16S/18S/ITS扩增子高通量测序引物的生物信息学评估和改进

In-silico evaluation and improvement on 16S/18S/ITS primers for amplicon high-throughput sequencing

摘要:

【背景】在过去的十几年里，基于核糖体RNA基因的扩增子测序技术被广泛用于各种生态系统中微生物群落的多样性检测。扩增子测序的使用极大地促进了土壤、水体、空气等环境中微生物生态的相关研究。【目的】随着高通量测序技术的不断发展和参考数据库的不断更新，针对不同的环境样本的引物选择和改进仍然需要更深入的校验。【方法】本文收集了目前在微生物群落研究中被广泛采用的标记基因扩增通用引物，包括16S rRNA基因扩增常用的8对通用引物和2对古菌引物、9对真菌转录间隔区(internal transcribed spacer，ITS)基因扩增引物，以及18S rRNA基因扩增的4对真核微生物通用引物和1对真菌特异性引物。这些引物中包括了地球微生物组计划(Earth Microbiome Project，EMP)推荐的2对16S rRNA基因扩增引物、1对ITS1基因扩增引物和1对18S rRNA基因扩增引物。采用最近更新的标准数据库对这些引物进行了覆盖度和特异性评价。【结果】 EMP推荐的引物依然具有较高的覆盖度，而其他引物在覆盖度及对特定环境或类群的特异性上也各有特点。此外，最近有研究对这些通用引物进行了一些改进，而我们也发现，一个碱基的变化都可能会导致评价结果或扩增产物发生明显变化，简并碱基的引入既可以覆盖更多的物种，但同时也会在一定程度上降低关注物种的特异性。【结论】研究结果为微生态研究中标记基因的引物选择提供了一个广泛的指导，但在关注具体科学问题时，引物的选择仍需数据指导与实验尝试。

关键词:扩增子测序，16S rRNA基因，ITS基因，18S rRNA基因，PCR扩增，引物，引物覆盖度，引物特异性，扩增产物片段长度

Abstract:

[Background] In the past decade, high-throughput sequencing technology on ribosomal RNA amplicons has been widely used to explore the diversity and composition of microbial communities in a variety of ecosystems. [Objective] With the development of sequencing technology and the expanding of reference databases, commonly adopted primers still need to be cautiously verified for different environmental samples. [Methods] We collected several marker gene amplification primers for microbial communities (i.e. bacteria, archaea, fungi and other eukaryotic microorganisms), including 8 universal primer sets and 2 archaea primer sets of 16S rRNA amplification, 9 universal primer sets for internal transcribed spacer (ITS) amplification, 4 universal primer sets and 1 fungal universal primer set for 18S rRNA amplification, which contained two 16S primer sets, one ITS1 primer set and one 18S primer set recommended by the Earth Microbiome Project (EMP). These primers were evaluated for the coverage and specificity using a recently updated standard reference databases. [Results] The EMP recommended primers all still have high coverage in current databases, while other primers have their own advantages in coverage and specificity to specific environments or taxonomic groups. In addition, recent studies have made some improvements on these universal primers. Changes in one single base may lead to changes in evaluation results or amplification products. The single degenerate base added to primers may cover more species, but it may also reduce the species specificity to some extent. [Conclusion] For different environmental samples, the selection of primers and experimental process still need more detailed verification. Our results could be a guide for the selection and improvement of amplified primers in microbial ecological studies.

Key words:Amplicon sequencing, 16S rRNA gene, Internal transcribed spacer (ITS) gene, 18S rRNA gene, PCR amplification, Primer, Primer coverage, Primer specificity, Amplification product fragment length