Gut Microbiome Analysis by 16S Sequencing

High-throughput sequencing of 16S DNA was carried out according to a previously described protocol [24 (link)]. Briefly, 30 adult female fly midguts were dissected into ice-cold PBS, and frozen samples of intestines were sent to the Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Total bacterial DNA extraction and sequencing were conducted according to standard protocols. DNA was amplified using the 515f/806r primer set (515f : 5′-GTG CCA GCM GCC GCG GTA A-3′, 806r: 5′-XXX XXX GGA CTA CHV GGG TWT CTA AT-3′), which targets the V4 region of the bacterial 16S rDNA. Pyrosequencing was conducted on an Illumina MiSeq 2 × 250 platform (San Diego, CA) according to published protocols. Sample reads were assembled using mothur v1.32. Chimeric sequences were removed using the USEARCH software based on the UCHIME algorithm. The microbial diversity was analyzed using the QIIME software with Python scripts. Operational Taxonomic Units (OTUs) were picked using the de novo OTU picking protocol, with a 97% similarity threshold.