**4. Data analysis**

Data analysis is a critical step of NGS tests. This analysis consist of a primary analysis, in which the base pairs are called and quality score are generated; a secondary analysis, numerous reads are aligned to the human reference sequence; and a tertiary analysis which consists of variant calling and annotation [117]. Many databases are useful for helping the variant annotation, such as the 1000 Genome Project [118], dbSNP database [119], Clinvar—NCBI [120], LOVD—Leiden Open Variation Database [121], The Cancer Genome Atlas (TCGA) [122] and others. However, information from these sources can contain ambiguous and insufficient information. Variants detected should be reported according to Human Genome Variation Society (HGVS) recommendations, with information of the human reference genome version and transcript information used to variant description [117]. The reference coding sequence should be preferably from the RefSeq database [123].

especially in de novo and highly repetitive regions sequencing; (D) data processing steps or bioinformatics is one major bottleneck for the implementation of NGS; (E) routine analysis of NGS data requires multidisciplinary teams; (F) it is critical to standardize the quality metrics for the NGS data generated. These include validation and comparison among platforms, data reliability, robustness and reproducibility, and quality of assemblers; (G) it is crucial to have a complete knowledge of family and personal history of the patient to help define the ideal analysis method, the analysis of the results obtained, and the post-test counselling and

Application of Next-Generation Sequencing in the Era of Precision Medicine

http://dx.doi.org/10.5772/intechopen.69337

305

Despite some challenges, it is hard not to be optimistic about the future of personalized genome sequencing and its potential impact on patient care and the advancement of knowl-

With the advancement of gene-sequencing technologies, numerous opportunities have arisen in the genetic diagnostic, preventive medicine and other areas of human health. As a result, several life science companies and clinical laboratories started their activities in this field offering equipment and supplies as well as molecular tests using the new-generation (parallel massive) sequencing methodology. However, most manufacturers do not market IVD products (in vitro diagnostic), but, in general, these products are classified as RUO (research use only). In practice, this difference in the classification of products and reagents represents serious implications on health. Products classified as IVD are regulated and therefore follow technical standards in their production and use, and consequently the efficiency must be guaranteed by the manufacturer. The ISO 13485 [128] is often used to ensure the quality of medical products, but other regulatory agencies such as the US Food and Drug Administration (FDA) may require other tests to prove this product is safe and effective, which is necessary for the product be classified as IVD and be commercialized on the American market. The same applies to the CE-IVD Marking in the European Economic Area (EEA). These requirements are part of an effort to ensure that users of these services and devices do not seek unnecessary treatment, delay their treatment or are exposed to inappropriate therapies. In the case of RUO products, none of these situations can be guaranteed, so the manufacturer will only be obliged to replace the product or its cost if it is performing improperly. In fact, some manufacturers may use standards of good manufacturing practice in the production of RUO equipment and supplies, but rarely perform tests to prove their efficiency in a particular case

In some cases due to the need to respond quickly to the market, especially in areas where the technological advance exceeds the regulatory capacity, some agencies allow the use of tests developed by clinical laboratories. The regulation in these cases is very simpler and favours the development of new technologies as the case of new-generation sequencing (NGS). However, these tests should also be used with caution, and the laboratories must prove its accuracy, or otherwise there may be the same hazards of products classified as RUO. In 2013, the US FDA agency required to genetic testing company 23andME to suspend the marketing of its products until it receives clearance from the agency. In a letter addressed to one of its

management [124–127].

edge of human biology and disease.

**5.1. Regulation on NGS tests**

of diagnostic.

All pathogenic, likely pathogenic and VUS variants have to be reported. Secondary or incidental finding (IF) is one significant matter, especially for WES, WGS and multi-gene panels, and its report will depend on local practice [38].

An in-house database containing all relevant variants identified in the laboratory provides an important tool in order to allow for further annotations, which greatly streamline the diagnostic process. Furthermore, an in-house database, linking patients and variants can help when a variant is re-classified. In this case, the laboratory is responsible for re-contacting the clinicians of the patients that are possibly affected by the new status of the variant [38].

### **4.1. Sanger sequencing validation**

Concerning the limitations of technology, the false positive rate for NGS, a second method, as Sanger sequencing, is required to confirm any findings with possible clinical significance. The laboratory must be able to guarantee that report variants are true variants; therefore, it is essential to mention that the variant reports were confirmed by Sanger method. An NGS technology will likely evolve, and within a few years confirmation might prove to be unnecessary [34, 39].

In some cases, mainly in large panels, complementing NGS testing with Sanger sequencing is inevitable. This limitation of NGS is dependent on the platform and on the enrichment methods, once that there are a number of strategies available with advantages and disadvantages. Sanger sequencing can also be used to fill regions that fail to amplify for having sequence complexities, such as sequence homology with pseudo genes, highly repetitive regions, GC-rich content, allelic dropout, or regions that are supported by an insufficient number of reads to call variants confidently [34]. However, in practice, the laboratories can opt to apply different settings for NGS tests. Three kinds of tests of multi-genes panel are identified: (A) the lab informs that more than 99% of interest region are covered, and all the gaps are filled with Sanger sequencing; (B) the lab describes which regions are sequenced and fills some specific gaps (core genes) with Sanger sequencing; and (C) no additional Sanger sequencing is offered [38]. It is essential to mention the horizontal coverage acquired in the test and the limitations of these tests in a disclaimer [39].
