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Next-generation
sequencing applied to
molecular diagnostics
Expert Rev. Mol. Diagn. 11(4), 425–444 (2011)

Rachael Natrajan†1 and
Jorge S Reis-Filho1
1
Breakthrough Breast Cancer Research
Centre, The Institute of Cancer
Research, 237 Fulham Road, London,
SW3 6JB, UK
†
Author for correspondence:
Tel.: +44 207 153 5529
Fax: +44 207 153 5340
rachael.natrajan@icr.ac.uk
Next-generation sequencing technologies have begun to revolutionize the field of cancer genetics
through rapid and accurate assessment of a patient’s DNA makeup with minimal cost. These
technologies have already led to the realization of the inter- and intra-tumor genetic heterogeneity
and the identification of novel mutations and chimeric genes, however, several challenges lie
ahead. Given the low number of recurrent somatic genetic aberrations in common types of
cancer, the identification of ‘driver’ genetic aberrations has proven challenging. Furthermore,
implementation of next-generation sequencing and/or some of its derivatives into routine practice
as diagnostic tests will require in-depth understanding of the pitfalls of these technologies and
a great degree of bioinformatic expertise. This article focuses on the contribution of next-generation
sequencing technologies to diagnosis and cancer prognostication and prediction.

Keywords : cancer • exome • fusion genes • genetics • massively parallel sequencing • mutations
• third-generation sequencing

First-generation DNA sequencing (Sanger second-generation sequencing has provided an

sequencing) was first described by Sanger, and
Maxam and Gilbert over 30 years ago [1,2] . Since
then, modifications to the Sanger sequenc-
ing methodology have provided incremental
improvements in its accuracy and efficiency,
enabling increased automation and incremen-
tal increases in its throughput. However, this
technology is still limited to targeted sequenc-
ing of known genomic sequences, usually up
to 1000 bp per sequencing reaction. In the last
few years, second-generation massively paral-
lel sequencing (also known as next-generation
sequencing) has emerged. It is based on method-
ologies that overcome the limitations of Sanger
sequencing. As a result of these technological
advances, it is now possible to sequence com-
plete genomes [3–12] , expressed genes (transcrip-
tomes) [13,14] and known exons (exomes) [15–17]
of patient samples. As well as providing whole
genomic sequencing coverage, second-genera-
tion sequencing is much more cost effective
than the first generation. For example, an entire
genome can now be sequenced for approximately
US$4000 at 30–40-times coverage [18] .
These cost reductions in sequencing in tandem
with the promise of the US$1000 genome [19]
now make it possible for clinical laborato-
ries to adopt this technology. Clinical use of
interesting alternative to identify the cause of
hereditary diseases through genome-wide asso-
ciation studies, as well as aiding the identification
of mutations that cause and promote the progres-
sion of cancer. Next-generation sequencing also
holds the promise of increasing our understand-
ing of cancer genetics, epigenetics and pharmaco-
genetics, and also detecting novel pathogens that
cause human diseases. In fact, next-generation
sequencing and/or one of its derivatives is likely
to replace microarrays for the molecular charac-
terization of cancer for diagnostic, prognostic and
predictive purposes [20,21] .
Implementing next-generation sequencing
into routine clinical diagnostic laboratories will
require clear guidelines, robust protocols and
standard operating procedures not only for sam-
ple preparation and sequencing (wet laboratory),
but also for the computational assessment (dry
laboratory) of the results. The interpretation of
billions of sequence reads is by no means trivial,
and improvements in the availability of ‘user-
friendly’ software and enhancements in com-
puter and data storage infrastructure will be of
paramount importance for the realization of the
potential of these technologies. Massively paral-
lel sequencing has already begun to revolution-
ize medical genetics as we know it, but the vast

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 Review Natrajan & Reis-Filho

amounts of personal genomic data raise ethical considerations that
will also have to be considered [22] . Indeed, the era of personalized
medicine is now a step closer than before and routine screening to
guide medical treatments of individuals throughout their lifetime
is becoming a reality.
Overview of second-generation
sequencing technologies
Massively parallel sequencing methods overcome the limita-
tions of scalability of traditional Sanger sequencing by eliminat-
ing the requirement of electrophoresis for the separation of the
sequences [23] . Most of the second-generation sequencing methods
work by ‘sequencing by synthesis’ by either creating micro­reactors
and/or attaching DNA molecules to be sequenced to solid sur-
faces or beads, allowing for millions of sequencing reactions to be
performed in parallel at the same time. A number of next genera-
tion DNA sequencers are commercially available, including the
Genome Analyzer and Hi-Seq™ (Illumina), 454-FLX (Roche),
SOLiD™ (Applied Biosystems) and Heliscope™ (Helicos), and
a number of third-generation single molecule sequencers are in
development (Table 1, for reviews see [24–32]). Second-generation
sequencing technologies involve the production of an adaptor-
modified random collection of DNA fragments that have been
clonally amplified (Figure 1) . These are subsequently sequenced by
repeated cycles of polymerase-based nucleotide extension or by
cycles of oligonucleotide ligation [26] . Next-generation sequenc-
ing results in the generation of a huge amount of sequence data
in the range of megabases to gigabases. Complete genomics offer
a ‘one-stop shop’ for whole-genome sequencing, using a propri-
etary sequencing technology based on DNA nanoballs involving
sample preparation, sequencing and data analysis. The advantage
of this technology lies in its custom alignment algorithm and data
analysis pipeline designed for human whole-genome sequence
comparisons, which have led to a substantial reduction in cost [18] .
Second-generation sequencing technologies have, therefore,
provided a new revolution for genetic analysis and have begun
to allow resequencing at single-nucleotide resolution of entire
normal human and cancer genomes at a relatively low cost. These
revolutionary methods hold the promise of identifying activat-
ing and inactivating mutations in key disease genes, assessment
of gene expression and novel transcript variants in both coding
and noncoding genes, and in tandem are able to identify somatic
structural rearrangements (i.e., translocations) and genomic
copy-number alterations.
Whole-genome sequencing
The first human whole genome to be sequenced by next-generation
sequencing technologies was James Watson’s genome in 2008 at an
average depth of 7.4-fold in 2 months, costing one hundredth of
the cost of conventional Sanger sequencing technologies [33] . Since
then the number of whole genomes sequenced has begun to rise
dramatically, now with up to 30-fold average coverage and includ-
ing individuals from different ethnicities [3–5] . As well as ‘normal’
genomes, the first cancer genome to be sequenced was reported
in 2008, in which the full DNA sequence of a patient with acute
myeloid leukemia was compared with the germ-line DNA from the
same individual [6] . In the past few years, more complete sequences
of cancer genomes compared with their normal genomes have
been published (Table 2) [8–12] . These whole-genome sequencing
studies have enabled the assessment of cancer-specific mutations

Table 1. Comparisons of second-generation sequencing technologies.

Sequencing Amplification Sequence Read Run Data Advantages Disadvantages
platform reaction length time yield
(bp) (days) (Gb)
454 Ti Roche™ Emulsion PCR Pyrosequencing 400 0.4 0.5 Short run times. High reagent cost. Higher
Longer reads improve error rates in repeat
mapping in repetitive sequences
regions. Ability to
detect large structural
variations
Illumina HiSeq™ Bridge PCR Reverse 100 4†, 8‡ 150– The most popular Short reads may miss
2000 terminator 200 platform larger structural
variations. Low
multiplexing capacity
of samples
ABI 5500 Emulsion PCR Ligation 75 3.5†, 7‡ 90 Good base call Short read length
sequencing accuracy. Good
multiplexing capability
Complete PCR on DNA Combinatorial 35 12 200 Low error rate. Analysis Large amounts of input
Genomics nanoballs probe-anchor and management DNA needed. Whole-
ligation handled by company genome DNA sequencing
only
Single-end runs.
†

Paired-end runs.
‡

Gb: Gigabyte of data.

426 Expert Rev. Mol. Diagn. 11(4), (2011)

 Next-generation sequencing applied to molecular diagnostics
by removing germ-line variants from the normal DNA sequence;
however, this process required sequencing of the germ-line DNA
of the same patient. Identification of somatic rather than germ-line
mutations is crucial for the cataloguing of the genetic events that
took place during the development and progression of disease.
Shah et al. used this approach to look at the mutational evolution
of a lobular breast carcinoma [11] . A comparative analysis of the
repertoire of mutations found in the metastatic deposits compared
with that from the primary tumor revealed that only five of 32
nonsynonymous coding mutations present in the metastasis were
also present in the primary tumor. Importantly, 19 mutations
present in the metastasis were not detected in the primary tumor.
Given the interval between primary tumor and metastasis and
the fact that the patient received radio- and chemo-therapy prior
to the resection of the metastatic deposit, these results are not
conclusive as to whether the metastasis-restricted mutations are
related to tumor progression, a consequence of chemo-/radio-
therapy or a combination of the two phenomena. In a subsequent
study, Ding et al. used whole-genome paired end sequencing to
determine the genetic alterations associated with tumor progres-
sion in a patient with a metaplastic breast carcinoma of basal-like
phenotype by sequencing the primary tumor, a metastatic deposit
in the brain, a xenograft derived from the primary tumor and
the germ-line DNA from the patient [12] . These analyses demon-
strated that although the primary tumor, the metastatic deposit
and the xenograft were remarkably similar in their repertoire of
somatic mutations, rearrangements and copy number aberrations,
important differences were found in terms of the percentage of
cells harboring a given mutation in each of these samples and the
presence of mutations restricted to one of these samples. In fact,
the somatic gene aberrations found in the metastatic deposit were
more similar to those of the xenograft than those of the primary
tumor. The xenograft and metastasis displayed an expansion in
the prevalence of 26 mutations and a decrease in prevalence of two
mutations over the primary tumor; in addition, novel mutations
of SNED1, FLNC and a 26.9 kb deletion of MECR were restricted
to the cancer cells in the metastatic deposit. Taken together, these
results suggest that metastatic deposits may arise from a number
of small clonal populations within the primary cancer [12] .
In addition to revealing the repertoire of point mutations in can-
cers, whole-genome sequencing can also provide important biologi-
cal insights in relation to the types of and patterns of DNA insults
involved in the development of a tumor. Disease-specific muta-
tional signatures in distinct types of cancer have recently been iden-
tified. The mutational signature of lung cancer patients associated
with tobacco exposure has an enrichment of G>T; C>A transver-
sions [7,10] , whereas the mutational spectrum in a melanoma patient
showed a UV exposure signature with an enrichment of C>T; G>A
transitions [9] . These efforts may lead to a better understanding of
the types of genetic damage caused by specific carcinogenic insults
and the characterization of the genetic lesions caused by specific
types of dysfunctional DNA repair pathways in cancers.
Whole-genome sequencing also has the advantage of detect-
ing structural rearrangements – that is, fusion genes present in
hematological malignancies and now known to be common in
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Review
solid cancers [34,35] . By investigating the read pairs that do not
align to the genome as would have been expected in two lung
cancer cell lines, Campbell et al. identified 103 somatic structural
variants. They observed that many of the somatic rearrange-
ments mapped to regions of amplification with a proportion
leading to expressed fusion transcripts [36] . A low-depth whole-
genome sequencing approach in 24 breast cancers identified a
large number of fusion genes and, most importantly, the exist-
ence of a subgroup of breast cancers characterized by the pres-
ence of multiple tandem repeats inserted in their genomes. The
term mutator phenotype was coined to describe this genomic
pattern, which is more prevalent in ER-/HER2-negative than
in ER-positive breast cancers [37] . The mechanism leading to
this mutator phenotype is yet to be determined; however, this
phenomenon may possibly be linked to the high proliferative
potential of a subgroup of ER-/HER2-negative cancers and the
slippage of replication forks. Further analyses to characterize this
fascinating phenotype are warranted. Although 29 expressed
in-frame fusions were detected in that study [37] , all of them
proved to be private (i.e., only found in the index case). Lee
et al. also used this approach to identify novel fusion genes in
a lung cancer patient present in the tumor but not the matched
normal tissue [7] . The large majority of the fusions identified
mapped to intergenic regions, suggesting that they are likely to
be passenger events; however, the functional consequences of
such rearrangements undoubtedly require further investigation.
A similar analysis of pancreatic primary cancers and metastatic
deposits led to the identification of 381 somatic rearrangements,
of which a proportion were also found in the metastasis, suggest-
ing the presence of the expansion of a specific clonal populations
in the metastatic process [38] .
As well as detecting alterations in known regions of the
genome, whole genome next-generation sequencing can also
be useful for the detection of mutations in gene promoter and
enhancer regions, as well as in introns, noncoding RNA species
and retro­transposons. It is becoming more apparent that hot long
interspersed LINE-1 elements (L1’s) account for a proportion
of our inter-individual genetic variation, and may contribute to
disease [39,40] . L1 is a class of mobile elements that generate new
retro­transposon insertions in human genomes. Using a combi-
nation of locus junction-specific PCR and 454 next-generation
sequencing, Iskow et al. developed a method to detect new and
otherwise young retrotransposon insertions in humans [41] . This
approach led to the observation of somatic L1 insertions in 30%
of lung cancer genomes. Methylation analysis revealed a hypo­
methylation signature that was able to distinguish lung cancers
with L1-permissive from non-L1-permissive lung cancers [41] .
Another useful application of next-generation sequencing lies
in the detection of pathogens that are present in the human
genome. Known as ‘metagenomics’, samples of human tissue can
be sequenced and reads aligned to microbial or viral genomes to
identify which pathogenic organism is present in the primary
sample. As the sequence reads are present in proportion to the
population frequency of each pathogen, information regard-
ing the relative abundance can also be inferred [42] . The human
427
 Review Natrajan & Reis-Filho

A A
C C G
C T
A T
G
T C
G
C

DNA fragmentation and Attachment to Cluster generation by Single-base extension with Imaging
adaptor ligation flow cell bridge amplification of incorporation of fluorescently
DNA fragments labeled nucleotides

T
A

DNA fragmentation and Coupling of DNA fragment to an amplification Single-base extension and
bead and amplification of DNA fragments generation of chemiluminescent Imaging
adaptor ligation
by emulsion PCR signal

nnnCTzzz
nnnCGzzz
ligase
nnnGAzzz
nnnCAzzz

DNA fragmentation and Ligation of fragments to magnetic beads Amplified DNA is attached Single-base extension
adaptor ligation and amplification of DNA fragments by to a glass slide and imaging
emulsion PCR

nnnTzzz
nnnCzzz

Anchor nnnGzzz
nnnGzzz nnnAzzz
GATCATTCCGACGGAA
Matching probe binds
Adaptor Genomic DNA
to anchor DNA
Generation of DNBs DNBs are adhered to a silicon chip Combinatorial probe anchor ligation with fluorescently
labeled oligonucleotides and image detection

Figure 1. Summary of second-generation sequencing technology workflows. (A) Illumina. DNA molecules are fragmented and 5´
and 3´ universal adaptors are added to the DNA. DNA molecules are then immobilized to a glass slide (flow cell) and the DNA molecules
are copied in situ via bridge amplification. This generates clonal clusters that are then denatured and annealed with a sequencing primer
and subjected to sequencing by synthesis using 3´ chemically inactive labeled nucleotides, which ensure a single base is incorporated at
each step. Each base-incorporation cycle is imaged and then the blocked fluorescence group is removed and the next base is
incorporated and imaged. (B) Roche 454. Library construction involves the ligation of special 454 5´ and 3´ adaptors to fragmented DNA.
One DNA fragment is then coupled to a single amplification bead, and emulsion PCR proceeds, where fragments are amplified before
sequencing. The beads are then loaded into picotiter plates. The pyrosequencing reaction then proceeds, which involves the addition of
sequencing enzymes. The sequencing instrument then flows individual nucleotides in a fixed order across the hundreds of thousands of
wells containing one bead each. The addition of one nucleotide complementary to the template strand results in a chemiluminescent
signal recorded by the charge-coupled-device camera within the instrument. (C) ABI SOLID. Oligonucleotide adaptor-linked DNA
fragments are ligated to magnetic beads that hold complementary oligonucleotides on them. Emulsion PCR is used to copy the DNA,
which is subsequently attached to the surface of a glass slide with the aid of a 3´ modification (purple) to allow covalent bonding to the
slide. The slide is placed into a fluidics cassette within the sequencer and sequencing occurs with the hybridization of primers to the
adapter molecules on the beads and a set of four 8mer di-base probes compete for ligation to the sequencing primer. A matched probe
is then ligated to the DNA, and a fluorescent readout of the fifth base is achieved. Chemical cleavage removes the fluorescent group
enabling subsequent rounds of ligation to occur. Two-base encoding aids the discrimination of base calling errors from true mutations.
(D) Complete Genomics. DNA is fragmented and configured in a head-to-tail manner connecting all copies together that form into a
DNB. These nanoballs contain hundreds of copies of a 70 base pair sequence. DNBs are adhered to spots on silicon chips, with each spot
containing a single DNB. Combinatorial probe anchor ligation accurately distinguishes between the different bases to attach fluorescent
molecules that emit signals when detected.
DNB: DNA nanoball.

428 Expert Rev. Mol. Diagn. 11(4), (2011)

 Next-generation sequencing applied to molecular diagnostics Review

Table 2. Next-generation sequencing studies of cancer genomes.

Study (year) Method Cancer type Samples Results Ref.
sequenced
(n)
Ley et al. (2008) Whole-genome high Acute myeloid 1 Ten acquired mutations in the tumor [6]
coverage leukemia
Campbell et al. Whole-genome low Lung 2 Identification of somatically acquired [36]
(2008) coverage structural rearrangements
Stephens et al. Whole-genome low Breast 24 Identification of structural variations and [37]
(2009) coverage fusion genes
Pleasance et al. Whole-genome high Melanoma 1 Catalogue of somatic mutations related to [9]
(2010) coverage UV-induced DNA damage
Pleasance et al. Whole-genome high Small-cell lung 1 Catalogue of somatic mutations related to [10]
(2010) coverage tobacco-induced DNA damage
Mardis et al. Whole-genome high Acute myeloid 1 Recurrent mutations in IDH1 [8]
(2009) coverage leukemia
Shah et al. Whole-genome high Breast 1 Identification of somatic mutations present  [11]
(2009) coverage in primary tumors and metastases
Ding et al. Whole-genome high Breast 1 Comparison of mutation spectrum in  [12]
(2010) coverage primary tumor, metastasis and xenograft
Lee et al. (2010) Whole-genome high Lung 1 Identification of mutation spectrum and  [7]
coverage structural variations in a paired normal and
tumor genome
Stephens et al. Whole-genome low Chronic lymphocytic 34 Identification of structural variations and  [99]
(2011) coverage leukemia and bone fusion genes
Zhao et al. Targeted exome Breast 1 Identification of known and novel tumor-  [58]
(2010) sequencing suppressor genes
Harbour et al. Targeted exome Uveal melanoma 2 Identification of recurrent inactivating  [59]
(2010) sequencing mutations in BAP1
Jiao et al. (2011) Targeted exome Pancreatic 10 Identification of mutations in the mTOR  [60]
sequencing neuroendocrine pathway
Timmerman Targeted exome Colorectal 2 Prediction of microsatellite instability  [69]
et al. (2010) sequencing
Jones et al. Targeted exome Pancreatic 5 Identification of PALB2 mutations  [63]
(2009) sequencing
Kan et al. (2010) Targeted exome Breast, ovarian, lung 441 Identification of differential mutation spectra  [62]
sequencing and prostate
Cheng et al. Targeted exome Colorectal 27 Identification of novel SNPs in APC  [66]
(2010) sequencing
Parsons et al. Targeted PCR on Pediatric 22 Identification of MLL2 and MLL3 mutations  [61]
(2011) Illumina GAII medulloblastoma in 16% of patients
and Sanger
sequencing
Villarroel et al. Targeted exome Pancreatic 1 Identification of a PALB2 mutation leading  [65]
(2010) sequencing to resistance to therapy
Varela et al. Targeted exome Renal cell 7 Truncating mutations of PBRM1  [199]
(2011) sequencing

microbiome (collection of organisms that live within the human disease [43] . These include the characterization of the microbial
body) is increasingly being studied to understand the microbial component of the human gut [44,45] and oral cavity [46] and is
components of the human genetic and metabolic landscape and being used to elucidate mechanisms associated with obesity [47]
how they contribute to normal physiology and predisposition to and pneumococcal evolution [48] . Furthermore, analysis of genetic

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 Review Natrajan & Reis-Filho

fragments present in matched diseased and healthy tissues has
the potential to identify viral agents in cancer and autoimmune
diseases, among others.
Targeted ‘exomic’ sequencing
Whilst whole-genome sequencing provides a comprehensive
catalogue of mutations, copy number variations and structural
rearrangements in a single experiment, it requires the greatest
amount of sequencing and poses significant logistic challenges
in relation to data handling and storage [49] . Given that protein-
coding genes only comprise approximately 1–2% of the genome,
harbor the majority of known disease-related mutations and most,
if not all, mutations that can currently be addressed therapeuti-
cally, approaches that selectively sequence just these regions of
the genome (i.e., the ‘exome’) have the ability to expedite the
characterization of genetic aberrations in different diseases [15–
17] . Importantly, targeted exomic sequencing can currently be
performed in relatively large collections of cancers. This approach
involves the capture of the target sequences by molecular inversion
probes or capture onto DNA or RNA ‘baits’ by arrayed probes
on a solid surface or by bead capture in solution (Figure 2) [50] . Any
regions of the genome can be selectively targeted for sequenc-
ing by capture methods and custom capture baits provided by
the companies selling this technology are becoming increasingly
more popular. Microfluidic approaches such as RainDance and
Fluidigm technology for sequence enrichment are also becom-
ing more popular. RainDance uses a library of PCR primers
that enable the amplification of thousands of genomic loci in
a single tube by using picoliter volume PCR reactions at a rate
of 10 million reactions/h. This has the advantage of high-speed
sample processing, avoiding the bias introduced by capture based
technologies [51] . An additional PCR-based enrichment strategy
is available through the Access Array platform (Fluidigm, CA,
USA). This technology uses nanoscale non-emulsion-based

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Gene 1 Gene 2 Gene 3 Gene 4 Gene 5
gDNA gDNA

Fragmentation and nick

Fragmentation of DNA
translation of DNA

Hybridization to baits
Combine droplet
primers and droplet Primers DNA
fragments in a
microfluidic chamber

Elute DNA

Droplet PCR

gDNA purification

Sequencing

Sequencing

Figure 2. Overview of capture-based technologies. (A) Schematic diagram of hybrid selection to capture specific regions of the
human genome. DNA is sheared and hybridized to tagged oligonucelotide ‘baits’ that are specific for the region of interest or exons of
genes. Captured DNA is subsequently eluted and libraries prepared for sequencing. (B) Schematic diagram of microfluidic technologies
for sequence capture. DNA is fragmented and purified. A microfluidic chip encapsulates primer pairs and primer pair droplets are mixed
together to create a primer library. Primers and DNA droplets with PCR components (buffer, deoxyribonucleotide triphosphates and DNA
polymerase) are mixed together where they are merged into a single droplet as they pass through the channel of the microfluidic chip.
Following PCR, the DNA is purified and prepared for sequencing.
gDNA: Genomic DNA.

430 Expert Rev. Mol. Diagn. 11(4), (2011)

 Next-generation sequencing applied to molecular diagnostics
PCR in microfluidic chips in chambers that are separated by
valves, allowing up to 48 samples per array. This has the advan-
tage of performing long-range PCR, but is less scalable than
RainDance [52] .
The sequencing of selected sets of exons has already been
proven to be an effective strategy for the identification of somatic
mutations for which effective therapies have been developed in
cancer, such as BRAF mutations in melanoma [53] and EGFR
mutations in lung cancer [54–56] . The first whole exome sequenc-
ing study of human cancers was performed using conventional
Sanger sequencing surveying 22 breast and colorectal tumors
for 14,661  different coding sequence transcripts and involv-
ing 135,483 primer pairs and encompassing 21 Mb of genomic
sequence [57] . Since the advent of capture technologies and mas-
sively parallel sequencing, sequencing the exome can be per-
formed within a matter of weeks. Exome capture and transcrip-
tomic sequencing were employed to compare the sequences from
a breast cancer cell line and its matched normal in regions of loss
of heterozygozity [58] . Allele-specific expression analysis led to the
identification of known and potential novel tumor-suppressor
genes [58] . Exomic sequencing of two uveal melanomas harboring
chromosome 3 monosomy and corresponding germ-line DNA
from the patients, revealed recurrent inactivating mutations of
the BAP1 gene, which maps to chromosome 3 and was shown
to be the likeliest target of chromosome  3 deletions in uveal
melanomas. Importantly, there is evidence to suggest that BAP1
gene inactivation plays a pivotal role in the metastatic potential
of this tumor type and is a potential therapeutic target [59] . The
exomic sequencing of ten pancreatic neuroendocrine tumors
has identified mutations in mTOR pathway genes, indicating a
potential avenue for therapeutic intervention in these patients [60] .
A recent publication, using a combination of high-throughput
Sanger sequencing and targeted next-generation sequencing to
survey the genetic landscape of childhood medullo­blastoma,
identified recurrent inactivating mutations of the histone-­lysine
N-methyltransferase genes MLL2 or MLL3 in 16% of patients,
demonstrating distinct differences in the prevalence of somatic
mutations in adult and pediatric cancers [61] .
A distinct perspective emerged from a recent study of the
exomes of 441 common types of epithelial malignancies, includ-
ing breast, ovarian, lung and prostate cancer types [62] . Unlike
rare cancer types, these tumors were characterized by numerous
mutations, but at low prevalence, suggesting that these common
types of cancer are heterogeneous in terms of the constellations
of genetic aberrations they harbor [62] .
Recently, PALB2 has been identified as a pancreatic cancer
susceptibility gene through exomic sequencing in which truncat-
ing mutations were identified [63] . Previous studies had already
demonstrated that PALB2 germ-line mutations may also be
associated with increased breast cancer risk [64] . Importantly,
a combination of functional analysis of xenografts derived
from primary pancreatic cancers that were subjected to exomic
sequencing [65] provided an exciting proof of principle of how
to individualize therapy according to the mutation repertoire
of the tumor.
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A combination of exon-capture and long-range PCR (to
enhance capture of GC regions) has been used to sequence the
adenomatous polyposis coli (APC) gene in 27 colorectal cancers
and adjacent normal tissues, identifying 69 novel SNPs including
one novel exonic SNP, not previously reported [66] . This brings to
our attention one of the drawbacks of whole genome and exomic
sequencing of primary cancer samples: the need for the matched
DNA to determine whether a mutation is somatic or germ-line
and to eliminate the huge number of private SNPs and copy-
number polymorphisms. Many of the samples in tumor banks do
not have matched germ-line DNA, hence making the evaluation
of novel SNPs and more complex genetic aberrations (e.g., indels)
challenging. However, the completion of the 1000  Genomes
Projects [67,68] will provide a more compete catalogue of germ-line
variations in the general population and aid in the identification
of disease-specific mutations.
Finally, exomic sequencing has also been shown to predict the
microsatellite instability status of colorectal cancers [69] . One
can envisage that similar approaches can also be employed to
identify patients who have deficient homologous recombination
DNA repair defects and may benefit from specific chemotherapy
agents (e.g., bifunctional alkylating agents) and inhibitors of the
poly(ADP) ribose polymerase (PARP).
New advances in exome sequencing will certainly make the
technology more suitable to be applied to the clinical setting.
The reduction in the amount of starting DNA material down to
50 ng will allow the sequencing of samples with limited amounts
of material, including clinical biopsies [70] . Furthermore, tech-
nologies that decrease the target capture area and increase the
multiplexing of patients make exomic sequencing significantly
more affordable and estimates suggest that the price of a single
exome sequence should reduce by a quarter of the current cost to
approximately US$500 within the next 12 months [71] .
RNA sequencing
Second-generation RNA sequencing (RNA-seq) is a power-
ful approach to analyze the expression of mRNA, total RNA
or micro- and noncoding RNAs [72–74] . Typically, RNA is
fragmented and converted into cDNA before the addition of
adaptor molecules for sequencing. Although studies show that
RNA-seq does recapitulate microarray-based measurements to
some extent [75,76] , RNA-seq has several advantages. Given that
there is no required prior knowledge of the transcript sequences
or variants, RNA-seq has a major advantage over microarrays,
and can be used to identify novel transcripts and splice vari-
ants [77] . Expression values are now digital rather than analogue
and are calculated based on the number of reads mapping to
the transcript of interest. This increase in dynamic range is well
beyond that achieved by microarray-based expression analysis and
resembles more accurately the range of transcript frequencies in
the cell. RNA-seq also has the ability to refine transcript struc-
tures, including read-throughs and chimeric/fusion genes [13,14] .
Given the abundance of some of the more ubiquitously expressed
transcripts, such as housekeeping genes, many sequencing reads
can be taken up by such genes, leading to lower coverage for lower
431
 Review Natrajan & Reis-Filho
expressed transcripts that may have biological importance. This
is in contrast to microarrays, where the dynamic range for each
gene is independent from one other. Strategies that can remove
highly abundant transcripts before sequencing will enable a better
characterization of such transcripts [78] .
RNA-sequencing can be used in differential expression analysis
between samples [79] , to look at allele-specific expression and novel
imprinted genes [73,80,81] . Analysis of RNA-seq can also be used
to detect somatic mutations; however, finding a matched normal
sample for disease tissues can be problematic, and given that nor-
mal tissue is unlikely to express genes to the same level, analysis
of somatic variants is challenging. Despite the challenges posed
by the identification of somatic mutations by RNA-seq, this tech-
nique has led to the identification of recurrent mutations in the
FOXL2 gene in granulosa cell tumors of the ovary and ARID1A
mutations in clear-cell and endometrioid ovarian cancers [82–84] . A
survey of ten melanomas by transcriptomic sequencing identified
721 novel nonsynonymous coding variants, including SRRM2 in
30% of the samples [85] . Of note, however, owing to variances in
the expression levels of genes, the authors were unable to identify
the known BRAF mutations in these samples [85] , which high-
lights the limitations of this approach for the identification of
somatic mutations. Combined RNA and DNA sequencing also
has the ability to identify RNA editing events, such as transcript
editing of COG3 and SRP9 genes in a lobular breast cancer [11] .
Next-generation cytogenetics
As well as using DNA sequencing technologies to study complex
rearrangements in cancer, translocations can also be inferred by
using paired-end RNA sequencing. Recurrent fusion genes have
recently been shown to be significantly more pervasive than antic-
ipated in carcinomas. In fact, recurrent fusion genes involving
ETS (erythroblastosis virus E26 transformation-specific) fam-
ily members have been shown to be a common genetic aberra-
tion in prostate cancer [35] , challenging the concept that fusion
events are only a feature in lymphomas, leukemias and sarcomas.
Recent studies have shown that some special types of breast cancer
also harbor recurrent chromosomal translocations, such as the
ETV6–NTRK3 oncogenic fusion gene in secretory carcinomas [86]
and the MYB–NFIB fusion gene in adenoid cystic carcinomas [87] .
Furthermore, recurrent fusion genes have been described in several
types of salivary gland and renal cancers [88–94] . Gene fusions and
other chimeric transcripts have been identified through RNA-seq
in prostate, breast and melanoma cancer primary tumors and cell
lines [13,14,85,95,96] , although the majority of these fusions appear to
be ‘private’ events (i.e., fusion genes only found in the index case).
However, recent studies using paired-end mRNA sequencing have
identified recurrent BRAF and CRAF fusions in melanoma and
gastric cancers, which have been shown to drive the oncogenic
activity of these genes. Furthermore, cancer cells harboring these
fusion events were shown to be particularly sensitive to treatment
with the small molecule inhibitor sorafenib [95] . Although these
fusion events were found in a small subset of tumors, targeting
these events in patients with small molecule inhibitors that are
currently available is a tantalizing prospect. It is possible that once
432
more recurrent fusion genes that bear clinical significance are
identified, commercially available assays will become available for
clinical use. One of the main clinical applications of these private
fusion genes is for the assessment of disease burden and monitor-
ing of residual disease for patients with solid malignancies after
primary treatment [97,98] . The primary challenge for the translation
of these observations into clinical practice lies in the identifica-
tion of recurrent ‘driver’ fusion events that can be targeted with
available drugs.
It is now becoming evident that the majority of fusion events
may in fact be byproducts (i.e., passenger events) of the evolution
of amplifications in cancer genomes, as intra- and inter-chromo-
somal rearrangements are more frequently found in amplified
regions of the genome [7,36,37,85] . Furthermore, next-generation
sequencing has recently demonstrated that some cancers, in par-
ticular bone tumors, may undergo a cellular catastrophe termed
‘chromothripsis’, whereby tens to hundreds of chromosomal
rearrangements involving a few localized genomic regions can be
acquired in a single genetic remodeling event, leading to multiple
oncogenic lesions [99] .
Other applications of next-generation sequencing
MiRNAs have been shown to play a number of important roles
in cancer and disease and recent efforts have begun to focus on
miRNA applications using next-generation sequencing [100–103] .
Recent studies have identified not only differences in the expres-
sion levels of miRNAs in cancer and normal samples, but also
370 novel miRNA species [104] , the miRNA expression profiles of
androgen-independent and -dependent prostate cancers [105] , and
several differentially expressed miRNAs that may contribute to
progression of acute myeloid leukemia [106] . Combined miRNA
and mRNA next-generation sequencing has identified a somatic
mutation in the 3´-UTR of TNFAIP2, a known target of the
PML–RARA oncogene in acute myeloid leukemia, resulting in
translational repression in a Dicer-dependent fashion [107] . These
results suggest that somatic mutations in miRNA binding sites are
another mechanism that can affect gene expression. Furthermore,
paired analysis of small RNAs from samples of normal and tumor-
adjacent breast tissue has identified 361 novel miRNA precursors,
10% of which were located in amplified regions of the genome.
These included a novel miRNA, miR-4728–3p, predicted to medi-
ate GTPase cellular transduction, found to span the 5´ splice site of
intron 24 within the ERBB2 gene, suggesting that its processing
may be dependent on splicing of ERBB2 itself [108] .
Modifications of protocols for massively parallel sequencing also
allow for a complete genome-wide assessment of histone modifica-
tions and related chromatin structures (for an excellent review on
this topic see [109]). Also known as Chip-seq, studies have begun
to look at gene regulation, global binding regions and functional
elements in cancer [110–113] . Studies to date have used this approach
to identify Runx1 as a novel tethering factor in estrogen-receptor-
mediated transcriptional activation in breast cancer [114] , to iden-
tify b-catenin binding regions in HCT116 human colon cancer
cells [115] and to identify novel CDX2 binding sites in intestinal
epithelial cells [116] . In addition to revealing that enzymes that
Expert Rev. Mol. Diagn. 11(4), (2011)
 Next-generation sequencing applied to molecular diagnostics
regulate methylation may be mutated in some types of cancer (e.g.,
DNMT3A mutations have recently been reported in acute myeloid
leukemia [117]), next-generation sequencing can also be used for an
unbiased genome-wide assessment of cytosine methylation sites
by bisulphite treatment or MeDip [118–123] and can be a useful
tool for profiling DNA methylation in clinical samples [124,125] .
A combined methylation profiling with mutational analysis by
means of next-generation sequencing revealed commonly mutated
and methylated genes that are associated with poor prognosis in
cancer patients [126] . Assessment of gene promoter methylation by
barcoded 454 sequencing of DNA from endometrial cancers has
led to the identification of a new class of microsatellite instabil-
ity (MSI+) endometrial cancers with heterogeneous patterns of
MLH1 promoter methylation [127] . The aforementioned approaches
may be used in the future to identify molecular subtypes of cancer
that are defined by distinct constellations of epigenetic defects.
Importantly, methylation markers have been put forward as useful
for both noninvasive early disease detection and disease monitor-
ing [128] . Li et al. have recently provided an elegant proof of prin-
ciple of the potential clinical utility of methylation markers for
the diagnosis and monitoring of colorectal cancer patients [129] .
Based on the observation that exon  1 of the vimentin (VIM)
gene is methyl­ated in colorectal carcinomas, a method for digital
assessment of VIM gene methylation based on ‘methyl-BEAMing’
(Beads, Emulsion, Amplification and Magnetics) and massively
parallel sequencing was devised. Plasma level VIM gene methyla-
tion as determined by methyl-BEAMing was shown to have a far
superior sensitivity and specificity than that offered by the assess-
ment of carcino-embryonic antigen (i.e., the only biomarker for
colorectal cancer that is currently used in clinical practice) for the
diagnosis of early-stage colorectal cancer, and to be more strongly
associated with residual disease than levels of carcino-embryonic
antigen. Methyl-BEAMing VIM gene methylation levels in feces
also showed promise as potential biomarkers for the diagnosis of
colorectal cancer [129] . These studies do provide evidence to suggest
that the assessment of cancer-specific methylation patterns can be
used clinically for disease diagnosis and monitoring after therapy.
Next-generation sequencing technology is quickly becoming the
method of choice for epigenomic profiling, and will lead to a more
comprehensive understanding of the epigenetic contributions to
human disease.
Next-generation sequencing for the identification of
causes of hereditary disease
One of the main applications of massively parallel sequencing is
to study germ-line DNA for genome-wide association studies to
identify mutations that cause rare Mendelian disorders. This is
exemplified by the identification of mutations in SH3TC2 caus-
ing Charcot–Marie–Tooth neuropathy [130] , the identification of
MHY3 germ-line mutations that cause Freeman–Sheldon syndrome
through exomic targeted sequencing of four diseased and eight unre-
lated individuals [17] , and the identification of germ-line mutations
in DHODH that cause Miller syndrome, found through exomic
sequencing of four affected individuals in three independent kin-
dred [16] . Over the past year exome sequencing has identified many
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Review
of the ‘low-hanging fruits’ in a number of rare hereditary disorders
for which no causal gene has been identified (Table 3) . Although
these diseases only affect a small number of patients, these results
have clear clinical implications, as these hereditary diseases can now
be objectively diagnosed. Genes responsible for more complex dis-
eases are now being discovered through targeted sequencing, such
as the identification of nonsense mutations in ANGPTL3 causing
hypolipidemia [131] and GPSM2 mutations in nonsyndromic hear-
ing loss [132] . Larger studies of more common complex phenotypes
have already begun to emerge, such as the identification of a poly-
morphism in EPAS1 in inhabitants of the high-altitude Tibetan
plateau [133] , and the identification of novel nonsynonymous coding
SNPs in a population of 200 individuals from Denmark [134] .
Next-generation sequencing of fetal DNA from the mater-
nal plasma [135] may also constitute a way forward for prenatal
noninvasive diagnosis of fetal genetic disorders. In addition, the
use of massively parallel genomic sequencing of maternal plasma
for the noninvasive prenatal diagnosis of fetal chromosomal aneu-
plodies has also been reported [136] . In particular, two independent
validation studies have looked at the use of multiplexed massively
parallel sequencing for the identification of trisomy 21. Using a
four-plex and duplex approach, in 449 and 713 high-risk pregnant
women, respectively [137,138] , trisomy 21 was detected with 100%
sensitivity and 99.7 and 97.9% specificity, respectively. It is antici-
pated that the development of haplotyping (distinguishing which
mutation is inherited from which parent) using sequencing alone
will be essential in predicting disease risk [139,140] .
Next-generation sequencing in the clinic
Second-generation sequencing and its derivatives have already
begun to be used for the management of cancer patients in proof-
of-concept studies. For instance, a patient with gemcitabine-­
resistant metastatic pancreatic cancer had tumor grown as a
xenograft, which was shown to be sensitive to chemotherapy
agents that cause DNA double strand breaks (i.e., mitomycin C
and cisplatin) [65] . In parallel, the primary tumor was subjected to
exomic sequencing, which revealed the presence of biallelic somatic
inactivation of PALB2. Inactivation of PALB2 has been shown
to lead to homologous recombination DNA repair defects [141] ;
the presence of these mutations is likely to explain the sensitivity
of the xenograft and patient to mitomycin C and cisplatin [65] . A
patient with a metastatic rare form of tongue carcinoma was treated
with sorafenib based on signaling pathways identified through
combined whole genome and transcriptomic sequencing of the
primary tumor [142] . Furthermore, exomic sequencing was suc-
cessfully used in the diagnosis and management of a child with
Crohn’s-like disease, as it led to the identification of a hemizygous
missense mutation in XIAP (a known cause of X-linked lympho­
proliferative syndrome and a risk factor for hemophagocytic lym-
phohistiocytosis). These results enabled the patient to receive an
allogeneic hematopoietic progenitor cell transplant to treat not
only the gastrointestinal symptoms, but also this life-threatening
condition [143] . These studies demonstrate the enormous power of
next-generation sequencing to diagnose and tailor the treatment
to individual patients.
433
 Review Natrajan & Reis-Filho

amplification [148] . Likewise, resistance to

Table 3. Next-generation sequencing studies of Mendelian disorders.
imatinib mesylate in patients with gas-
Study (year) Method Disease type Gene Ref. trointestinal stromal tumors appears to
associated be mediated by the selection of nonmodal
with disease clones harboring secondary KIT gene muta-
Bilguvar et al. (2010) Exome Severe brain WDR62  [200] tions [149,150] . Even in the case of synthetic
malformations lethality-based therapeutic approaches,
Bolze et al. (2010) Exome Autoimmune FADD  [201] clonal selection appears to be the mecha-
lymphoproliferative nism of resistance. Tumors that harbor
syndrome BRCA1 and BRCA2 mutations show exqui-
Bowden et al. (2010) Exome Insulin resistance ADIPOQ  [202] site sensitivity to PARP inhibitors. In vitro
atherosclerosis models have demonstrated that resistance to
Byun et al. (2010) Exome Fatal classic Kaposi STIM1  [203] these agents in BRCA2 mutant pancreatic
sarcoma cell lines is mediated by the selection of can-
Caliskan et al. (2011) Exome Nonsyndromic mental TECR  [204]
cer cells that harbor intragenic deletions or
retardation secondary mutations that remove the initial
truncating mutation and restore the open
Gilissen et al. (2010) Exome Sensenbrenner WDR35  [205]
reading frame of BRCA1 and BRCA2 [151–
syndrome
154] . These results were further corroborated
Haack et al. (2010) Exome Complex I deficiency ACAD9  [206] by the sequencing of recurrences of BRCA1
Hoischen et al. (2010) Exome Schinzel–Giedion SETBP1  [207] or BRCA2 mutant human ovarian cancers
syndrome treated with platinum salts [151–154] . Next-
Johnson et al. (2010) Exome Brown–Vialetto–van C20orf54  [208] generation sequencing-based approaches
Laere syndrome may help expedite the identification of
Johnson et al. (2010) Exome Amyotrophic lateral VCP  [208] mutations that confer resistance to specific
sclerosis therapeutic agents. If primary tumors are
Krawitz et al. (2010) Exome Hyperphosphatasia PIGV  [209]
sequenced at an increased depth, specific
mental retardation mutations in minor populations of the cells
syndrome can be detected, paving the way for more
Lalonde et al. (2010) Exome Fowler syndrome FLVCR2  [210]
accurate therapeutic predictions.
Lupski et al. (2010) Whole Charcot–Marie–Tooth SH3TC2  [130]
Genetic testing using
genome neuropathy
next-generation sequencing
Musunuru et al. (2010) Exome Familial combined ANGPTL3  [131]
Based on the results achieved in the last
hypolipidemia 5  years, one could argue that it is only a
Ng et al. (2009) Exome Freeman–Sheldon MHY3  [17] matter of time before genetic tests based
syndrome on next-generation sequencing or one of its
Ng et al. (2010) Exome Kabuki syndrome MLL2  [15] derivatives are incorporated into the routine
Ng et al. (2010) Exome Miller syndrome DHODH  [16] of genetic clinics. An increasing number of
sequence-based diagnostic tests for many
Otto et al. (2010) Exome Retinal–renal SDCCAG8  [211]
hereditary disorders, including X-linked
ciliopathy
mental retardation, Parkinson’s disease, epi-
Walsh et al. (2010) Exome Nonsyndromic GPSM2  [132] lepsy and mitochondrial disorders, are being
hearing loss (DFNB82)
developed (Table 4) . One strategy to introduce
Wang et al. (2010) Exome Spinocerebellar TGM6  [212] routine genetic testing in clinical laborato-
ataxias ries is the use of ‘tagged’ PCR products from
a pool of patients [155] . This technique uses
There are now several lines of evidence to suggest that a given long-range PCR followed by standard library preparation and has
cancer is composed of mosaic of cells with distinct genetic aber- been developed for routine genetic testing of BRCA1 and TP53
rations in addition to the founder genetic events found in all mutations in hereditary breast cancer and Li-Fraumeni families,
cancer cells (see previously), and that this intra-tumor genetic where it has been shown to be more sensitive than conventional
heterogeneity may mediate resistance to systemic therapies. Sanger methods [156] . This approach may be of particular inter-
Resistance to EGFR small molecule inhibitors is to some extent est for screening a panel of breast cancer susceptibility genes in
mediated by selection of nonmodal resistant clones harboring families with non-BRCA1/BRCA2 hereditary breast cancer [157,158] .
the T790M EGFR ‘gatekeeper’ mutation [144–147] or MET gene Automated and scalable methods to capture specific ‘exomes’, such

434 Expert Rev. Mol. Diagn. 11(4), (2011)

 Next-generation sequencing applied to molecular diagnostics Review

Table 4. Summary of diagnostic tests using next-generation sequencing.

Company Genetic test Sequencing Platform Ref.
Emory University X-linked intellectual disability, congenital muscular Targeted capture ABI SOLID  [314]
dystrophy and congenital disorders of glycosylation
Ambry Genetics Ambry Screen (covers several hundred well- Barcoded PCR Illumina  [315]
understood childhood disease-causing mutations) Genome
Analyzer
German sequencing service 28 different panels, including epilepsy, amyotrophic Targeted capture ABI SOLID  [316]
provider (CeGaT) lateral sclerosis, Parkinson’s, metabolic disorders and
hereditary eye diseases
National Centre for 448 genetic diseases, a mix of autosomal recessive and Targeted capture Illumina Hiseq™  [317]
Genome Resources X-linked recessive disorders
Good Start Genetics Prepregnancy genetic tests Targeted capture Illumina  [318]
Genome
Analyzer
GeneDx 20 genetic diseases including mitochondrial disorders, Targeted capture ABI SOLID  [319]
cystic fibrosis, sickle cell anemia and Tay-Sachs disease
University of Leeds Institute BRCA1/2 and TP53 hereditary breast, ovarian cancer Barcoded PCR Illumina  [320]
of Molecular Medicine and Li-Fraumeni syndrome Genome
Analyzer

as a 480 Kb cancer-exome subset of 115 cancer-related genes, has
been developed using microfluidic DNA arrays [71] . The develop-
ment and commercialization of these tests (Table 4) is remarkable,
especially if one considers the time it has taken for microarray-based
tests to be incorporated into clinical practice.
Third-generation sequencing technologies
Second-generation sequencing, whilst identifying many novel
mutations and translocations that are of clinical benefit in cancer,
can only detect clonal mutations in a tumor. There is now direct
evidence of the genetic heterogeneity within tumors (i.e., that
tumors are composed of a mosaic of nonmodal populations harbor-
ing genetic aberrations in addition to the initiating events) [159,160]
and recurrences may be caused by the outgrowth of nonmodal
populations of cancer cells that harbor specific genetic aberra-
tions [12,148,161] . The development of third-­generation sequencing
technologies can circumvent this limitation of second-generation
platforms by the use of single-molecule sequencing (which avoids
PCR-based amplification bias) that promises to improve data qual-
ity and increase the types of data produced. A number of third-
generation sequencing technologies are currently in development
(Table 5 ; for a detailed description of these technologies, the readers
are referred to references [32,162]). The Helicos Heliscope [301] was
the first third-generation sequencer to be commercialized. The
Helicos true single-molecule sequencing technology uses labeled
nucleotides admixed with nucleic acid templates that are immobi-
lized on a flow cell. Fluorescent signals are emitted in real time as
each additional base is incorporated and detected by the HeliScope
Genetic Analysis System. This system can sequence up to 180 mb/h
with an average read length of 35 bases [163–165] . The Helicos true
single-molecule sequencing technology allows whole-genome
sequencing, targeted resequencing, digital gene expression, DNA
barcoding, mRNA-seq, Chip-seq and protein quantification, and
has digital copy number and small RNA sequencing applications
under development [164–172] . Pacific Biosciences [302] have developed
the single-molecule real-time (SMRT) technology (PACBIO RS)
that incorporates thousands of zero-mode waveguides (ZMWs)
on SMRT cells to which a single DNA polymerase molecule is
immobilized. This allows the instrument to detect the addition
of each individual fluorophore-labeled phospho-linked nucleotide
in real time [173,174] . This technology has the advantage of produc-
ing very long read lengths of >1000 bp up to 10,000 bp and short
run times (i.e., between 30 min to 1 h). Owing to the sample
preparation procedure [174] , SMRT sequencing is suitable for a
number of applications that are currently not available with other
technologies, such as the ability to capture kinetic information,
the incorporation of methylated residues [175] and real time transi-
tion of tRNAs on a single ribosome as it translates mRNA [176] .
Oxford Nanopore Technologies [303] are currently in the process
of developing an electronic-based single-molecule sequencing tech-
nology, which measures the current change as a DNA polymerase
molecule passes through a nanopore, identifying the DNA base
on the molecule in sequence [177–179] . This enables single strands of
DNA to be sequenced and assessment of the incorporated bases by
electrics rather than optics. This novel approach is highly scalable,
with negligible sequencing costs and provides simpler data process-
ing and storage. In addition to the applications currently feasible
with second-generation platforms, Nanopore sequencing technol-
ogy can be used for numerous other applications, including the
identification of epigenetic alterations [180] and protein analysis [181] .
The benchtop Ion Torrent PGM [304] sequencer, which has
already been commercialized, uses a series of semiconductor sen-
sors on a single chip that measure hydrogen ions that are pro-
duced by the process of DNA replication (i.e., proton sequenc-
ing). The ion semiconductor sequencing chip is the machine,
which houses a high-density array of wells, providing millions

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 Review Natrajan & Reis-Filho

Table 5. Summary of third-generation sequencing technologies.

Platform Company TGS Read Run time Applications Ref.
technology length
HeliScope Helicos tSMS 35 bp 8 days Whole-genome sequencing,  [321]
targeted resequencing, digital gene
expression, DNA barcoding,
mRNA-seq, Chip-seq, protein
quantification
PacBio RS Pacific SMRT zero-mode 1000– 1h Single-molecule RNA, DNA  [322]
Biosciences waveguides 10,000 bp sequencing, identification of
epigenetic alterations, RNA
modifications, mRNA translation
Ion Torrent PGM Life Technologies Semiconductor 100–200 bp 2h Targeted DNA sequencing  [323]
Oxford Nanopore Oxford Nanopore Exonuclease DNA 50,000 bp 24 h Single-molecule RNA, DNA  [324]
Technologies sequencing sequencing
Life Technologies Life Technologies Qdot® >100 kb 20 min Single-molecule RNA, DNA  [325]
FRET nanocrystals and sequencing
FRET
FRET: Förster resonance energy transfer; PGM: Personal genome machine; Qdot ®: Quantum dot; SMRT: Single-molecule real time; TGS: Third-generation sequencing;
tSMS: True single-molecule sequencing.

of individual reactors allowing sequencing runs to be performed involves quantification of reads, mutation calling, copy number
in approximately 2 h that generate an ‘ionogram’, which is con- analysis and analysis of structural variations. A plethora of algo-
verted into DNA base calls by the software provided with the rithms have been developed for this purpose (for recent reviews
PGM sequencer. At present, this technology is only suitable for on this topic, see [49,185,186]), including a number of open-source
targeted sequencing and still requires PCR amplification of the packages in Bioconductor [306] . One of the difficulties for the trans-
DNA template and termination in order to monitor base incor- lation of bioinformatic analysis into the clinical setting is that the
poration. Given that the Ion Torrent PGM is relatively cheap and majority of these algorithms need some programming expertise and
provides a complete workflow from sample preparation to sample specialized servers to handle and store all the data. Commercial
analysis, it is envisaged that many clinical laboratories will take software that allows for the analysis of next-generation sequenc-
advantage of the technology for routine resequencing. ing data are available (e.g., NextGene®  [307] , Genesifter ® [308] ,
Another approach to SMRT sequencing uses fluorescence reso- GenomeQuest [309] , and DNAnexus [310]); however, these can be
nance energy transfer (FRET). This technology is being devel- relatively expensive and are relatively limited in the scope of analy-
oped by Life Technologies [305] , and uses a Qdot nanocrystal as the ses they can perform. Freely available visualization tools, such as
FRET donor coupled to a DNA polymerase enzyme. Introduction the Broad’s Integrative Genomics Viewer (IGV) [311] , have recently
of a nucleotide interrupts the Qdot fluorescence and energy is trans- been released and are relatively user friendly.
ferred, leading to the release of the fluorophore label on the nucle- Most researchers align the next-generation sequencing reads to
otide. This technology, while still in its infancy, has the potential a known reference, such as the most current build of the genome,
to sequence millions of bases per second, with a run taking less employing known SNP databases to filter out alterations that
than 20 min and generating hundreds of megabases of sequence. are germ-line polymorphisms. As the 1000 Genomes Project
progresses, a catalogue of germ-line variants will be available to help
Challenges for next-generation sequencing filter out such SNPs [67,68] . In the analysis of Mendelian diseases,
One of the main challenges facing the implementation of next-­ use of unaffected family members provides a useful background
generation sequencing into clinical practice is the bioinformatic in which to identify novel disease-causing mutations. However,
analysis and data storage. The most computationally intensive in cancer research one must also sequence the normal germ-line
part of the basic bioinformatic pipeline is the conversion of the DNA to reliably only look at somatic variations. Somatic mutation
image data into sequence reads, a step called ‘base calling’. This calling in cancer is much more complicated than germ-line due
generates FASTQ files, which are usually obtained using platform- to the variance in ploidy and purity of tumor DNA, and is also
specific pipelines. Once the sequence reads have been generated, strongly dependent on the allelic fraction [49] . Elimination of false-
they are aligned to a known reference sequence or assembled positives and -negatives is also a major challenge in next-generation
de novo [182–184] . The presence of short reads in massively parallel sequencing analysis. Elimination of false-positives can be achieved
sequencing makes alignment more difficult than with conventional through validation by conventional PCR and Sanger sequencing
Sanger sequencing, with the choice of method depending on the and now in a more high-throughput fashion through the use of
sequencing platform used, data type and computational resources. Sequenom massARRAY technology [187] ; however, elimination of
Depending on the application of the sequencing, the next step false-negatives is much more difficult.

436 Expert Rev. Mol. Diagn. 11(4), (2011)

 Next-generation sequencing applied to molecular diagnostics
Typically a single sequencing run can generate up to 15 tera-
bytes of information and we are currently in a climate where we
are generating more sequencing data than we have storage for.
The next-generation sequencing data deluge is one of the chal-
lenges that are yet to be met. Given that image files require the
most storage capacity, many researchers are opting to delete these
files once base calling has been achieved and storing FASTQ
or just the post-alignment files. Clinical laboratories will have
to implement an efficient way of tracking data and keeping the
necessary files to go back to if needed. One way of overcoming
the need to purchase expensive servers is to use cloud computing.
In fact both GenomeQuest and DNAnexus have cloud-based
pipelines, eliminating the need for dedicated computer hardware
and software.
Albeit challenging, assigning reliable mutation calls, insertions/
deletions and structural variations is only a small part of the story.
The main challenge lies in the distinction of what constitutes a
‘driver’ mutation event versus a ‘passenger’ event (i.e., has no bio-
logical significance on the cell harboring its mutation at a given
point in time). This is particularly true for complex heterogene-
ous passenger events. There are a number of algorithms that exist
that predict the functional effect of a mutation of interest on a
protein (e.g., SIFT, PolyPhen, CanPredict and CHASM) [188–191]
that can be used to prioritize mutations for follow-up studies,
and more recently algorithms that predict the pathogenicity of
somatic mutations based on the selection pressure and type of
mutation have been developed [192,193] . However, novel predicted
‘drivers’ still need to be functionally investigated in appropriate
model systems before they can be definitively defined as driver
events. However, these approaches do not take into account the
issue of intra-tumor genetic heterogeneity. Given that sequence
reads represent an average of all cells within a tumor, rare muta-
tions that predict resistance to therapy (e.g., secondary KIT
mutations and EGFR T790M mutations) but are at very low
prevalence within the tumor (e.g., 1 or 0.1%) may be missed
by current approaches. This is particularly problematic if the
prevalence of the mutation within the tumor population is simi-
lar to the frequency of sequencing error rates of the sequencing
technology employed. This in part may be overcome through the
development of increasingly sensitive techniques that will allow
sequencing of single cells with third-generation single-molecule
sequencing technologies.
Ethical considerations
Massively parallel next-generation sequencing is already enter-
ing clinical practice, and with it raises important ethical con-
siderations, that need to be fully resolved before routine imple-
mentation, in particular with whole genome/exome sequencing
of patients DNA as these techniques produce huge amounts of
personal medical information. Clarity, in terms of how to ensure
the appropriate storage and anonymity of the data from patients,
has been provided by the ethics committees of the International
Cancer Genome Consortium (ICGC) [312] and The Cancer
Genome Atlas TCGA [313] . In the case of a patient who requests
to have their whole genome sequenced because of a family history
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Review
of a given genetic disorder, there is a great likelihood of the dis-
covery of other risk factors affecting their health. In addition,
variants of unknown significance are inevitably going to cause
the patient anxiety and stress. Therefore, several questions are
crucial: how much of the sequence information should be offered
to the patient? How should the results from minors be handled,
in particular carriers of adult-onset diseases? How will we han-
dle genetic discrimination? It may be the case that consent will
have to be completely different for whole-genome sequencing
than for gene locus-specific sequencing tests. This may involve
patients having to receive counseling before consent is agreed
and also throughout the testing and analysis process. For an in-
depth discussion of the ethical considerations of next-generation
sequencing, the readers are referred to recent reviews on this
topic [22,25,27,194] .
Expert commentary
It is arguably inevitable that improvements in sequencing tech-
nologies as we head towards third-generation sequencing, and
a reduction in sequencing costs will allow the eagerly antici-
pated US$1000 genome to become a reality. Large-scale projects
aimed at sequencing more individuals, such as the 1000 Genomes
Project [195] and the Personal Genome Project [196] will lead to a
greater understanding of the landscape of the human genome. In
tandem, the advent of initiatives to identify somatic alterations
in cancer, such as TCGA [197] and ICGC [198] , hold the excit-
ing prospect of revealing a number of key oncogenic mutations
that define clinically relevant subtypes for the development of
new cancer therapies [198] . The contribution of next-generation
sequencing in diagnosis and treatment decision has already been
illustrated in proof-of-principle studies. It is anticipated that fur-
ther studies comprising a larger number of samples will provide
increasingly more coherent data and potentially pave the way for
the next generation of diagnostic genetic tests.
Five-year view
Next-generation sequencing technologies are advancing at an
unprecedented speed. It is envisaged that 5  years from now,
sequencing of a person’s DNA will be routine clinical practice
for the diagnosis of rare and complex hereditary diseases. Targeted
sequencing of therapeutically relevant mutations will become part
of the diagnostic repertoire of molecular pathology laboratories for
the management of cancer patients. Furthermore, genome-wide
or targeted sequencing of cancers may be introduced at least in
the context of clinical trials designed to determine the mecha-
nisms of resistance to specific therapeutic agents and to develop
predictive markers.
However, it should be noted that numerous challenges lie
ahead. Despite the decreasing cost of sequencing, genetic associa-
tion studies will require incredibly large sample sizes and innova-
tive statistical approaches. Studies designed to define next-gener-
ation sequencing-based predictive markers for cancer therapeutics
will not only require large sample sizes, but also a much greater
understanding of the sources of biases stemming from these
approaches. Studies published so far are little more than proofs of
437
 Review Natrajan & Reis-Filho

principle; thorough analyses to determine the generalizability of Acknowledgements

their findings are eagerly awaited. The bioinformatic challenges
posed by the output of next-generation sequencers are orders of
magnitude greater than those posed by the analysis microarrays in
the last decade. Although bioinformatics and statistical analysis
methods for the analysis of next-generation sequencing data are
being developed at an unprecedented pace, further standard­
ization of bioinformatic algorithms/tools for data analysis will be
required to translate the output from sequencers into benefit for
patients. Data sharing to maximize the output of next-generation
sequencing studies needs to be facilitated and embraced by the
academic community. Finally, ethical issues will also require
careful consideration.
The authors would like to thank Iwanka Kozarewa for her
helpful discussions.
Financial & competing interests disclosure
This work was supported by The Breakthrough Breast Cancer charity. Rachael
Natrajan and Jorge S Reis-Filho are funded in part by Breakthrough Breast
Cancer. Jorge S Reis-Filho is the recipient of the 2010 CRUK Future Leaders
Prize. The authors have no other relevant affiliations or financial involvement
with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the manuscript apart
from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Key issues
• Next-generation sequencing technologies have uncovered a wide range of genetic aberrations that contribute to cancer development
and progression.
• Challenges lie in the identification of ‘driver’ versus ‘passenger’ events.
• Exomic sequencing has begun to emerge as a tool of choice for the identification of the causes of rare hereditary disorders.
• Next-generation sequencing data require an availability of high-performance computing and bioinformatic support that is beyond most
research laboratories.
• Issues of quality control and standardization of protocols need to be addressed before routine clinical implementation.
• The ethical aspects need to be carefully considered by the community before next-generation sequencing can be applied to large
populations of patients.

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