Next-generation sequencing approach

for the diagnosis of human diseases:

open challenges and new opportunities
Chiara Di Resta1,2, Silvia Galbiati2, Paola Carrera2,3, Maurizio Ferrari1,2,3
1
Vita-Salute San Raffaele University, Milan, Italy
2
Genomic Unit for the Diagnosis of Human Disorders, Division of Genetics and Cell Biology,
IRCCS San Raffaele Hospital, Milan, Italy
3
Laboratory of Clinical Molecular Biology and Cytogenetics, IRCCS San Raffaele Hospital, Milan, Italy

ARTICLE INFO ABSTRACT

Corresponding author: The rapid evolution and widespread use of next gen-
Maurizio Ferrari eration sequencing (NGS) in clinical laboratories has
Genomic Unit for the Diagnosis
of Human Pathologies allowed an incredible progress in the genetic diagnos-
Division of Genetics and Cellular Biology tics of several inherited disorders. However, the new
IRCCS San Raffaele Hospital technologies have brought new challenges. In this
Via Olgettina 60
20132 Milan review we consider the important issue of NGS data
Italy analysis, as well as the interpretation of unknown ge-
Phone: 02-26432303 netic variants and the management of the incidental
Fax: 02-26434351
E-mail: ferrari.maurizio@hsr.it findings. Moreover, we focus the attention on the
new professional figure of bioinformatics and the new
Key words: role of medical geneticists in clinical management of
next generation sequencing, genetics,
inherited disorders, causative mutations, patients. Furthermore, we consider some of the main
sequence depth, coverage, clinical applications of NGS, taking into consideration
incidental findings, variants interpretation, that there will be a growing progress in this field in the
diagnostics, genetic medicine
forthcoming future.
Disclosures:
The authors declare no conflict of interest.

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Next-generation sequencing approach for the diagnosis of human diseases

INTRODUCTION dNTP chemistry that releases hydrogen ions dur-

ing base incorporation by DNA polymerase and
The next-generation sequencing (NGS) has been
a modified silicon chip detecting the pH modifi-
introduced in genomic laboratories about 10
cation [15], while Illumina technology is based
years ago. Its impact on technological revolution
on the existing Solexa sequencing by synthesis
has important implications in human biology
chemistry with the use of very small flow-cells,
and medicine [1]. After improvements in accura-
reduced imaging time and fast sequencing pro-
cy, robustness and handling, it became a widely
cess [14].
used and an alternative approach to the direct
Sanger sequencing [2,3].
NGS APPROACH IN CLINICAL
The progress of NGS is leading to the increase LABORATORIES
of discovery of number of genes associated to
human inherited disorders and to the elucida- The increase in number of causative genes asso-
tion of molecular basis of complex disease [4]. ciated with human inherited disorders is directly
Moreover, since on NGS platforms it is possible associated with the implementation of NGS.
to perform a parallel sequencing of different tar- Until now Sanger sequencing has been the gold
get regions, NGS is widely used in diagnostics. standard in clinical laboratories for single-gene
Recently, the use of NGS in clinical laboratories tests and it serves as the standard methods by
has became increasingly widespread, used in which NGS data should be compared and validat-
diagnostics of infectious diseases, immune dis- ed [16]. However, Sanger sequencing achieves
orders, human hereditary disorders and in non- the diagnostic goal when there is a clear pheno-
invasive prenatal diagnosis, and, more recently, typic indication of a classical Mendelian disorder
in the therapeutic decision making for somatic and the single-gene test approach is preferred.
cancers [5–12]. It eliminates the problem of incidental findings,
A great advantage of NGS approach is based on that we will discuss later, but it may push the
its ability to deliver clinical diagnosis in a short patients into a “diagnostic odyssey”, where they
time [3]. could be evaluated by multiple providers, some-
times for years, without a genetic diagnosis [13].
Currently, there are several NGS platforms avail-
able for routine diagnostic applications. These Today there is a different scenario, in which ge-
sequencers allow performing an high-through- nomic technologies can be very useful to detect
put analysis within few days, considerably de- genetic variations in patients with a high accura-
creasing costs [13]. These new technologies are cy and an important reduction of costs, thanks
different from Sanger sequencing because they to the first-generation sequencing approach. In
are based on a massively parallel analysis and particular, next-generation sequencing will in-
high throughput. Today two different NGS tech- creasingly be used for clinically heterogeneous
nologies are mainly used in clinical laboratories: inherited disorders, resulting in an increase in
Ion Torrent and Illumina systems [14]. The Ion number of reported disease-causing genes [6].
Torrent Personal Genome Machine (PGM) was Indeed, in the majority of human inherited
launched in 2011, while the widely used Illumina diseases not merely one gene but a number
benchtops for diagnostic purpose are MiSeq, of genes may interact leading to overlapping
marketed in 2011, MiniSeq, launched in 2016, pathological phenotypes [2]. NGS approach is
or iSeq100, debuting in the end of 2017. The Ion tempting when there is a genetic contribution
Torrent exploited the emulsion PCR using native in heterogeneous and complex diseases, such as

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Next-generation sequencing approach for the diagnosis of human diseases

in cardiomyopathies, in cardiac arrhythmias, in Syndrome and the dominant Schinzel-Giedion

connective tissue disorders, in mental retarda- Syndrome [25]. However it is important to keep
tion or autism, where a large number of genes in mind that about 10% of targeted bases se-
are involved in a large phenotypic spectrum quenced in WES do not get the 20 read depth
[10,11,17]. In these cases, NGS approaches al- [26], required for clinical confidence and inter-
lows to test a large number of genes simultane- pretation, and approximately only 85% of genes
ously in a cost-effective manner [13]. An impor- associated to human diseases into the principle
tant issue is to decide which kind of NGS testing database (OMIM) receive the adequate coverage
strategy is best suited for each clinical case. Two [27]. Poor coverage in WES can due to several fac-
options are currently available: targeted gene tors: probes that are not tiled for particular genes
panels or whole-exome sequencing (WES) [13]. probably not included during assay development
Targeted sequencing of selected genes offers or because repetitive sequences prevented in-
a good coverage (mean 300X, depending on clusion or poorly performing probes owing to
platforms and number of analyzed samples) GC-richness and low mapping quality [6].
for the entire analyzed panel and specific re- However it is important to consider that both of
gions refractory to NGS can be sequenced by these approaches can significantly reduce costs
Sanger sequencing, in order to cover the gap and turn-around time for a genetic test [13].
and to validate the NGS data [18,19]. So far,
targeted resequencing has been adopted to de- THE MAIN ISSUE OF NGS:
velop tests for genetic disorders, such as non- THE INTERPRETATION OF GENETIC
syndromic deafness [20,21], common and het- DATA FOR A CLINICAL UTILITY
erogeneous diseases, such as hypertension and
diabetes [22], or in traditional cytogenetic and In the NGS process one limiting step is without
Mendelian disorder diagnosis [23,24]. The main doubt the complexity of genetic variation inter-
limitation of targeted sequencing is the rigid- pretation in whole exome, due to the presence
ity of testing only a selected number of genes. of thousands of rare single nucleotide varia-
Since the genetic field is rapidly evolving, new tions without pathogenic effect. Moreover, in
genes may be associated with a clinical pheno- the majority of human diseases the pathologi-
type and as such redesigning and revalidation cal phenotype may be caused by a pathogenic
of the panel is needed [13,16]. On the contrary rare mutation with a strong effect or it may be
a clear advantage of the use of targeted panel is caused by a co-presence of multiple genetic
the reduction of number of incidental findings variations [28][29].
and/or the number of variants of unknown sig- Reliable interpretation of the multiple and de
nificance, that will be discuss later in this review. novo variants identified through NGS will re-
On the other hand, the benefit of WES is testing quire additional experience and validation be-
a greater number of genes, even if, in practice, fore it reaches the clinical stage on a large scale,
complete coverage of all coding exons is infea- particularly for diagnosis of complex traits [30].
sible. The WES application may be useful, for In the recent past, genetic data did not drive di-
example, in negative cases in targeted sequenc- agnosis but had a primarily confirmatory role.
ing or in a rare disease, especially in exploiting Today the major challenge is to convert patho-
trios approach. Indeed, it allowed the identifi- genic genetic data into a primary diagnostic tool
cation of genes responsible for the dominant that can shape clinical decisions and patients
Freeman-Sheldon syndrome, the recessive Miller management [31].

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Next-generation sequencing approach for the diagnosis of human diseases

Actually, the interpretation of genetic variants are present in each individual’s genome and to-
is based on criteria published by the American day their prioritization remains a primary chal-
college of medical genetics and genomics lenge [35].
(ACMG). The ACMG recommends that the vari- In some cases, the interpretation of VUS can be
ants be allocated to one of the categories re- useful in commencing the segregation analysis
ported below [32]: in large families including affected members or
a. disease causing (class V): the sequence vari- the identification of the occurrence of de novo
ation is previously reported and recognized variation in the affected patient. Unfortunately,
as causative of the disorder; in many cases the interpretation of VUS re-
b. likely disease causing (class IV): the sequence mains unresolved and its identification cannot
variation is not previously reported as ex- be used for the clinical management of patients
pected to cause the disorder, frequently in a and families [29,36].
known disease gene; Until now few clear guidelines are published
c. variant of unknown clinical significance (VUS; for the VUS interpretation [36]. Today, in order
class III): the sequence variation is unknown to try to assign a pathological score to VUS, it
or expected to be causative of disease and is important to consider, for example, its al-
is found to be connected with a clinical lelic frequency in a control population (1000
presentation; Genomes or exome sequencing project con-
sortium [ExAC]), the amino acidic conservation,
d. likely not disease causing (class II): the se- the predicted effect on protein function and the
quence variation is not previously report- results of published functional assay [37,38].
ed and it is probably not causative of the
pathology; Up to now in silico prediction algorithms, such
as Polyphen, Sift, Mutation Taster or UMD
e. not disease causing (class I): the sequence predictor, have been developed and they are
variation is already reported and document- widely used for the missense variants inter-
ed as neutral variant. pretation [37]. However, they present some
Moreover, most of these classes of variants are intrinsic caveat and limitations, affecting their
subject to supplementary interpretation focus- specificity and sensitivity, that can lead to pos-
ing on literature reported, population frequen- sible false-positive and false-negative interpre-
cies, clinical findings, mutation databases and tations [39]. Another existing problem involves
possibly case-specific research data [31]. The the allelic frequency, that is mainly estimated
principal human variant databases are useful from the 1000 Genome project and ExAC, that
to annotate both common and pathogenic vari- represents only a fraction of the worldwide
ants, such as dbSNP, gnomAD or ExAC database population, so the declared allelic frequency
(Exome Aggregation Consortium) [33], and to available is not stratified according to the real
classify variants previously associated with hu- population groups [29].
man disorders, such as Human Gene Mutation Since the problem of the management of VUSs
Database (HGMD) [34] and ClinVar. is not yet resolved, it would be fundamental
The variants of unknown significance (VUS) rep- to collect and share VUSs and available clinical
resent a problem for the interpretative process. data, allowing a progressive and definitive clas-
Indeed it is known that hundreds of loss of func- sification of these variants, as deleterious (class
tion variants with unknown clinical significance V) or neutral ones (class I) [29,30].

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Another important challenge of the use of NGS to be described on the report for clinicians and
approach in clinical diagnostic is the manage- patients. In particular, it is needed to declare
ment of the amount of data generated [40]. the sensitivity and specificity of the techniques
Indeed generation, analysis and also storage of used considering both technical and bioinfor-
NGS data require sophisticated bioinformatics matics parameters. It is important to report
infrastructure [41]. which target region was not sequenced, the
A skilled bioinformatics staff is needed to man- number of reads obtained, the quality of the se-
age and analyze NGS data, and so both com- quence, the limitations of the chosen sequenc-
puting infrastructure and manpower impact ing method and of the settings of used bioinfor-
on costs of NGS applications in clinical diag- matics pipeline [16,45].
nostics. Bioinformaticians are to be mandatory
in the organization chart of clinical laborato- ETHICAL CONSIDERATIONS AND
ries in the NGS era, where they have to closely MANAGEMENT OF INCIDENTAL FINDINGS
collaborate with clinicians and laboratory staff The development and the widespread use of
to optimize the panel testing and the NGS data NGS in clinical laboratories are paired with de-
analyses [42]. bate on the ethics for reporting incidental find-
Bioinformatics has been recently defined as the ings [46,47]. In 2013 the ACMG has highlighted
discipline that develops and applies advanced the question of the incidental findings (IF), de-
computational tools to manage and analyze the fining them as “genetic variations identified by
NGS data. Bioinformatics pipeline developed for genomic sequencing but not related to the dis-
NGS are aimed to convert the raw sequencing ease being investigated” [48].
signals to data, data to information, and infor- According to the European Society of Human
mation to knowledge [43]. Genetics (ESHG) guidelines, the targeted diag-
This process can be developed in three different nostic testing should be performed minimizing
steps - primary, secondary, and tertiary analy- the likelihood of detecting incidental findings,
ses [44]: focusing only on genes clinically actionable [49].
It means that genetic testing should aim to ana-
• The primary analysis is the process of raw lyze the causative genes associated to the prima-
data produced by NGS instruments, ry clinical questions, even if a broader panel of
• the secondary analysis is the alignment to a genes or the whole exome sequencing has been
reference sequence and the calling variants performed [49]. It is the role of responsible clini-
and, finally, cians requesting the test to disclose an incidental
• the tertiary analysis is the confirmation or finding to a patient, not the role of the clinical
validation of detected variants, providing laboratory.
evidence to facilitate interpretation [41]. The impact of the IF determines how the genetic
All clinical bioinformatics systems require these finding should be disclosed or not to a patient,
three steps that should be properly validated also to avoid unwarranted psychological stress.
and documented. In particular, it requires de- In particular, if it can bring minor consequences
termination of variant calling sensitivity, speci- or if a clinical intervention is possible, then the
ficity, accuracy and precision for all variants variant should be reported.
reported in the clinical assay [44]. The quality On the contrary, if the variant is associated to a
criteria of the performed sequencing test have late onset disorder or has major consequences,

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Next-generation sequencing approach for the diagnosis of human diseases

counselling and consent will determine if and to patients’ families may not be appropriate and
when the variant can and should be reported to the genetic information may be reconsider later
the patient [36]. This implies that genetic tests in baby’s life. Similarly, the report of IFs may be
should be ordered by medical professionals who postponed in cases where parents or patients
are capable of performing appropriate counsel- are given a diagnosis linked to poor prognosis or
ling [50]. For that reason, the counselling and in case of post-mortem genetic testing.
the informed consent are critical steps. Additional contexts in which the reporting of in-
There is a difference between recording and cidental findings may have an influence on the
reporting a variant, as well as between who re- patients management are carrier testing, pre-
ceives this information, clinicians or patients, natal diagnosis, pharmacogenetics testing and
and when. When a variant is reported to a additional non-diagnostic testing such as medi-
clinician, it does not mean that it will be re- cal research (dependent on the study design),
vealed to patient. Indeed, the clinician should forensic testing, parental and genealogical test-
evaluate the impossible clinical implication of ing. In conclusion, the issue of IFs requires an
this information, based on the clinical history appropriate pre and post counselling to correct-
of patient. For example, the impact of an IF ly inform the patient [16].
in a case without a known family history for a The widespread implementation of NGS ap-
specific disorder is different from the case in proach in diagnosis of human pathologies raises
which the patient is already aware of a preex- the problem of management of IFs and VUSs and
isting familial condition. it is needed to have clear guidelines for the han-
Another interesting example is the acute neona- dling of NGS data in the diagnostics approach
tal care, in which immediate reporting of all IFs (Figure 1).

Figure 1 Advantages and challenges of the use

of gene panel NGS testing and WES

So far the application of WES in clinical diagnostics presents more open challenges (B) than targeted sequencing (A).

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Next-generation sequencing approach for the diagnosis of human diseases

CONCLUSIONS time taken for direct Sanger sequencing and the

odyssey lived by some patients before to under-
Until now Sanger sequencing has been the
stand the cause of their rare disorder [6].
gold standard in molecular diagnostics and
it has been used in clinical testing method for This strategy of approaching the clinical evalua-
Mendelian disorders, in which most of causative tion has also economically beneficial in patients
variants are identified in the principal causative without diagnosis [52].
genes. Since the rapid and incremental improve- The euphoria of the widespread use of the NGS
ments in instrumentations, methodologies and applications to the clinical diagnosis is combined
throughput and the significant reduction of with the awareness of emerged challenges, such
costs, the NGS technologies are being integrat- as the validation of large number of genetic vari-
ed into patient care and clinical management. ations detected, that can be IF or VUSs, the use
NGS allows sequencing of all genes relevant to of standardization processes in clinical diagnos-
a given phenotype starting from a small amount tics, the management of terabytes of data and
of total DNA. In that way, the limitation factors variants interpretation.
are no longer the size of the gene or its causative In the NGS approach, the analysis of data re-
contribution but the actual knowledge of the ge- quires the development of a standard pipeline to
netic basis of patient’s disease [6]. process sequencing data. The flow chart analysis
In the past, clinicians considered genetic tests includes mapping, variant calling and annotation.
with a marginal diagnostic value, only if a de- Today there are various public database, such as
finitive diagnosis was not yielded or if it had im- dnSNP [53], the 1000 Genome Project [54], ExAC,
plications on future family planning. Often the as well as several internal control databases.
positive genetic test results did not influence Targeted panel sequencing or clinical exome
clinical management of the patient. sequencing identifies several variations in each
However today, with the potentiality of NGS, the person, but as far there are no clear guidelines
parallel sequencing of large multi-genes panel, to filter variants and to delineate their possible
that may describe a broader range of pheno- pathological meanings. For this reason, the
types, the clinicians are changing their point of pathogenic validation may be the limiting step.
view on the role of the genetics in patients care. Because of these considerations, it is important
Indeed, nowadays the genetic testing may be to apply the NGS approach in clinical diagnostics
for that disorders of which the main causative
useful for the evaluation of a clinical case and, if
genes have been identified. Indeed, in this case
the result were to be positive, it may save time
the genetic tests can successfully reveal a useful
and money in identifying the etiology.
result.
Today physicians often begin their clinical evalu-
Moreover, another consideration involves the
ations with the genetic tests. For example, the
fundamental change of the figure of medical ge-
evaluation of patients with left ventricular hy-
neticist in the NGS era. Indeed, the NGS applica-
pertrophy begins with genetic testing, given that
tions into diagnostic field can lead to useful re-
the genetic diagnosis is achieved in about 80%
sults for patient’s care with genetic disorders. As
of hypertrophic cardiomyopathy cases [51]. such, the geneticists will become a pivotal part
The results of most targeted genetic tests may of the collaborative team of clinicians and their
be available for clinicians in 2-8 weeks, which role will be fundamental for the clinical inter-
is an impressive improvement compared to the pretation of NGS data to guide patient care [25].

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Consequently, clinical medical geneticists have sequencing of nucleic acid composition directly
to complement their skills with expertise in the in fixed cells and tissues providing a throughput
clinical interpretation of NGS data. analysis, opening great opportunity mainly for
Moreover we have to keep in mind that the the analysis of tumor cells variability in situ [58].
medical geneticist has an important and crucial In forthcoming future, it holds exciting prospec-
role also in the pre-test counseling, to deliver tive for research and new insights regarding ge-
reliable information to patients [29]. Indeed it nomic diagnostics.
is important to clearly explain to the patient
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