Comparison of iSeq and Miseq in 16S rRNA sequencing-based human gut microbiome analysis

Illuminas MiSeq platform is a common approach in 16S-based microbiome analysis. Such usage is self-perpetuating in that many studies seek to employ widely used approaches to facilitate comparison of their results to existing literature. Yet, a range of factors, including cost and equipment availability can necessitate alternate approaches. For example, use of a Nano kit, lowers reagent costs by over 60% while others may only have access to entry-level sequencers such as Illuminas iSeq. The extent to which these approaches would impact results and subsequently conclusions is unknown. Attempting to address this question from the literature is complicated in that various studies not only use distinct cohorts but also differ in the reagents/methodologies to used to isolate DNA and generate sequencing libraries. Hence, we sequenced a single 16S rRNA gene amplicon library derived from 60 fecal samples, collected during a dietary supplement intervention study via MiSeq, MiseqNano, and iSeq. We evaluated platform performance by several key measurements: alpha diversity, beta diversity, taxonomic composition, and differential taxonomic abundance analysis. We found that iSeq outperformed MiSeq-Nano in alpha diversity and differential abundance detection, while MiSeq-Nano provided better taxonomic resolution than iSeq. Most importantly, all platforms showed similar core biological patterns in alpha and beta diversity and overall taxonomic composition. Thus, MiSeq, MiseqNano, and iSeq are likely to yield the same biological conclusions although specific questions and logistical consideration may favor one of these approaches.