An integrative protocol for one-step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data
An integrative protocol for one-step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data
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DOI：
10.1007/s42995-023-00182-1
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发表时间：
2023-08
影响因子：
5.7
通讯作者：
Jiahao Ni;Jiaoyi Pan;Yaohai Wang;Tianhao Chen;Xinshi Feng;Yichen Li-;Tongtong Lin;Michael Lynch;Hongan Long;Weiyi Li
中科院分区：
文献类型：
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作者：
Jiahao Ni;Jiaoyi Pan;Yaohai Wang;Tianhao Chen;Xinshi Feng;Yichen Li-;Tongtong Lin;Michael Lynch;Hongan Long;Weiyi Li
扩增子的高通量测序已广泛用于精确、高效地鉴定物种组成和分析群落结构，极大地促进了涉及大量复杂样品的生物学研究，特别是涉及环境和病原体监测的研究。用于扩增子测序的商业文库制备试剂盒通常需要多个步骤，包括适配器连接和索引，价格昂贵且耗时，特别是用于大规模应用。为了克服这些限制，先前提出了一种“一步PCR方法”，用于使用长融合引物构建扩增子文库。然而，目标基因的高效扩增和测序数据的精确解复用仍有待解决。为了解决这些问题，我们提出了一步PCR扩增文库构建（OSPALC）的综合方案。通过这种方法产生了高质量的读数，以可靠地识别模拟细菌群落和环境样品的物种组成。使用该方案，扩增子文库通过一个带长引物的常规PCR构建，并且每个DNA/cDNA样本的总成本通过多步PCR方法降低到仅为典型成本的7%。本文以16S rDNA V4区OSPALC文库的构建为例，对引物进行了实证检验，并优化了PCR条件。工具设计引物针对任何基因组区域也提出。原则上，OSPALC可以很容易地利用DNA或RNA样本构建任何目标基因的扩增子文库，并将促进许多领域的研究。
High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. Commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. To overcome these limitations, a “one-step PCR approach” has been previously proposed for constructions of amplicon libraries using long fusion primers. However, efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed. To tackle these, we present an integrative protocol for one-step PCR amplicon library construction (OSPALC). High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library is constructed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 7% of the typical cost via the multi-step PCR approach. Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Tools to design primers targeting at any genomic regions are also presented. In principle, OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples, and will facilitate research in numerous fields.