SRX2036445 16s rRNA SRP080236 PRJNA335705 The recently proposed guidelines for standardizing the description of next-generation sequencing datasets in publications as described by Nilsson et al. (2008) were followed. Genomic DNA was extracted from 8 g of soil subsample after mixing the various samples of each site using the Mo Bio Power soil kit following the manufacturer’s instructions. The default primer sequences to be used for 16S amplicon library preparation 27F (GAGTTTGATCMTGGCTCAG) and 518R (WTTACCGCGGCTGCTGG), and the final Fusion Primer set was In case of Lib-L Forward primer (Primer A-Key): 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG?{MID}? GAGTTTGATCMTGGCTCAG ?3’ and Reverse primer (Primer B-Key): 5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG?{MID}- WTTACCGCGGCTGCTGG ?3’. A robust set of ten decamer Multiplex Identifier (MID) sequences was designed to facilitate library multiplexing in the 454 sequencing system. A length of ten bases is sufficient to ascertain that, for the large number of reads involved and the design parameters considered, the chances of mis-assigning reads is extremely low. Amplicon libraries may be generated using this primer design strategy. The PCR Master Mix was prepared (Forward Primer (10 µM) 1 µl, Reverse Primer (10 µM) 1 µl, dNTP mix (10 mM each) 0.5 µl, FastStart 10× Buffer #2 2.5 µl, FastStart HiFi Polymerase (5 U/µl) 0.25 µl and Molecular Biology Grade Water 18.75 µl to give total 24 µl. Genomic DNA was diluted to 5 – 20 ng/µl and to each 24 µl of PCR Master Mix, add 1 µl of a diluted DNA sample. The PCR program was run as follow; 94°C, 3 min, then 25 to 35 cycle: 94°C, 15 sec 55 – 65°C, 45 sec 72°C, 1 min, then 72°C, 8 min and hold on 4°C. Pyrosequencing of the PCR Product was performed by Macrogen (Korea) using a 454 GS FLX Titanium system (Roche, Germany). SRS1627270 SAMN05534236 16s rRNA AMPLICON METAGENOMIC PCR 454 GS FLX Titanium