Under the concept of massive sequencing or NGS (next generation sequencing), there are multiple highly complex diagnostic techniques that have significantly increased the diagnostic possibilities in pediatric neurology. We can classify them based on several criteria.

Historical criterion (generation).
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1.
Gupta N, Verma VK. Next-Generation Sequencing and Its Application: Empowering in Public Health Beyond Reality. In: Arora PK, editor. Microbial Technology for the Welfare of Society [Internet]. Singapore: Springer; 2019 [cited 2021 May 2]. p. 313–41. (Microorganisms for Sustainability). Available from: https://doi.org/10.1007/978-981-13-8844-6_15
  • Second generation sequencing methods are based on the principle of «shotgun sequencing«, and require fragmentation of the DNA under study, but unfortunately the length of the reads is short (short-read sequencing), so certain types of mutations are not identifiable (STR, deletions or duplications, etc.).
  • Third- and fourth-generation methods have been developed to overcome this limitation, which is why they are also called long-read sequencing.
Capture type.

The type of capture used is of fundamental clinical importance, since it will condition, almost more than the type of technology used, the clinical decisions that we must make in case we do not obtain definitive diagnostic results.

Gene panel.
  • It involves the analysis of a maximum of approximately 200 genes.
  • It allows the study of diseases with genotypic variability, but requires the generation of a prior hypothesis (phenotype-first approach).
  • It significantly limits the number of findings unrelated to the problem under study, as well as variants of uncertain significance.
Clinical/targeted exome sequencing/mendeliome study
  • It consists of the analysis of all coding regions (coding regions account for 1% of the genome and accumulate 80% of known pathogenic variants) for which disease-related genes have been previously described (those genes with unknown function do not provide clinically relevant information even if we discover pathogenic variants).
  • It allows the study without raising a prior hypothesis (genotype-first approach), and involves the analysis of more than 4000 genes (depending on the evolution of scientific knowledge, currently in OMIM they already exceed 6000), which is equivalent to a size of about 10 Mb.
  • It allows the detection of flanking non-coding variants (variants in 3′ or 5′ UTR).
  • It does not allow the research use of genes with unknown function, as they are not included, nor will it allow the reanalysis of those genes since their sequence has not been obtained.
  • Reanalysis of data after several years may be of interest, since the number of genes studied means that pathogenic variants that will be discovered in the future may not have been described yet.
  • It offers a good balance between the risk of variants of uncertain significance and diagnostic yield without skyrocketing the financial and labor costs of a whole exome or genome analysis.
Exome sequencing study.
  • It allows the detection of variants in protein-coding regions, both in genes already related to pathology and in those in which it has not been described, thus involving the analysis of more than 20,000 genes, which is equivalent to a size of about 100 Mb.
  • It allows the detection of flanking non-coding variants (variants in 3′ or 5′ UTR).
  • It allows the formulation of research hypotheses and the discovery of new diseases and unknown functions of genes that are not yet well described.
  • It allows the reanalysis of data in search of new knowledge several years after the study was carried out without the need to obtain new samples.
  • It significantly increases the financial and labor cost.
  • It significantly increases the probability of findings of uncertain significance and unexpected findings, so it is recommended to carry out the trio study to cross family information in an automated way.
Genome sequencing study.
  • It allows the detection of all variants in protein-coding regions.
  • It allows the detection of all non-coding variants (deep intronic, variants in 3′ or 5′ UTR).
  • It allows the detection of variants in regulatory regions (enhancers, silencers, insulators).
  • It allows the detection of small deletions or duplications (single exon).
  • It allows the detection of structural variants (translocations, insertions, deletions, and their breakpoints).
  • It is the most expensive process in financial and labor terms.
  • It significantly increases the probability of findings of uncertain significance and unexpected findings, so it is recommended to carry out the trio study to cross family information in an automated way.
  • It corresponds approximately to a size of 3 Gb.

You can consult the different commercialized capture types to know their scope.

Type of sequencing technology
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1.
Gupta N, Verma VK. Next-Generation Sequencing and Its Application: Empowering in Public Health Beyond Reality. In: Arora PK, editor. Microbial Technology for the Welfare of Society [Internet]. Singapore: Springer; 2019 [cited 2021 May 2]. p. 313–41. (Microorganisms for Sustainability). Available from: https://doi.org/10.1007/978-981-13-8844-6_15
  • It is interesting to note that methods that use amplification can induce a bias in the analysis of GC-rich sequences, due to the difficulties PCR encounters in these regions.

https://link.springer.com/chapter/10.1007/978-981-13-8844-6_15

https://info.abmgood.com/next-generation-sequencing-ngs