DNA was extracted from various black soldier fly tissues and developmental stages (Fig. 1) using the NucleoSpin Soil Kit (Macherey-Nagel, Düren, DE) following the manufacturer’s protocol:
The extracted DNA was quantified and quality-checked via spectophotometry (NanoDropTM 1000c, Thermo Scientific, Waltham, MA, US) and agarose gel electrophoresis.
The quality-checked DNA extracts were sent to Microsynth GmBH (Balgach, Switzerland) for enrichment and amplicon sequencing. After diluting the samples, the enrichment PCR with locus-specific primers for the V3-V4 (f-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNCCTACGGGNGGCWGCAG / r-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNGACTACHVGGGTATCTAATCC) and ITS2 region (f-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNGCATCGATGAAGAACGCAGC / r-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNTCCTCCGCTTATTGATATGC), followed by a 1-step PCR with locus-specific primers and Illumina overhang and a cleanbead purification was carried out. The final libraries were pooled and subjected to a final cleanbed purification of the pool (red = locus-specific sequences).
The amplicon sequencing was carried out on a Illumina MiSeq, following a 2 × 250 approach. The universal bacterial primers 341f/802r (5′-CCTACGGGRSGCAGCAG-3′ / 5′-TACNVGGGTATCTAATCC-3′) and the universal fungal primers ITS3f/ITS4r (5′-GCATCGATGAAGAACGCAGC-3′ / 5′-TCCTCCGCTTATTGATATGC-3′) were used to target the V3-V4 and ITS2 genetic regions, respectively. Library preparation was performed by the sequencing provider based on a Nextera two-step PCR including purification, quantification, and equimolar pooling.
All sequence data were processed using DADA2 (Callahan et al., 2018). Data analysis and visualization was mainly done using the R packages ggplot2 (Wickham, 2016), vegan (Oksanen et al., 2022), phyloseq (McMurdie and Holmes, 2013), ampvis2 (Andersen et al., 2018). For a detailed and reproducible description please continue here.