title: Characterization of the bacterial community composition in water of drinking water production and distribution systems in Flanders, Belgium
design: An amplicon library was created using the primer combination 515F (5-GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACVSGGGTATCTAAT-3) generating amplicons which cover the prokaryotic (bacterial and archaeal) V4 region of the 16S rRNA gene . This primer combination has been commonly used in diverse metagenomics studies, including water research. Fusion primers, required for the 454 pyrosequencing process, were designed according to the guidelines for 454 GS-FLX Titanium Lib-L sequencing and contained the Roche 454 pyrosequencing adapters and a sample-specific multiplex identifier (MID) sequence in between the adapter and the forward primer for sample-specific sequence tracking. A T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) was used for PCR amplification. The total reaction volume was 20 L and contained 1.0 L 10x diluted genomic DNA, 1.5 L dNTP mixture (2 mM stock; Invitrogen, Carlsbad, CA, USA), 0.5 L of each primer (20 M stock), 2.0 L 10x Titanium Taq PCR buffer, 0.4 L Titanium Taq DNA polymerase (Clontech Laboratories, Palo Alto, CA, USA) and 14.1 L nuclease-free water. The following PCR conditions were used: initial denaturation of 2 minutes at 94 C, followed by 30 cycles of 45 s at 94 C, 45 s at 59 C, and 1 min at 72 C, followed by a final extension phase of 10 min at 72 C. Following agarose gel electrophoresis, amplicons of the expected size range were excised and extracted from the gel using the QIAquick gel extraction kit, according to the manufacturers instructions (Qiagen, Hilden, Germany). Purified dsDNA amplicons were quantified using a Qubit 2.0 fluorometer and the high-sensitivity DNA reagent kit (Invitrogen, Carlsbad, CA, USA). Next, all samples were diluted to equimolar concentrations and an amplicon library containing 1.00*109 molecules/L per sample was prepared. A final quality check was done on an Agilent Bioanalyzer 2100 with high-sensitivity chip (Agilent Technologies, Waldbronn, Germany), and the library was sequenced using the Roche GS-FLX instrument with Titanium chemistry according to manufacturers instructions (Roche Applied Science, Mannheim, Germany).