Better primer design for metagenomics applications by increasing taxonomic distinguishability

Abstract

Current methods of understanding microbiome composition and structure rely on accurately estimating the number of distinct species and their relative abundance. Most of these methods require an efficient PCR whose forward and reverse primers bind well to the same, large number of identifiable species, and produce amplicons that are unique. It is therefore not surprising that currently used universal primers designed many years ago are not as efficient and fail to bind to recently cataloged species. We propose an automated general method of designing PCR primer pairs that abide by primer design rules and uses current sequence database as input. Since the method is automated, primers can be designed for targeted microbial species or updated as species are added or deleted from the database. In silico experiments and laboratory experiments confirm the efficacy of the newly designed primers for metagenomics applications.

Figure 1

Comparing HMP and MJ primers: PCR amplification for one sample with 3 known HMP primer pairs and 8 newly designed primer pairs.

Figure 2

Comparing HMP and MJ primers: BioAnalyzer read length distribution for 8 different samples. Top row is for MJV68 and bottom one is for HMPV35.