title: Amplicon Sequencing of 16S rDNA from Bulk Soils
design: DNA was isolated from 250 mg of soil using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc, CA, USA). RNA was obtained from soil using Total RNA Powersoil from MoBio and first strand cDNA was synthesized using first-strand cDNA synthesis system kit from Roche. In a first round of PCR amplification, the V4 hyper variable region of the 16s rRNA gene was amplified from gDNA and cDNA samples using the RDP universal primers containing a TAG sequence at the 5´end of both, Forward and Reverse amplification primers. The Forward TAG sequence is CACGACGTTGTAAAACGAC and Reverse TAG is: CAGGAAACAGCTATGACC. The universal primer sequence is:  563F (5’-CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTAYTGGGYDTAAAGNG -3’, where ACGAGTGCGT is an example, different MIDs for each sample were used) and 802R (5’-CTATGCGCCTTGCCAGCCCGCTCAGTACCRGGGTHTCTAATCC, 5’-CTATGCGCCTTGCCAGCCCGCTCAGTACCAGAGTATCTAATTC, 5’-CTATGCGCCTTGCCAGCCCGCTCAGCTACDSRGGTMTCTAATC, 5’-CTATGCGCCTTGCCAGCCCGCTCAGTACNVGGGTATCTAATCC) obtained from RDP’s Pyrosequencing Pipeline: http://pyro.cme.msu.edu/pyro/help.                                                                                 A second round of PCR was performed using first PCR amplification products as templates. This PCR was done to add Barcode sequences to identify each sample. Primers for this step were constructed using the same TAG sequences in the 3´primer region in both Forward and Reverse Primers, followed at the 5´by a MID or barcode sequence (see below for the sequences) and the standard Roche A and B adapters for LibA amplification.                                                            The final structure of the amplicon was: 5´ RocheAadapter/MID/TAG/PrimerFW/AmpliconSequence/PrimerRV/TAG/RocheBadapter 3´.                                                                                                                                                         For each sample, two independent PCRs were performed. The PCR mix (final volume 25 ul) contained 2.5 ul FastStart High Fidelity 10X Reaction Buffer (Roche Applied Science, Mannheim, Germany), 20 ng of template DNA, 0.4 uM each primer, 1.25 U FastStart High Fidelity Enzyme Blend (Roche Applied Science), and 0.2 mM DNTPs. The PCR conditions were 95 °C for 5 minutes for initial denaturalization, followed by 95 °C for 45 seconds, 57 °C for 45 seconds, 72 °C for 60 seconds in 30 cycles, and a final elongation step at 72°C for 4 minutes. Two negative control reactions containing all components were performed without the template. Reactions were pooled in equal amounts for each site and purified using the MinElute PCR Purification Kit (Qiagen GmbH, Hilden, Germany). Quantification of the purified PCR products was performed by using Quant-iT PicoGreen dsDNA Kit (Invitrogen Molecular Probes Inc, Oregon, USA).  Purified PCR products were immobilized onto DNA capture beads, amplified through emulsion-based clonal amplification (emPCR), and sequenced together in one PicoTiterPlate device on a Genome Sequencer FLX (Roche Applied Science) according to the manufacturer’s instructions at INDEAR (Argentina) Genome sequencing facility.  Samples were multiplexed in a single 454 GS FLX Titanium run obtaining 158,164 filter passed reads with sequences with an average length of 287 bp.                                                                                       The QIIME mapping file used to demultiplex samples was (TAG sequence was considered as part of the Barcode, For a correct view of the mapping file copy and paste the following text and replace @ by a new line character).       #SampleID BarcodeSequence LinkerPrimerSequence ReversePrimer Replicate Site Area Management PlantType Depth LandUse Soil source Description  @anaBg.SD1.a ACGAGTGCGTCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil Agro Wheat 0-20cm NT Bulk anaBg.SD1.a  @anaBg.SD2.a ACGCTCGACACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil Agro Wheat 0-20cm NT Bulk anaBg.SD2.a  @anaBg.SD3.a AGACGCACTCCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil Agro Wheat 0-20cm NT Bulk anaBg.SD3.a  @anaBg.LC3.a AGCACTGTAGCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil Agro Wheat 0-20cm CT Bulk anaBg.LC3.a  @anaBg.LC1.a ATCAGACACGCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil Agro Wheat 0-20cm CT Bulk anaBg.LC1.a  @anaBg.LC2.a ATATCGCGAGCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil Agro Wheat 0-20cm CT Bulk anaBg.LC2.a  @anaBg.NA1.a CGTGTCTCTACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil NoAgro WheepingGrass 0-20cm NA Bulk anaBg.NA1.a  @anaBg.NA2.a CTCGCGTGTCCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil NoAgro WheepingGrass 0-20cm NA Bulk anaBg.NA2.a  @anaBg.NA3.a TCTCTATGCGCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC a INTA_Anguil Anguil NoAgro Grass 0-20cm NA Bulk anaBg.NA3.a  @anaBg.SD1.b TCACGCGAGACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil Agro Wheat 0-20cm NT Bulk anaBg.SD1.b  @anaBg.SD2.b TCGATAGTGACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil Agro Wheat 0-20cm NT Bulk anaBg.SD2.b  @anaBg.SD3.b TCGCTGCGTACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil Agro Wheat 0-20cm NT Bulk anaBg.SD3.b  @anaBg.LC3.b TCTGACGTCACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil Agro Wheat 0-20cm CT Bulk anaBg.LC3.b  @anaBg.LC1.b TGAGTCAGTACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil Agro Wheat 0-20cm CT Bulk anaBg.LC1.b  @anaBg.LC2.b TGTAGTGTGACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil Agro Wheat 0-20cm CT Bulk anaBg.LC2.b  @anaBg.NA1.b TGTCACACGACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil NoAgro WheepingGrass 0-20cm NA Bulk anaBg.NA1.b  @anaBg.NA2.b TGTCGTCGCACACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil NoAgro WheepingGrass 0-20cm NA Bulk anaBg.NA2.b  @anaBg.NA3.b ACACATACGCCACGACGTTGTAAAACGAC AYTGGGYDTAAAGNG CAGGAAACAGCTATGACC b INTA_Anguil Anguil NoAgro Grass 0-20cm NA Bulk anaBg.NA3.b