phototroph, bacterial and fungal communities at the surface of Gartenacker soil
The bTEFAP method was performed by the Research and Testing Laboratory (Lubbock, TX). All DNA samples were adjusted to 100 ng/µl. A 100 ng (1 µl) aliquot of each samples DNA was used for a 50 µl PCR reaction. HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) was used for PCR. Amplicons were purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA). In preparation for FLX sequencing (Roche, Nutley, New Jersey), the DNA fragments size and concentration were accurately measured by using DNA chips under a Bio-Rad Experion Automated Electrophoresis Station (Bio-Rad Laboratories, CA, USA) and a TBS-380 Fluorometer (Turner Biosystems, CA, USA). A 9.6 E+06 sample of double-stranded DNA molecules/µl were combined with 9.6 million DNA capture beads, and then amplified by emulsion PCR. After bead recovery and bead enrichment, the bead-attached DNAs were denatured with NaOH, and sequencing primers were annealed. A two-region 454 sequencing run was performed on a 70×75 GS PicoTiterPlate (PTP) by using a Genome Sequencer Titanium FLX System (Roche, Nutley, New Jersey). It should be noted that 100 total samples were run within this same FLX 2-region sequencing reaction. The additional 79 tagged samples were associated with unrelated studies. All FLX related procedures were performed following Genome Sequencer FLX System manufacturers instructions (Roche, Nutley, New Jersey).