**3.6. LEA-seq (low error amplicon sequencing)**

The LEA-seq technique (low error amplicon sequencing) emerged in 2013 and was developed and patented by Gordon and Faith (2014) [53]. This method was created to improve the quality and depth of sequencing runs, since the massive amount of data produced by NGS has caused a high error rate in the sequencing, due to problems with the algorithms or platform reading lengths [53].

LEA-seq is a nucleic acid sequencing technique that identifies events that occur at low frequency, seeking to understand mutation events. The three basic steps for implementing this technique are: (1) linear PCR, (2) exponential PCR and (3) sequencing. This technique is performed based on bacterial 16S sequencing in which PCR carries numerous times and each amplified PCR uses specific primers for each linear molecule [53].

The LEA-seq technique is a quantitative method that has the advantages of generating and reading. This permits the formation of a consensus and the elimination of errors for each molecule. Currently, the available techniques do not support error detection in sequencing or identification of whether there is a real variation in the sequence of that microorganism. The multiple sequencing, using the LEA-seq technique, supports better quality and precision about the organism.

The study by Faith et al. (2013) aimed to identify the composition of the faecal microbiota of adults and to understand the role of these bacterial species and their therapeutic potential for intestinal diseases. This technique allowed them to work with a large number of samples (over 500 isolates), as well as to achieve a fast and accurate analysis of the data [54].

Researchers have a continuing interest in improving this technique, since it can be used for clinical investigation due to its high accuracy: for example, in patients with genetic mutations or somatic mutations. LEA-seq can assist in the search for knowledge about intestinal micro‐ biota, as it may reveal their composition, opening up prospects for the diagnosis, treatment and prevention of gastrointestinal tract diseases.

#### **3.7. CRISPR (clustered regularly interspaced short palindromic repeats)**

Ishino et al. (1987) were the first to describe CRISPR [55]. This system has been identified in 40% of bacterial genomes so far [56] and they are defined as short repetitions of grouped bases. The determination of the CRISPR locus and the characterization of adjacent genes, known as *cas* genes, responsible for the function of CRISPR, only occurred in 2002 [57]. The CRISPR/Cas system uses small non-coding RNAs in association with Cas proteins. Cas9 is a nuclease which cleaves DNA in the selected region, so that the CRISPR system/Cas9 can be used to edit genomes.

CRISPR/Cas activity involves three main mechanisms: (1) acquisition, the step in which the DNA fragment is inserted into the CRISPR locus in the genome of interest; (2) transcription, in which the CRISPR locus is transcribed and processed; (3) interference, in which the ejection of nucleic acids occurs. All those mechanisms contribute to bacterial persistence in the environment [58, 59]. Furthermore, CRISPR provides mechanisms to limit the spread of antibiotic resistance or virulence factors. However, Gophna et al. (2015) demonstrated that, even though there are different measurements to evaluate horizontal gene transfer, it is not possible to identify a correlation between the CRISPR/Cas system and the evolution of the species. Changes occur only at the population level [60].

RNA-seq helped in the annotation transcription of regions, mainly non-coding, and also enabled the identification of CRISPR elements in prokaryotes [61]. The CRISPR system can also be used as a tool in studies centered on gene regulation, since this system is able to activate or repress genes.

Zoephel and Randau (2013) discuss how the structure of CRISPR can affect the maturation of RNA and, thus, influence the functionality of the CRISPR/Cas system [62]. The RNA-seq approach was used to evaluate differential gene expression in *S. aureus*, a pathogen of major importance. It was able to identify the CRISPR in these strains and helped in investigating their possible role, since these regions show an adaptive response to infection [63]. Thus, we see the importance of the use of the RNA-seq approach in the magnification of knowledge about function in prokaryotes.
