Amplicon sequencing of microorganisms from host Nezara virudula: Infected Egg
DNA extraction from individual tissues and organs was performed using the Genomic DNA protocol from Qiagen DNeasy PowerSoil kit according to manufacturers instructions. The 16S rRNA gene V4 variable region PCR primers 515F/806R (Caporaso et al. 2011, Caporaso et al. 2012, Apprill et al. 2015, Parada et al. 2016) were used for PCR.  The PCR was run using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 95C for 5 minutes, followed by 30-35 cycles of 95C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 10 minutes was performed.  After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Samples from multiple projects were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. The pooled and purified PCR product was used to prepare a multiplexed Illumina DNA library. Sequencing was performed on an Illumina Hiseq v 1.5 in 2x250 paired end mode following the manufacturers guidelines.  Deposited data has been demultiplexed, merges and adapter trimmed.