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in detectably altered current flow through the pore. During the library preparation step, fragmented DNA
is repaired using a PreCR step. Two adaptors are then added to the DNA, a Y adapter and a hairpin adaptor.
A motor protein unzips the double stranded DNA at the Y adapter and feeds the DNA as a single strand
through the nanopore (www.nanoporetech.com) [77,78] .

The BGI BGISEQ-500 sequencer uses a Probe-Anchor Synthesis (cPAS) and a DNA Nanoballs (DNB)
technology. The cPAS chemistry works by incorporating a fluorescent probe into a DNA anchor on the DNB,
followed by high-resolution digital imaging. This combination of linear amplification and DNB technology
reduces the error rate while enhancing the signal. In addition, the size of the DNB is controlled in such a
way that only one DNB is bound per active site (www.bgi.com).

VALIDATION OF NGS FINDINGS
The rapid evolution of NGS technology keeps enhancing throughput and reducing run time and costs.
Thus, NGS appears ready to offer opportunities for implementation in clinical procedures. On the other
hand, though, the inevitably labor-intensive and time-demanding clinical experimentation is vastly lagging
behind, thus fostering ‘home-made’ adoption of genomic tests (www.23andme.com/en-int/), in the virtual
absence of experimentally-validated guidelines. Genetic information demands may create further health
[27]
care disparities because of the high cost of these technologies and even pose health care risks if results are
[27]
prematurely translated to the consumer market outside of regulatory protection (www.genomeweb.com/
dxpgx/fda-warns-consumer-genomics-firms-illumina-selling-unapproved-dx-products#.XBLPfBNKi-U).
As such, these practices are devoid of validated medical status (www.nytimes.com/2013/11/26/business/fda-
demands-a-halt-to-a-dna-test-kits-marketing.html).

GUIDANCE FROM REGULATORY AGENCIES
FDA and EMA have established recommendations to adopt pharmacogenetic and pharmacogenomic
methods in research and diagnostics. Adequately numbered studies are highly recommended for achieving
meaningful screening power, particularly when target mutations are rare (www.fda.gov/ucm/groups/fdagov-
public/@fdagov-meddev-gen/documents/document/ucm071075.pdf). Regulatory agencies correspondingly
urge to create public databases, that would include global pharmacogenetic data to provide key scientific
input to both basic and clinical research (www.fda.gov/ucm/groups/fdagov-public/@fdagov-meddev-gen/
documents/document/ucm509837.pdf ).

Pharmacogenetic and pharmacogenomic tests will play ever more important roles in efforts to prevent
adverse drug reactions. In this case, drug-metabolizing enzymes, drug transporters and drug targets are
potential target genes, as they may alter the drug action or metabolism. HLA typing itself may be considered
a biomarker of drug response, as it is associated to distinct haplotypes/populations with different prevalence
of specific mutations in actionable genes. According to EMA, the evaluation of HLA can be carried out
in drug developmental programs, in order to discover new predictive HLA biomarkers. The use of whole
exome sequencing for the HLA region is strongly recommended by EMA, and is proposed to become
the gold standard for HLA typing (www.ema.europa.eu/documents/scientific-guideline/guideline-good-
pharmacogenomic-practice-first-version_en.pdf). Technical recommendations have also been issued, as
EMA recommends that the technical predictive value of NGS should be at least 99.9%. In germline genetics,
a minimum coverage of > 30× is desirable. A higher one should be pursued if a genetic variant is uncommon
(www.ema.europa.eu/documents/scientific-guideline/guideline-good-pharmacogenomic-practice-first-
version_en.pdf). It is expected, though, that improvements in current technologies will rapidly superseed
these thresholds.

IMPACT OF NGS - SUCCESSFUL STRATEGIES AND PROOF-OF-CONCEPT ACHIEVEMENTS
Rapid progress in deciphering cancer genomes is being achieved through ongoing international efforts,
including The Cancer Genome Atlas and the International Cancer Genome Consortium. These collaborative