Quick dna fecal and soil microbe kit

Obtained honeybee gut pools were well homogenized with a pestle, after which was added 1,400 μl of lysis solution containing 60 μl proteinase K per pool (20 mg/ml concentration) and glass beads. Total destruction of gut epithelial tissues was obtained after 1 h incubation at 55°C. Only 1/4 of the resulting sludge (450 μl) was used for gut genomic DNA extraction with Quick-DNA Fecal and Soil Microbe kit (Zymo Research, California, USA). The 16S rRNA gene amplification and libraries prepared for Illumina MiSeq platform sequencing were carried out according to Alberoni et al. (67 (link)). Briefly, the V3-V4 was amplified with KAPA Hi-Fi PCR Master Mix (Roche, Monza, Italy) with a maximum of 25 cycles. PCR products were purified with AMPure magnetic beads (Beckman Coulter, Milan, Italy) and indexed with i7 and i5 Illumina adapters (Illumina, Milan, Italy). NGS sequencing was performed with the addition of 22% PhiX to the sample pool (Illumina, Milan, Italy). Bioinformatic analyses were performed with Qiime1, and representative operational taxonomic units (OTUs) were subjected to BLAST search against the most updated SILVA database release 132. OTUs with less than 0.1% abundance were discarded. The α–diversity was evaluated using Chao1, observed OTU, and PD whole tree metrics, whereas β–diversity was evaluated using both weighted and unweighted UniFrac.

Obtained honeybee gut pools were well homogenized with a pestle, after which was added 1,400 μl of lysis solution containing 60 μl proteinase K per pool (20 mg/ml concentration) and glass beads. Total destruction of gut epithelial tissues was obtained after 1 h incubation at 55°C. Only 1/4 of the resulting sludge (450 μl) was used for gut genomic DNA extraction with Quick-DNA Fecal and Soil Microbe kit (Zymo Research, California, USA). The 16S rRNA gene amplification and libraries prepared for Illumina MiSeq platform sequencing were carried out according to Alberoni et al. (67 (link)). Briefly, the V3-V4 was amplified with KAPA Hi-Fi PCR Master Mix (Roche, Monza, Italy) with a maximum of 25 cycles. PCR products were purified with AMPure magnetic beads (Beckman Coulter, Milan, Italy) and indexed with i7 and i5 Illumina adapters (Illumina, Milan, Italy). NGS sequencing was performed with the addition of 22% PhiX to the sample pool (Illumina, Milan, Italy). Bioinformatic analyses were performed with Qiime1, and representative operational taxonomic units (OTUs) were subjected to BLAST search against the most updated SILVA database release 132. OTUs with less than 0.1% abundance were discarded. The α–diversity was evaluated using Chao1, observed OTU, and PD whole tree metrics, whereas β–diversity was evaluated using both weighted and unweighted UniFrac.

Obtained gut content pools were homogenised with a pestle, mixed with 1400 μL lysis buffer (Zymo Research, Tustin, California, USA) and 60 μL proteinase K (AppliChem GmbH, Darmstadt, Germany) at a final concentration of 20 mg/mL. Samples were further broken down with glass beads shaking at 50 Hz, and followed by 1-h incubation at 55 °C. 450 μL of the resulting sludge was used for gut genomic DNA extraction with Quick-DNA Fecal and Soil Microbe Kit (Zymo Research, California, USA). A total of 36 samples were subjected to NGS analysis on Illumina MiSeq platform (BioFab s.r.l, Rome, Italy). The amplification of V3-V4 region of 16S rRNA gene, libraries preparation for Illumina MiSeq platform and sequencing were performed according to Alberoni et al. [35 (link)] as well as bioinformatic analyses, which relied on the most updated SILVA database release 132. The database was implemented inserting full length 16S rRNA sequences of administered bacteria. OTUs with less than 0.1% abundance were discarded. alpha–diversity was evaluated using Chao1, Observed OTU and PD whole tree metrics, whereas beta–diversity was evaluated using both weighted and unweighted UniFrac.