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Updated: May 5, 2026
Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
Published on: April 14, 2016
Challenge of validation in whole-cell spike-in amplicon sequencing to comprehensively quantify food lactic acid
Mugihito Oshiro1, Keisuke Nakamura1, Yukihiro Tashiro1
1Laboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Abstract:
Lactic acid bacteria (LAB) shape diverse communities in fermented foods. Developing comprehensive quantification methods for community structure will revolutionize our understanding of food LAB microbiome. For this purpose, 16S rRNA gene amplicon-based quantification, using spiked exogenous bacterial cells as an internal standard, shows potential for comprehensiveness and accuracy. We validated cell spike-in amplicon sequencing for quantifying LAB communities in food. Low efficiency of LAB DNA extraction underscores the importance of compensating for DNA loss by spiking internal standard cells. Quantitative equations generated using 15 selected LAB mock species showed positive relationships between the ratio of MiSeq read counts and the expected 16S rRNA gene copy numbers, with coefficients of determination (R2) ≥ 0.6823. The fold differences between observed and expected 16S copy numbers were within the range of 1/3 to 3-fold. Our validation highlights that accurate preparation of the LAB mock community is crucial for cell spike-in amplicon sequencing accuracy.
