MDA.1
The DNA template used in this experiment was subjected to prior whole genome amplification (WGA): MDA (Multiple Displacement Amplification) and it is the replicate 1. The DNA amplicon library construction involved a PCR amplification step targeting genomic bacterial DNA of the SSU rRNA gene V5-V7 hypervariable region. The PCR reaction allowed the tagging of amplicons with identification tags or barcodes and was performed with forward primer 785F (GGATTAGATACCCBRGTAGTC) and reverse primer 1175R (ACGTCRTCCCCDCCTTCCTC). These primers also included at the 5’ end the 454 Life Sciences (Branford, CT, USA) adapter A (in the forward primer) and B (in the reverse primer) plus a unique 10 nucleotides long barcode in the forward primer. The PCR program consisted of an initial denaturation at 95°C for 120 s, 9 cycles (95°C for 30 s, 53°C for 30 s and 72°C for 80 s), 29 cycles (95°C for 30 s, 62°C for 30 s and 72°C for 80 s) and a final elongation step at 72°C for 180 s. A total volume of 50 μl was used in each PCR reaction, containing 0.4 μM forward and reverse primers, 25 μl GoTaq Colorless Master Mix (2x) (Promega, Madison, WI, USA), 13.4 μl nuclease-free water (Promega, Madison, WI, USA) and 6 μl template. Bands were cut from 1.2% agarose gels, purified with MinElute Gel Extraction kit (QIAGEN, Hilden, Germany), analysed with Experion Automated Electrophoresis System (Bio-Rad Laboratories, USA) and quantified with PicoGreen. All amplicons were pooled together in equimolar quantities (20 ng each). This mixture was dried at room temperature overnight by using a Centrivap concentrator Labconco (Beun De Ronde, Abcoude, the Netherlands) connected to an automatic freeze-dryer (Virtis, Gardiner, N.Y.; Cenco Instrumenten, Breda, The Netherlands) and resuspended to a final concentration of 20 ng DNA/ μl of nuclease-free water. The size and quality of the amplicon mixture was analyzed with a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and sequenced with a 454 GS FLX Titanium system (Roche, Basel, Switzerland) and amplicon processed at Macrogen Inc. (Seoul, South-Korea).
VU University Amsterdam
MDA.1