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evolution from hotspot panels, they still do not guarantee complete coverage of the target genes (no intronic
or regulatory sequences are included) and remain anchored to preexisting knowledge (only genes already
known to be involved in drugs’ action are included).

Disease-focused panels
Similarly to the actionable panels, disease-focused panels include sets of genes (exomes) known to be
involved in a specific disease. These are mainly used as a screening panel to evaluate the hereditary risk of
developing specific tumors, e.g., BRCA-1, -2-driven breast and ovarian cancers. This type of panel is more
useful for screening programs and in early/preventive management of cancer.

Comprehensive panels
The difficulty in recruiting adequately large sample groups of homogeneous tumors within realistic time-
frames rarely justifies the costs of production of different disease focused panels. This has led life science
companies to create larger, more comprehensive panels in which multiple genes that correlate with multiple
diseases are included. These panels can include thousands of genes (exons only) which are selected among
the ones that are most commonly tested. Like other targeted panels, comprehensive panels depend on pre-
existing knowledge on the correlation between genes and the investigated phenotype.

Validated panels
As of November 27th, 2018, ~1677 NGS-based clinical tests for cancer were available across the world (search
terms “NGS” AND “Cancer” in the NIH Genetic Testing Registry, website www.ncbi.nlm.nih.gov).

Whole exome sequencing
Despite the ever-increasing number of genes that are included in targeted panels, the risk of not including
elements that are possibly correlated with the investigated phenotype remains high. To overcome this
limitation increasing numbers of researchers resort to whole exome sequencing (WES). This approach allows
to identify both known and unknown alterations that occur within coding regions. Some groups estimate
[63]
that WES may cover ≈85% of disease-related variants . This estimate should be taken with great caution, as
approximately 98% of the genome remains uncovered. Such regions include regulatory elements, non-coding
mRNA, splice sites, anchor sites to the nuclear membrane, chromatin modifiers, and other regions we simply
don’t know enough about.

Whole genome sequencing
Whole genome sequencing (WGS) provides a global coverage of the human genome. This approach
represents the last and most comprehensive step in genomic analysis. The feasibility of this approach in
recent years was aided by the introduction of higher-processivity NGS technologies (higher speed, lower
costs). The main limitation of this approach is essentially related to the complexity of data post-analysis, and
related statistics, with considerable risk of both false positives and false negatives.

RNA sequencing
With a coverage comparable to that of WES, RNA sequencing profiles the transcriptome of a tumor.
Differently from WES, whose target is the subject’s DNA, RNA provides a dynamic/functional image of a
cancer. It has the advantage of detecting the presence of fusion (onco)genes, which are often found in cancer
cases [8,68,69] . It may aid clinicians in the choice of drug therapy and basic science for the identification of novel
potential drug targets.

Chromatin immunoprecipitation sequencing
This approach is normally used to understand DNA-protein binding interactions, e.g., regulatory protein-
binding sites within a DNA region. This also has the capability to detect epigenetic alterations [70,71] . The