The total number of microbial cells on earth is estimated to be about 1030 (Turnbaugh and Gordon, 2008), but 99% of all microorganisms in almost every environment on earth remain, as yet, uncultured (Amann et al., 1990; Curtis et al., 2002). Culture-independent analyses arose to overcome the well-known limitations of the classical culture-based approach (Ercolini, 2013): since most of the microorganisms are not able to grow in the common laboratory media, when we use the classical cultivation methods, we select only a part of the microbiota (those microbes which are able to grow in the medium used), and we can get only a partial picture of the microbial diversity of that environment. For this reason, in the past 20 years, we moved to the culture-independent approach, which allows analyzing the microbiota by using the nucleic acids directly extracted from the matrix, without applying any selection. Culture-independent techniques completely revolutionized our way of studying microbial ecology and we started to consider microbial populations as consortia of microbes, leading to a real cultural evolution (Cocolin and Ercolini, 2015). The advent of next-generation sequencing technologies in 2004 provided unprecedented sampling depth compared to traditional culture-independent approaches, such as denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism analysis, or Sanger sequencing of 16S rRNA gene clone libraries. The possible applications of high-throughput sequencing (HTS) in food microbial ecology are summarized in Figure 1. HTS studies in microbial ecology can be grouped into two fields: target-gene surveys (so-called amplicon metagenomics), based on the sequencing of libraries of amplicons of a gene of interest, and shotgun metagenomics, where libraries of randomly isolated DNA fragments are sequenced (Figure 1). In the first case, a polymerase chain reaction (PCR) step is performed after total DNA extraction (RNA has to be retrotranscribed to complementary DNA), in order to select the gene to be sequenced. In the shotgun approach, no PCR selection is performed and total DNA or cDNA are fragmented and directly sequenced. Moreover, HTS is uniquely quantitative: the number of reads obtained for each operational taxonomic unit (OTU) is proportional to the abundance of that OTU in the sample sequenced, therefore we can estimate the occurrence (%) of different OTUs (Ercolini, 2013).