Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform

TLDR

Sequencing technology has evolved from clone libraries of a few hundred 16S rRNA fragments to millions of reads per study with next‑generation platforms such as 454 and Illumina, reshaping microbial ecology research. The authors aim to evaluate the strengths, weaknesses, and overall suitability of Illumina MiSeq for interrogating microbial communities. They developed an improved MiSeq protocol that sequences variable regions of the 16S rRNA gene (V34, V4, V45) using paired 250‑nt reads, tested it on mock and natural samples, and demonstrated that many samples can be multiplexed alongside shotgun metagenomes. Titrating amplicon concentration and applying a quality‑score correction reduced error rates by up to two orders of magnitude, and the resulting data matched or exceeded 454 performance while delivering higher coverage at a fraction of the cost.

Abstract

Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.