Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products

TL;DR

This paper introduces a cost-effective, high-throughput microbiome profiling method using combinatorial sequence-tagged PCR and Illumina sequencing, enabling detailed analysis of microbial communities with minimal primers.

Contribution

The authors developed a novel combinatorial tagging approach for PCR primers that significantly reduces primer requirements while allowing deep sequencing of microbiomes.

Findings

PCR errors dominate non-organism variants in sequencing data.

Short reads can reliably classify organisms to genus or species level.

Method is applicable to various short, informative genetic regions.

Abstract

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads al- lowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an ob- servation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bac- terial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.