A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes
A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes
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DOI：
10.3389/fmicb.2015.00731
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发表时间：
2015-07-16
影响因子：
5.2
通讯作者：
Loy, Alexander
中科院分区：
文献类型：
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作者：
Herbold, Craig W.;Pelikan, Claus;Loy, Alexander
系统发育和功能基因扩增子的高通量测序为复杂微生物群落的结构和功能潜力提供了巨大的洞察力。在这里，我们介绍了一种高度适应性和经济的PCR方法，以条形码和池库的许多目标基因。在这种方法中，我们用通用的、可互换的条形码引物取代了基因和测序平台特异性融合引物，从而实现了几乎无限的定制条形码引物组合。与使用长融合引物的条形码化相比，我们的多靶基因方法更经济，因为它总体上需要较少数量的引物，并且基于合成和纯化成本通常较低的短引物。为了突出我们的方法，我们将从各种环境或宿主相关微生物群落样品中获得的900多个不同的小亚基rRNA和功能基因扩增子文库汇集到一个单一的配对末端Illumine MiSeq运行中。尽管扩增子区域的大小范围为约290至720 bp，但我们未发现与扩增子长度或基因靶相关的显著系统性测序偏倚。我们的研究结果表明，这种灵活的多路复用方法产生了大量的，多样化的，高质量的扩增子序列数据集，为现代微生物生态学研究。
High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumine MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.