A Review on the Applications of Next Generation Sequencing Technologies as Applied to Food-Related Microbiome Studies

Abstract

The development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods.

Targeting the 16S rRNA Gene for Bacterial Identification in Complex Mixed Samples: Comparative Evaluation of Second (Illumina) and Third (Oxford Nanopore Technologies) Generation Sequencing Technologies

“…Selecting regions spanning multiple 16S rRNA gene variable regions and combining information collected over different regions, substantially improved accuracy. In our analysis, the sequences spanning variable regions V4-V6 and V1-V3 yielded the best results, although other studies have recommended other regions [17]. Other confounding factors included amplification efficiency, dominant species present in other samples of the same sequencing run, potential contaminants of the Illumina sequencing workflow, and reference database employed.…”

Heterogeneity Spacers in 16S rDNA Primers Improve Analysis of Mouse Gut Microbiomes via Greater Nucleotide Diversity

“…Two other studies have utilized the concept of the heterogeneity spacers (one for V3–V4 primers with Illumina MiSeq for clinical samples and the other using Illumina HiSeq for mock microbial communities and environmental samples) along with multiple in-line indexing; both demonstrated an increased quality of sequences and total number of reads [14,15]. Mock microbial communities in 16S rDNA sequencing acts as potential internal controls to test primer effectiveness against a known bacterial composition [22], and in the study with HiSeq, heterogeneity spacers and triple indexing more accurately identified the expected bacterial composition of the mock community [15]. Our study shows that heterogeneity spacers alone can improve sequence quality in the conserved mouse microbiome compared to our controls, Run 1.…”

Diversity and Succession of Microbiota during Fermentation of the Traditional Indian Food Idli

“…Along with the isolation of cultivable organisms, the role and behavior of microorganisms in food fermentation have been elucidated using advanced techniques involving genomics, transcriptomics, proteomics, and metabolomics (13)(14)(15)(16)(17). The recently developed high-throughput DNA sequencing methods have been employed to find out the different microorganisms and their proportion and succession in complex food fermentations.…”