title: Pooled DNA from soil Samples Type IV
design: To assign phylogenetic affiliation, 454 pyrosequencing was conducted. In order to reduce the number of samples, equal amounts of DNA from each of the 9 samples were pooled according to type. Multiplex identifiers were added to the primers with a 4 base tag for each sample. The V3 16S rRNA genes were amplified using the primers 341F (5’-CCTACGGGAGGCAGCAG-3’) and 805R (5’- CCGTCAATTCCTTTRAGTTT-3’), containing the 454 FLX adaptors, with the sample-specific multiple identifier.    A PCR mixture contained each primer at 0.5 umol l-1, each deoxynucleoside triphosphate at 0.25 mmol-1, 1.25 U of Phusion High-Fidelity DNA Polymerase DNA Polymerase and HF buffer (Finzymes). PCRs were conducted in triplicate and pooled, purified using the NucleoSpin Kit™ (Clonetech, Japan)and quantified using the Picogreen assay kit(Invitrogen)Samples were then submitted to the GATC (Germany) for pyrosequencing on a 454 GS FLX.  Hypolith Type I: Barcode CATA TitA_8-28fB 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGCATAAGAGTTTGATCCTGGCTCAG-3’  Hypolith Type II: ACAG TitA_8-28fC 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGACAGAGAGTTTGATCCTGGCTCAG-3’   Hypolith Type III: TTCA TitA_8-28fE 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCAAGAGTTTGATCCTGGCTCAG-3’   Open Soil Type IV AGCG TitA_8-28fF 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAGAGCGAGAGTTTGATCCTGGCTCAG-3’  