DNA-seq of fruiting body of P pulmonarious
resh, mature sporophores were collected from the mushroom beds for DNA extraction. The fruiting bodies of P. pulmonarius (MTCC 1805) were surface sterilized, and DNA was extracted using the Xploregen kit. A total of 40 ng of extracted DNA was utilized for amplification with 10 pM of each primer (F: 5-AGAGTTTGATGMTGGCTCAG-3 and R: 5-TTACCGCGGCMGCSGGCAC-3). The extracted DNA and the amplified  PCR products were purified and subjected to quality control checks using gel electrophoresis and Nanodrop.The purified amplicons were cleaned with Ampure beads to remove residual primers. Subsequently, sequencing libraries were prepared by adding Illumina barcoded adapters through an additional 8 cycles of PCR. These libraries were further purified using Ampure beads and quantitated with the Qubit dsDNA High Sensitivity assay kit. Sequencing was performed using the Illumina MiSeq platform with the 2x300PE V3-V4 sequencing kit.The 16S  rRNA V3-V4 region reference sequences were retrieved from the NCBI database. Base Call File (bcl) data from the sequencer was de-multiplexed into raw fastq files. Quality assessment of the data was carried out using FastQC (v0.11.9) and MultiQC (v1.10.1). Samples passing the quality control metrics were processed using the in-house Biokart Pipeline for metagenomic analysis, including 16S metagenomics. The pipeline output included a raw Operational Taxonomic Units (OTU) table, which served as the foundation for subsequent analyses. Abundance feature tables were constructed, and the top ten organisms in each sample were identified and visualized. This workflow ensured a comprehensive evaluation of the microbiome associated with the mushroom sporophore.