Challenge of validation in whole-cell spike-in amplicon sequencing to comprehensively quantify food lactic acid bacteriota.

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 one-third to three-fold. Our validation highlights that accurate preparation of the LAB mock community is crucial for cell spike-in amplicon sequencing accuracy.