Table_1_An Economical and Flexible Dual Barcoding, Two-Step PCR Approach for Highly Multiplexed Amplicon Sequencing.XLSX

In microbiome research, phylogenetic and functional marker gene amplicon sequencing is the most commonly-used community profiling approach. Consequently, a plethora of protocols for the preparation and multiplexing of samples for amplicon sequencing have been developed. Here, we present two economical high-throughput gene amplification and sequencing workflows that are implemented as standard operating procedures at the Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna. These workflows are based on a previously-published two-step PCR approach, but have been updated to either increase the accuracy of results, or alternatively to achieve orders of magnitude higher numbers of samples to be multiplexed in a single sequencing run. The high-accuracy workflow relies on unique dual sample barcoding. It allows the same level of sample multiplexing as the previously-published two-step PCR approach, but effectively eliminates residual read missasignments between samples (crosstalk) which are inherent to single barcoding approaches. The high-multiplexing workflow is based on combinatorial dual sample barcoding, which theoretically allows for multiplexing up to 299,756 amplicon libraries of the same target gene in a single massively-parallelized amplicon sequencing run. Both workflows presented here are highly economical, easy to implement, and can, without significant modifications or cost, be applied to any target gene of interest.在微生物组研究中，系统发育与功能标记基因扩增子测序（amplicon sequencing）是最常用的群落谱分析手段。因此，针对扩增子测序的样本制备与多重复合方案已层出不穷。 本研究介绍了两种经济高效的高通量基因扩增与测序流程，目前已作为标准操作规程（standard operating procedures，SOP）在维也纳医科大学与维也纳大学联合微生物组实验室（Joint Microbiome Facility）落地执行。这两种流程均基于已发表的两步聚合酶链式反应（PCR）方案，但经过升级优化，前者可提升分析结果的准确性，后者则可将单次测序运行中可复合的样本数量提升数个数量级。 高精度流程采用独特双样本条形码标记技术，其可实现与已发表的两步PCR方案同等水平的样本复合能力，但可有效消除单条形码标记方案固有的样本间残余读段错配分配问题（即串扰）。高复合能力流程则采用组合式双样本条形码标记技术，理论上可在单次大规模并行扩增子测序运行中，对多达299756个同一靶基因的扩增子文库进行复合测序。 本研究介绍的两种流程均具备极高的经济性与易实施性，且无需进行大幅修改或额外成本投入，即可应用于任意感兴趣的靶基因研究。