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Supplementary Information
Materials and Methods
PCR reactions were performed in triplicate with the primer sets 349f/806r (Lenchi et al.,
2013), 27f/519r (Amann et al., 1995) and ITS3/ITS4 (White et al., 1990) to amplify the
hypervariable regions of the archaeal V3-V5 and bacterial V1-V3 16S genes as well as
the ITS2 region of the hypervariable fungal ribosomal operon, respectively. The PCR
conditions to amplify both archaeal and bacterial 16S were similar (initial denaturation at
95°C for 15 min; 30 cycles of denaturation at 94°C for 40 s and elongation at 72°C for 1
min; final elongation at 72°C for 10 min) but differed in their annealing temperature
(archaeal 16S: 57°C for 40 s; bacterial 16S: 60°C for 40 s). The PCR conditions to
amplify the fungal ITS2 region consisted of an initial denaturation step at 95°C for 15
min followed by 40 cycles of denaturation (94°C for 40 s), annealing (56°C for 40 s) and
elongation (72°C for 1 min) steps with a final elongation step at 72°C for 10 min.
Sequence curation
Reads with less than 360 flows, more than two mismatches to the target specific primers and
one mismatch to the barcode sequences were discarded while reads with flowgrams bigger than
720 were trimmed to 720 flows. Erroneous reads obtained due to homopolymeric signal
misinterpretation were removed with PyroNoise (Quince et al., 2009). Target primer and barcode
sequences were trimmed. V-Xtractor (Hartmann et al., 2010) and ITSx (Bengtsson-Palme et al.,
2013) were used to extract phylogenetically comparable segments from the bacterial V1-V2 and
archaeal V3-V4 regions of the 16S ribosomal sequence as well as the fungal ITS-2 region.
Errors introduced during PCR amplification were reduced with SeqNoise (Quince et al., 2011)
and chimeric sequences were removed with UCHIME (Edgar et al., 2011)
in MOTHUR.
Chimera-free sequences were clustered in operational taxonomic units (OTU) at 97% similarity
with CROP (Hao et al., 2011). Taxonomic assignment was performed in MOTHUR with a naïve
Bayesian classifier (bootstrap support > 60%) (Wang et al., 2007) comparing the curated
sequences with the GREENGENES (DeSantis et al., 2006; McDonald et al., 2012) and UNITE
(Abarenkov et al., 2010) reference databases for archaea, bacteria and fungi, respectively.
Consensus taxonomy data were generated in MOTHUR so that 80% of the sequences related to
an OTU were represented.
References
Amann R, Ludwig W, Schleifer K. (1995). Phylogenetic identification and in situ
detection of individual microbial cells without cultivation. Microbiol Rev 59:143–196.
Lenchi N, Inceoğlu O, Kebbouche-Gana S, Gana ML, Llirós M, Servais P, et al. (2013).
Diversity of microbial communities in production and injection waters of Algerian oilfields
revealed by 16S rRNA gene amplicon 454 pyrosequencing. PLoS One 8:e66588.
White T, Bruns T, Lee S, Taylor J. (1990). Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In:PCR protocols: a guide to methods and
applications, Innis, M, Gelfand, D, Sninsko, J, & White, T (eds), Academic Press: San
Diego, pp. 315–322.