Gs junior sequencing system

Eggs, larvae, pupae and adults were sampled and washed twice with 100% ethanol. Triplicate samples were taken for each insect stage. At each stage, G. mellonella populations were collected and rinsed with 100% ethanol and sterile water. For eggs, DNA isolation was performed after surface sterilization with 100% ethanol, and for larvae and pupae, gut samples were collected for subsequent analysis. DNA isolation from each sample was performed using a genomic DNA isolation kit (Wizard, USA). PCR amplification was carried out using primers targeting the V1 to V3 regions of the 16S rRNA gene. Sequencing was performed at Theragen (Bio Institute, Suwon, South Korea) and processed by the GS Junior sequencing system (Roche, Branford, CT, USA) according to the manufacturer's instructions. The readings obtained from each sample were sorted by the unique barcode of each sequence product. All readings containing two or more ambiguous nucleotides, low-quality scores (average score of <25), or readings below 300 bp were discarded. Potential chimeric sequences were removed by the Bellerophone method and then taxonomically assigned via the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net). The richness and evenness of the samples were determined by ACE, Chao1, Jackknife, NPShannon, Shannon, and Simpson indices at 3% distance.

Five hundred nanograms of the cDNA was processed for sequencing using Roche’s GS FLX Titanium chemistry following Roche’s Rapid Library Preparation and emPCR Lib-L method manual. The library was sequenced on Roche’s GS Junior sequencing system as per manufacturer’s instructions. Using Roche’s GS Reference Mapper, the sequence data obtained was used to perform a reference-guided alignment using the default parameters with the exception of the minimal overlap identity modified from 90% to 40%.

Bacterial DNA was extracted from feces using QIAamp DNA stool mini kits (Qiagen). PCR amplification was performed using primers targeting the segment from the V3 to V4 regions of the 16S rRNA gene with extracted DNA. For bacterial amplifications, we used barcoded primers of 341F (5′-CCTACGGGNBGCASCAG-3′) and 805R (5′-GACTACNVGGGTATCTAATCC-3′). The amplifications were performed under the following conditions: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, with final elongation at 72°C for 5 minutes. The QIAquick PCR purification kit (Qiagen) was used to purify the purification of the amplified products. Equal concentrations of purified products were pooled, and short fragments (non-target products) were removed with an AMPure bead kit (Agencourt, Beverly, MA). The quality and product size were assessed on a Bioanalyzer 2100 (Agilent) using a DNA 7500 chip. Mixed amplicon sequencing was conducted by emulsion PCR and then deposited on picotiter plates. The sequencing was carried out at Chunlab (Seoul, South Korea) on a GS Junior Sequencing System (Roche, Basel, Switzerland) per the manufacturer’s instructions. Taxonomic cladogram was analyzed using LEfSe with a threshold 2 on the logarithmic LDA score.⁶¹ (link)