pulp-mango-3
Total DNA was extracted from five samples of pulp (20 mg), larvae (one) or adults (two guts per sample) using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Amplicons of the variable V3 region of the 16S gene were amplified from 100 ng of total DNA samples using the primers described by Klindworth et al. (2013). PCRs (50 L) containing Qiagen buffer 1X, dNTPs 0.2 mM, 16s F&R 0.2 M, Taq polymerase 2.5 U were performed using the following amplification program: 94C/2 min of initial denaturation followed by 25 cycles of 94C/15 s denaturation, 55C/30 s annealing and 72C/1 min amplification, and a final amplification at 72C/5 min. Illumina libraries were constructed by adding Nextera XT adapters (Illumina Inc.). Initial amplification and adapter-ligation PCRs were performed using Kapa polymerase (Kappa Biosystems) and purified immediately after finishing each PCR using 0.8X Agencourt Ampure XP cleaning beads (Beckman Coulter). Library concentration was quantified using a DNA HS kit (Invitrogen) in a Qubit 2.0 fluorometer (Invitrogen). Library size was evaluated using a Bioanalyzer DNA HS chip (Agilent). Libraries were diluted and pooled to an equimolar concentration to be denatured and loaded into a MiSeq sequencer using a MiSeq 600-cycle Reagent kit V3 (Illumina Inc.)