) and 4 μM of forward and reverse primer in 50 μl. The cycle programme was 94°C for 1 min, 25 cycles of 94°C for 1 min, 42°C for 1 min, and 72°C for 3 min, followed by 60°C for 60 min. The PCR amplicons were purified on Sephadex columns (SephadexTM G-50 Superfine, Amersham Bioscience AB, Uppsala, Sweden) and approximately 10 ng DNA were cloned with a PCR 4.0-TOPO kit, following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Positive clones were selected by ampicillin resistance and the blue or white colony colour. The cloned and amplified 16S rRNA sequences were purified on Sephadex columns. The sequencing

reaction was determined using the ABI Dye Terminator technology and the Applied Biosystems 3130xl DNAsequencer (Applied Biosystems, Foster City, USA). The 16S rRNA gene sequences were analysed with Sequencing Analysis 5.2 (Applied Biosystems, Foster City, USA) and assembled with Sequencer 4.6 (Gene codes, Ann Arbor, MI). selleck chemical Bellerophon ( Huber et al. 2004), Chimera-Check ( DeSantis et al. 2006), DECIPHER ( Wright et al. 2012) and BLAST ( Zhang et al. 2000) were used to check for chimeras. From each full length 16S rRNA gene sequence

the primer sequences were removed. The initial phylogenetic affiliation was assigned using SeqMatch ( Wang et al. 2007) of the Ribosomal Database Project ( Cole et al. 2009). Sequences were aligned with the SINA online aligner tool (www.arb-sina.de) MEK inhibitor (Pruesse et al. 2012). The alignment was imported into the ARB and manually corrected. Sequences were incorporated into the 16S rRNA tree (SILVA rel 111) by the parsimony method. Phylogenetic affiliation was assigned based on information in the tree. Clones of phytoplankton plasmids (15 of all 101 submitted clones) were excluded from further analyses. The 16S rRNA gene sequences were deposited under Acc. No. KF596513 – KF596613. With Lasergene SeqBuilder (DNASTAR) the length of the in silico terminal restriction fragments (iTRF) of 16S rRNA gene sequences were determined by (i) trimming the sequences at the restriction recognition site of the restriction enzyme AluI, and (ii) adding the 20 nucleotides of the forward primer 27F to each sequence. The online programs MiCA 3 (http://mica.ibest.uidaho.edu, Shyu et al. 2007) and TRFragCalc ( Hahnke et al.