Exploring the diversity of extremely halophilic archaea in food-grade salts
A nested PCR was performed with two archaeal specific primer pairs to prepare adapter-ligated DNA fragments. The first amplification used primers 340F and 1000R (Gantner et al., 2011). As the amplicon concentration was low in the PCR product, a concentration step was subsequently performed. For each DNA extract, 3 x 100 µl of PCR products were adsorbed on silica-membrane spin column, and then eluted with 40 µl Tris-EDTA buffer. An archaeal 16S rRNA gene tag-encoded FLX amplicon pyrosequencing was subsequently performed. Samples were amplified using forward and reverse fusion primers. The forward primer was constructed with the Roche A linker, an 8-10 bp barcode and the 340F S-D-Arch-0340-a-S-18 primer. The reverse fusion primer was constructed with a biotin molecule, the Roche B linker and the 915R S-D-Arch-0915-a-A-20 primer (Stahl and Amann, 2011). Amplification products were visualized with eGels (Life Technologies, Grand Island, New-York, USA). Products were then pooled equimolar and each pool was cleaned with Diffinity RapidTip (Diffinity Genomics, West Henrietta, New-York, USA) and size selected using Agencourt AMPure XP (BeckmanCoulter, Indianapolis, Indiana, USA) following Roche 454 protocols (454 Life Sciences, Branford, CT USA). Size selected pools were then quantified and 150 ng of DNA were hybridized to Dynabeads M-270 (Life Technologies) to create single-stranded DNA (ssDNA) following Roche 454 protocols (454 Life Sciences). ssDNA was diluted and used in emPCR reactions, which were performed and subsequently enriched. Sequencing was performed following established manufacture protocols (454 Life Sciences).