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

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.