Heterogeneity spacers in 16S rDNA primers improve analysis of mouse gut microbiomes via greater nucleotide diversity.

Illumina-based amplicon sequencing suffers from the deleterious effects of highly homogenous nucleotide composition, limiting the number of high-quality reads generated per run. We attempted to alleviate this limitation by comparing the results obtained from 16S ribosomal DNA (16S rDNA) sequencing of mouse gut microbiomes using Illumina V3-V4 primers (Run 1) and custom primers that incorporate a heterogeneity spacer (0-7 nucleotides) upstream of the 16S priming region (Run 2). Overall, Run 2 had higher quality sequences, a more diverse microbial profile, and higher precision within, and variation between, experimental groups than Run 1. Our primer design offers a simple way to increase the quality of 16S rDNA sequencing and increases the number of useable reads generated per Illumina run.