A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) Internal Transcribed Spacer rDNA fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR utilizes flexible primer choices to construct library for Illumina has been applied several studies in forest agricultural systems. The protocol, while producing high-quality reads, often yields a large number (up 46%) of reads are unable be assigned specific sample according its barcode. Here, we improve this technique through an optimized two-step protocol. We tested compared improved meta-barcoding protocol against using four different pairs (fungal ITS: ITS1F-ITS2 ITS1F-ITS4, bacterial 16S: 515F-806R 341F-806R). demonstrate sequence quantity recovery rate were significantly with approach (fourfold more read counts per sample; determined ≈90% run) retaining high quality (Q30 > 80%). Given synthetic barcodes incorporated independently from any primers, can broadly adapted genomic regions organisms scientific interest.