16srRNA of rhizosphere bacteria of American ginseng under high flag shade sheds
Bacterial amplicon library was obtained for Illumina sequencing using the primer combination 338F (5'-ACTCCTACGGGAGGCAGCA-3')[50] and 806R (5'-GGACTACHVGGGTWTCTAAT-3') [50], which target the V3-V4 region of the 16S rRNA gene.Each 25 l PCR reaction mixture contained 0.25l Q5 High-Fidelity DNA Polymerase (New England Biolabs, USA), 5 l 5Q5 Reaction Buffer, 0.5l 10mM dNTP, with each primer in the reaction mixture being supplied at a concentration of 0.5M. DNA was added depending on concentrations as inferred from gel electrophoresis and was modified accordingly with ddH2O to reach the final volume. Cycling conditions included initial denaturation at 98C for 1 min; followed by 30 cycles of denaturation at 98C, each cyclelasting 15s, annealing at 55C for 30 s, and extension at 72C for 30 s; a final extensionphase was performed at 72C for 5 min. The PCR products were separated on a 2%(w/v) agarose gel, purifed using the AxyPrepTM DNA Gel Extraction Kit (Axygen, USA), and quantifed using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA).The amplicon libraries were constructed using TruSeq Nano DNA LT Library Prep Kit (Illumina) according to the manufacturers protocol. The qualified libraries were sequenced on an Illumina MiSeq platform and 300 bp paired-end reads were generated.