Open AccessHeterogeneity spacers in 16S rDNA primers improve analysis of mouse gut microbiomes via greater nucleotide diversity
Author(s) -
Elizabeth A. Jensen,
Darlene E. Berryman,
Erin R. Murphy,
Rachel Carroll,
Joshua Busken,
Edward O. List,
William H. Broach
Publication year - 2019
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2019-0025
Subject(s) - biology , amplicon , genetics , 16s ribosomal rna , primer (cosmetics) , illumina dye sequencing , amplicon sequencing , ribosomal dna , dna sequencing , microbiome , computational biology , polymerase chain reaction , dna , gene , phylogenetics , chemistry , organic chemistry
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.