454 gs flx titanium pyrosequencing system

The PowerLyzer Ultra Clean Microbial DNA isolation kit (Mo Bio Laboratories Inc., CA, United States) was used to extract total DNA from 1 mL of the bacterial cultures or 0.5 g from LH samples and sludge samples collected from industrial bioreactors (BR) treating perchlorate contaminated groundwaters. The V3–V5 region of the 16S rRNA gene was amplified using key-tagged eubacterial primers based on design by Sims et al. (2012) for bacterial cultures and BR samples and based on Chih-Ying et al. (2012) (link) for LH DNA. The 16S rRNA gene amplicons were sequenced by Roche 454-GS-FLX+-Titanium pyrosequencing system (Sequencing service, UC Berkeley, California). DNA sequences were deposited on NCBI Genebank under accession numbers detailed in Supplementary Table 1. Additionally, different isolates were obtained from high Arctic LH samples, although there were no perchlorate reducers among them. To confirm the presence of a cld gene, a PCR amplification was carried out using cld specific primers (Supplementary Table 2), and the amplicon was sequenced and compared against the NCBI database for sequence similarities.

Genomic DNA from oral bacteria was extracted from oral wash samples using the MoBio PowerSoil DNA Isolation Kit (Qiagen). 16Sv3-4 rRNA gene sequencing was performed on the extracted bacterial DNA and gene amplicon libraries were generated as reported previously (15, 43 (link)). Briefly, libraries were created to allow for sequencing covering variable regions V3 to V4 (Primers: 347F-5′GGAGGCAGCAGTAAGGAAT-3′ and 803R- 5′CTACCGGGGTATCTAATCC-3′). For 16Sv3-4 rRNA gene amplification preparation, 5 ng of genomic DNA was used as the template in 25 μL of PCR reaction buffer. The PCR amplicons were then purified using the Agencourt AMPure XP kit (Beckman Coulter) and purified by fluorometry using the Quant-I T PicoGreen dsDNA Assay Kit (Invitrogen; ref. 15 (link)). A total of 10⁷ molecules/μL of purified amplicons were pooled for sequencing using Roche 454 GS FLX Titanium pyrosequencing system.