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Updated: Jan 22, 2026

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers
Published on: October 5, 2017
Heterogeneity spacers in 16S rDNA primers improve analysis of mouse gut microbiomes via greater nucleotide diversity
Elizabeth A Jensen1,2,3, Darlene E Berryman1,3,4, Erin R Murphy1,4,5,6
1Department of Translational Biomedical Sciences, Graduate College, Ohio University, Athens, OH, USA.
Abstract:
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.
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