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

Elizabeth A Jensen; Darlene E Berryman; Erin R Murphy; Ronan K Carroll; Joshua Busken; Edward O List; William H Broach

doi:10.2144/btn-2019-0025

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

Biotechniques. 2019 Aug;67(2):55-62. doi: 10.2144/btn-2019-0025. Epub 2019 Jul 10.

Authors

Elizabeth A Jensen^{1

2

3}, Darlene E Berryman^{1

3

4}, Erin R Murphy^{1

4

5

6}, Ronan K Carroll^{1

7}, Joshua Busken^{3

8}, Edward O List^{1

3

9}, William H Broach¹⁰

Affiliations

¹ Department of Translational Biomedical Sciences, Graduate College, Ohio University, Athens, OH, USA.
² Ohio University Heritage College of Osteopathic Medicine, Athens, OH 45701, USA.
³ The Diabetes Institute, Konneker Research Center 108, Ohio University, Athens, OH 45701, USA.
⁴ Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH 45701, USA.
⁵ Molecular & Cellular Biology Program, Ohio University, Athens, OH 45701, USA.
⁶ Infectious & Tropical Disease Institute, Irvine 228, Ohio University, Athens, OH 45701, USA.
⁷ Department of Biological Sciences, College of Arts & Sciences, Ohio University, Athens, OH 45701, USA.
⁸ College of Health Sciences & Professions, Ohio University, Athens, OH 45701, USA.
⁹ Edison Biotechnology Institute, Athens, OH 45701, USA.
¹⁰ Ohio University Genomics Facility, Ohio University, Athens, OH 45701, USA.

PMID: 31290335
DOI: 10.2144/btn-2019-0025

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.

Keywords: 16S rDNA sequencing; V3–V4 region; microbiome; optimization; primer design.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
DNA Primers / genetics
Feces / microbiology*
Female
Gastrointestinal Microbiome*
Gene Library
Mice
Mice, Inbred C57BL
RNA, Ribosomal, 16S / genetics*
Sequence Analysis, DNA / methods*

Substances

DNA Primers
RNA, Ribosomal, 16S

Grants and funding

F32 GM069312/GM/NIGMS NIH HHS/United States