Metagenomic analysis of seed endophytic bacterial and fungal communities in wheat using 16S rRNA and ITS sequencing

This study presents the metagenomic analysis of the seed endophytic bacterial and fungal communities of wheat using 16S rRNA and ITS metagenomic sequencing. DNA was extracted from surface-sterilized wheat seeds using the Fast DNA SPIN Kit for Soil (MP Biomedicals, USA) and a FastPrep-24 homogenizer. Libraries targeting the V3-V4 hypervariable regions of the 16S rRNA gene were prepared according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol using region-specific primers compatible with Illumina adapters and indexes. Libraries targeting the fungal ITS region were generated using a PCR-based amplicon workflow with ITS-specific primers compatible with Illumina-type adapters and indexes. Amplicons were purified with AMPure XT magnetic beads, indexed in a second PCR with Nextera-style index primers, quantified with a Qubit dsDNA HS Assay Kit on a Qubit 2.0 Fluorometer, and sequenced on a FASTASeq 300 instrument (GeneMind, China) with 2x300 bp paired-end reads.