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

中文翻译：

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一种两步 PCR 方案，可为 Illumina MiSeq Meta-Barcoding 提供灵活的引物选择和高测序产量

主要针对 16S 核糖体 DNA (rDNA)（用于细菌和古细菌）和内部转录间隔区 rDNA（用于真菌）的高通量扩增子测序促进了不同环境中微生物群落的发现。利用灵活的引物选择来构建 Illumina 扩增子测序文库的三步 PCR 已应用于森林和农业系统的多项研究。三步 PCR 协​​议在产生高质量读数的同时，通常会产生大量（高达 46%）的读数，这些读数无法根据条形码分配给特定样本。在这里，我们通过优化的两步 PCR 协​​议改进了这项技术。我们使用四种不同的引物对（真菌 ITS：ITS1F-ITS2 和 ITS1F-ITS4，以及细菌 16S：515F-806R 和 341F-806R）。我们证明，使用两步 PCR 方法（每个样本的读取计数增加四倍；每次运行确​​定的读取≈90%）显着提高了序列数量和回收率，同时保持了高读取质量（Q30 > 80%）。鉴于合成条码独立于任何特定引物纳入，这种两步 PCR 协​​议可以广泛适用于不同的基因组区域和科学感兴趣的生物体。