honey bee hive microbiome: ITS-sample-19
Sample Collection  and  Preparation:Biological material was collected from honey bee colony air using BeeSafe samplers. Each sampler utilized a UV-sterilized filter surface cut from a Filtrete 1900 Smart Air Filter. DNA Extraction: Total DNA was extracted inside a Class II A2 Biological Safety Cabinet (BSC) using the QIAGEN DNeasy Blood and Tissue kit (Qiagen N.V., Germany), following the manufacturers protocol. Amplification and Library Preparation: Two target regions were amplified via PCR: Internal Transcribed Spacer 2 (ITS2) region using S2F/ITS4 primers. 16S rRNA V4 region using 515F/806R primers. Sequencing  and Primary Processing: Sequencing was performed on the Oxford Nanopore Technologies (ONT) platform. Basecalling: Performed with Dorado v0.8.2 using the dna_r10.4.1_e8.2_400bps_sup@v5.0.0 model. Data Conversion: Raw signals were converted to FASTQ format using SAMtools v1.20. Preprocessing: Reads were demultiplexed by Unique Molecular Identifiers (UMIs), trimmed of primers, and size-filtered using Cutadapt v4.9 based on expected amplicon lengths. Bioinformatics and Taxonomic Assignment. Downstream analysis was conducted in QIIME2 v2023.9. The pipeline included: Quality filtering and dereplication of reads. De novo clustering using VSEARCH. Chimera detection and removal with uchime-denovo. Taxonomic assignment using the QIIME2 feature-classifier plugin