title: 16S-Seq of citrus leafminer: YG
design: The primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTATCTAAT) with adapter sequence and barcodes combined to the end of reverse primer were applied to amplify the V3-V4 regions of bacterial 16S rRNA gene from each extracted DNA12. A 20 L mixture (0.4 L of FastPfu Polymerase, 4 L of 5  FastPfu Buffer, 0.8 L of each primer (5 M), 2 L of dNTPs (2.5 mM), and 11.8 L ddH2O) was used to perform the PCR reaction with the template DNA (10 ng). Thermal cycling consisted of initial denaturation at 95C for 2 min, followed by 30 cycles of denaturation at 95C for 5 s, annealing at 55C for 30 s, and elongation at 72C for 30 s, and a final extension at 72C for 6 min. All PCR products extracted and  purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) with 2% agarose gels. Then, Qubit3.0 (Life Invitrogen) was applied to quantify the concentrations of each purified PCR product and all of them were equally mixed to generate the amplicon libraries using TruSeq Nano DNA LT Library Prep Kit (Illumina, USA). Library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent, USA) and QuantiFluor dsDNA system (Promega, USA). Finally, an Illumina Novaseq6000 platform was applied to sequence these libraries with a 250 bp paired-end strategy.