IonTorrent Smagellanicum nifH amplicon library - sample TDF
Amplicon libraries of the nifH gene were generated by IonTorrent PGM sequencing. First A fragment of the nifH gene (360bp) was amplified by polymerase chain reaction (PCR) in a nested approach: the primary step included the forward primer 19F (5'-GCIWTYTAYGGIAARGGIGG-3'; Ueda et al. 1995) and the reverse primer nifH3 (5'-ATRTTRTTNGCNGCRTA-3; Zani et al. 2000); in the second step, the forward primer nifH1 (5'-TGYGAYCCNAARGCNGA-3) and the reverse primer nifH2                (5'-ANDGCCATCATYTCNCC-3; Zehr and McReynolds 1989) were used. For PCR, PerfCTa Quanta master mix (Quanta Biosciences, Gaithersburg, USA) was used. Both PCR protocols started with an initial denaturation step of 98 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min; completed by a final elongation step at 72 °C for 10 min. Nested PCR was performed with 1 μL PCR product of the first PCR. Correct length of both PCR products was checked with gel electrophoreses (GE) on an agarose gel (1%, runtime of 1 h at 60mV). Products of the nested PCR with correct lengths (360 base pairs (bp)) were used for amplicon sequencing.    Amplicon sequencing  Amplicon sequencing of nested PCR products was done using the Ion Torrent Personal Genome MachineTM (Life technologies, Carlsbad, USA). Products were prepared for sequencing following the manufacturers protocol (MAN0006846 Revision 3.0) using the Ion Plus Fragment Library Kit (Life technologies, Carlsbad, USA) and the Ion XpressTM Barcode Adapter Kits 1-2 (Life technologies, Carlsbad, USA). DNA products of several PCR-reactions of the same sample were pooled to obtain sufficient amounts of DNA. First, PCR products were purified using magnetic beads (Agencourt® AMPure® XP Kit, Beckman Coulter, Brae, USA) and magnetic rack (DynaMagTM-2 magnet). After each following step purification of the DNA fragments was performed using this method.     After determination of purity and concentration using the Bioanalyzer® 2100 and the High sensitivity DNA kit (Agilent Technologies, Santa Clara, USA), DNA fragments of the same sample were pooled in equimolar amounts, to a final concentration of 50 ng DNA μL-1. Next, DNA fragments were end-repaired, adapters containing specific barcodes (for each sample a different barcode) were ligated, followed by a nick-repair. Finally, amplification (6 cycles, according to manufacturer’s protocol) was performed, followed by quantification and a purity check of the product using the Bioanalyzer® 2100 and the High sensitivity DNA kit (Agilent Technologies, Santa Clara, USA). All barcoded samples for sequencing were pooled in equimolar amounts to a final concentration of 26 pM DNA. Pooled fragments were amplified on Ion Sphere particles using the Ion One TouchTM Instrument and Ion One TouchTM 400 Template Kit (Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. After enrichment of the Template-Positive Ion SphereTM Particles using the Ion One TouchTM ES (Life Technologies, Carlsbad, USA), the spheres were loaded on an Ion 314TM v2 Chip. Subsequently, DNA fragments were sequenced using the Ion PGMTM 400 Sequencing Kit according the manufacturer’s instructions (MAN0007242 Revision 2.0).  references:  Ueda T, Suga Y, Yahiro N, Matsuguchi T (1995) Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. J Bacteriol 177:1414–1417.  Zani S, Mellon MT, Collier JL, Zehr JP (2000) Expression of nifH Genes in Natural Microbial Assemblages in Lake George, New York, Detected by Reverse Transcriptase PCR. Appl Environ Microbiol 66:3119–3124. doi: 10.1128/AEM.66.7.3119-3124.2000  Zehr J, McReynolds L (1989) Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55:2522–2526.