title: Fly second-stage larvae metagenome
design: DNA from each frozen sample was extracted using the NucleoSpin tissue kit. DNA concentration and purity were measured using a NanoDrop Spectrophotometer. Polymerase chain reaction (PCR) amplification of the V3-V4 region of the 16S rRNA gene was performed using specific primers 341F (5'-CCT AYG GGR BGC ASC AG-3') and 806R (5'-GGA CTA CNN GGG TAT CTA AT-3'). The PCR products (400-450 bp) were generated. These PCR products with proper size were evaluated by 2% agarose gel electrophoresis and used for library preparation. The DNA fragments were end-repaired and A-tailed then further ligated with Illumina adapters. Library quality was evaluated using the Qubit 2.0 Fluorometer and the Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated.