A fragment of the 16S rRNA gene encompassing the V3 and V4 variable regions [29 (link)] was amplified with primers 357f (5′ CCT ACG GGA GGC AGC AG 3′, [30 ]) and 786r (5′ AC CAG GGT ATC TAA WCC 3′, this work) with overhangs required for pyrosequencing. The forward primer was used in three versions differing in the multiplex identifier (MID) sequence between the A adaptor and the actual primer. The reaction mix contained the following: 1 ng of template DNA, 10 pmol primers, 0.2 mM dNTP, 1.5 mM MgCl2, 0.2 U of Taq polymerase, and the appropriate 1× buffer (Fermentas, Lithuania). The cycling conditions were as follows: 95 ºC for 5 min, 30 cycles of: 95 ºC for 30 s, 52 ºC for 30 s, 72 ºC for 30 s; and finally 72 ºC for 2 min. The reactions were carried out in MasterCycler thermocycler (Eppendorf, Germany). Eight independent reactions were performed for each sample.
PCR products were purified with the DNA CleanUp kit (A&A Biotechnology, Poland), pooled in equimolar amounts, vacuum dried, and either sent for sequencing at Max Planck Molecular Genetics Institute sequencing service (Berlin, Germany—Olkusz samples) or sequenced at the Chair of Genetics, University of Silesia (Katowice, Poland—Alwernia ones). Pyrosequencing was performed with the use of Titanium chemistry on GS FLX and GS Junior instruments (Roche, USA) as described in the manufacturer’s protocols.
PCR products were purified with the DNA CleanUp kit (A&A Biotechnology, Poland), pooled in equimolar amounts, vacuum dried, and either sent for sequencing at Max Planck Molecular Genetics Institute sequencing service (Berlin, Germany—Olkusz samples) or sequenced at the Chair of Genetics, University of Silesia (Katowice, Poland—Alwernia ones). Pyrosequencing was performed with the use of Titanium chemistry on GS FLX and GS Junior instruments (Roche, USA) as described in the manufacturer’s protocols.