Practical innovations for high-throughput amplicon sequencing

Characterizing microbial communities by sequencing rRNA amplicons is a cornerstone of metagenomics. We present cost-saving and accuracy improvements for 16S rRNA amplicon sequencing on the Illumina MiSeq platform, many of which are generalizable to other amplicons and sequencing systems. First, we deploy a mix of frame-shifted primers as an alternative to the common, wasteful practice of re-sequencing 5 to 50% phiX174 genomic DNA to provide diversity at each sequencing cycle. Second, we produce amplicons by uniquely tagging all template molecules, and then amplifying the tagged templates in a PCR step. Consensus sequences built from sequences sharing an identical tag reconstruct the original template population, dramatically minimizing sequencing error and PCR bias. Third, the ability to mix-and-match various template-tagging primers with the same set of universal PCR-plus-barcode primers allows combinatorial barcoding and investigation of multiple amplicons. Last, because rRNA studies often involve characterization of microbial communities in association with their eukaryotic hosts, we deploy PCR clamps to block amplification of contaminating plant plastid and mitochondrial DNA sequences, yielding upwards of five-fold increase in microbial sequences without biasing the plant-associated microbial community. These techniques, in conjunction with the informatics pipeline we provide, improve both the efficiency and accuracy of rRNA amplicon sequencing, and may be deployed independently to suit user needs.