abstract

Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through
advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing
reaction has pro- foundly increased the total number of produced sequence reads per run. Current
sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA
samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of
biomedical research. In this chapter we review the technical background of the different commercially
available NGS platforms with respect to template generation and the sequencing reaction and take a small
step towards what the upcoming NGS technologies will bring. We close with an overview of different
implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From
Genome to Function.

1. Introduction
2. Sequence library preparation
The growing power and reducing cost sparked an enormous
range of applications of Next generation sequencing (NGS) All currently available sequencing platforms require some level
technology. Gradual- ly, sequencing is starting to become the of DNA pre-processing into a library suitable for sequencing. In
standard technology to apply, certainly at the first step where the general, these steps involve shearing of high molecular weight DNA
main question is “what's all involved”, “what's the basis”. It should into an ap- propriate platform-specific size range, followed by an
be realized that for many appli- cations sequencing would always end polishing step to generate blunt ended DNA fragments. Specific
have been the method of choice, yet it was science-fiction, adapters are ligat- ed to these fragments by either A/T overhang or
technically unthinkable and later possible but far too costly. We direct blunt ligation. A functional library requires having specific
perform genome-wide association studies (GWAS) using SNP- adapter sequences to be added to the 3′ and 5′ ends. Each of the
arrays simply because we cannot afford to perform whole- genome sequence platforms uses a differ- ent set of unique adapter
sequencing in ten-thousands of individuals. This is changing sequences to be compatible with the further steps of the process
rapidly and sequencing will become our molecular microscope, the (Fig. 1).
tool to get a first look. Although replication, transcription, Following adapter ligation Life Technologies (Solid, PGM,
translation, methylation and nuclear DNA folding are completely Proton) libraries require a nick translation step to get functional
different process- es, they can all be studied using sequencing. molecules while for the other technologies the sample is in principle
An important advantage of sequence data is its quality, robustness ready for loading immediately after ligation. One may then choose
and low noise. It should be noted that a successful NGS project requires to sequence these libraries directly as amplification free libraries or
expertise both at the wet lab as well as the bioinformatics side in order introduce a pre-amplification step prior to sequencing. It is
to warrant high quality data and data interpretation. The sequence itself important to realize that any step during pre-processing which
is hard evidence of its correctness. A sequencing system will not pro- involves amplification of the molecules [1] or which has been shown
duce “random” sequences and when it does this becomes evident im- to be sequence biased, like ligations [2], will impose a selection on
mediately from QC calls obtained from spike-in controls. Furthermore molecules that end up in the sequenceable libraries.
random sequences will have no match and can be easily discarded
3. Current sequencing technology

during data analysis and when their number exceeds a certain The different sequence platform vendors have devised different
threshold it is evident that there is a serious problem somewhere strategies to prepare the sequence libraries into suitable templates
in the study. as
 The final step is to remove one strand of the dsDNA fragments
using the cleavable site in the surface oligo (Fig. 1A; step 6) and to
block all 3′ ends with ddNTP to prevent the otherwise open 3′ ends
to act as sequencing primer sites on adjacent library molecules [4].
With optimal loading of library molecules one flow-cell lane will
yield approximately 800–1000 K clusters per mm2. Optimal
amounts depend not only on the concentration of the library, but
also on the length of the molecules. Short molecules yield clusters
with a small area that are denser and therefore generate more
intense signals. Loading a wide fragment size distribution will
generate clusters varying widely in size and signal strength which
may impair the number of passing filter reads.
Bridge amplification is not a very efficient method for clonal ampli-
fication, i.e., the 35 cycles of isothermal amplification yield a mere
~ 1000 copies of the initial molecule. Moreover, there will be predomi-
nantly outward growth of the clusters, there is a high probability of
the template strands to re-hybridize instead of annealing to a new prim-
er site on the glass surface and there is both an upper and a lower limit
to the length of the template molecules that can be reliably amplified. In
addition, DNA polymerases, which are known to have biases towards
specific DNA templates are used during the amplification processes.
The bridge amplification scheme that Illumina exploits yields a high
number of clusters, i.e., with good loading of the flow cell, the total
num- ber of reads generated per Hiseq2000 lane may reach ~ 180
million. With a paired-end 2 × 100 bp read format the total output of
Fig. 1. Structure of sequence library molecules for the different technologies. Linear one flow-cell lane is up to ~ 36 Gb. A full run of 2 flow cells sequencing
library molecules (Panel A) contain different adapter sequences at the 5′ [A] and 3′
in parallel may yield ~ 600 Gb of data.
[B] ends of the library inserts. Circular library molecules (Panel B) contain identical
adapter molecules at both ends of the insert. During sequencing, the polonies on the flow cell are read one nucle-
otide at a time in repetitive cycles. During these cycles, fluorescently la-
well as to detect the signal and ultimately read the DNA sequence. beled dNTPs are incorporated into the growing DNA chain. Each of the
For the Illumina, Solid, PGM and 454 systems a local clonal four dNTP species (A, C, T, G) has a single different fluorescent label
amplification of the initial template molecules into polonies [3] is which serves to identify the base and act as a reversible terminator
required to increase the signal-to-noise ratio because the systems to prevent multiple extension events. After imaging the fluorescent
are not sensitive enough to detect the extension of one base at the group is cleaved off, the reversible terminator is de-activated and the
individual DNA template mol- ecule level. On the other hand, the template strands are ready for the next incorporation cycle. The se-
Heliscope and PacBio SMRT systems do not need any pre- quence is read by following the fluorescent signal per extension step
amplification steps as these systems are sensitive enough to detect for each cluster. Under ideal circumstances, all bases within a cluster
individual single molecule template extensions. The different will be extended in phase. However, a small portion of the molecules
strategies to generate the sequence reads also lead to dif- ferences in do not extend properly and fall either behind (phasing) or advance a
the output capacity for the different platforms (Table 1). Below we base (pre-phasing). Over many cycles, these errors will accumulate
will focus on the newer sequencing platforms, being the Illumina, and decrease the signal to noise ratio per cluster, causing a decrease in
LifeTechnologies Semiconductor sequencing and PacBio. Other quality towards the ends of the reads.
older platforms will briefly be discussed in Online Supplement 1. The cycle time for the HiSeq2000 is approximately 1 h. The
major contributor is the imaging of the flow-cell. The enzymatic
reactions take very little time at all. By reducing the imaging time,
3.1. Illumina technology the whole sequencing process can be sped up considerably. This is
implemented in the miSeq and Hiseq2500 platforms by providing
All of the enzymatic processes and imaging steps of the Illumina the option to decrease the total surface area to be imaged. In rapid
tech- nology take place in a flow cell. Depending on the specific mode, cycle time can thereby be reduced to 5 and 10 min for the
Illumina platform it may be partitioned into 1 (miSeq), 2 miSeq and Hiseq 2500, respectively. Furthermore, with optimized
(HiSeq2500) or 8 (HiSeq2000, HiSeq2500) separate lanes. The reagent kits for these short cycle times it is possible to achieve a 2 ×
Illumina platform uses bridge amplification for polony generation 300 bp paired end run on the miSeq, with 85% of data points
and a sequencing by synthesis (SBS) approach (Fig. 2A). Forward above Q30 and run times of
and reverse oligos for amplification (one with a cleavable site), ~ 65 h. However, the increased sequencing speed does come at a price.
complementary to the adapter sequences in- troduced during the With the decreased surface area, the total number of data points that
library preparation steps, are attached to the entire inside surface of can be generated per run will reduce, increasing sequencing cost per
the flow cell lanes. The first step for loading the library onto the flow- nucleotide significantly.
cell is denaturation of the dsDNA fragments into individual ssDNA Early 2014, Illumina has announced the release of two new
molecules. When on the flow-cell, these hybridize to the oligo nu- sequencer models, i.e., the NextSeq 500 and the HiSeq X Ten. The
cleotides on the surface (Fig. 1A; step 1) which are used as primers former system was designed to be a highly flexible, smaller version of
to form an initial copy of the individual sequencing template the HiSeq2500, provid- ing both medium (40 Gb) and a high output
molecule (Fig. 1A; step 2). The initial library molecules are removed (120 Gb) modes both with run times under 30 h. The HiSeq X Ten
and the copied, flow cell-attached fragments are used to generate a was designed for one main purpose: enabling whole human genome
cluster of identical template molecules using isothermal sequencing and reaching the
amplification. This is done through cyclic alternations of three specific $1000 genome in run costs. The main advancement enabling this is
buffers that mediate the denaturation, annealing and extension steps the introduction of the patterned flowcells. In contrast to the spatial
at 60 °C. During these steps the 3′ end of the copied library molecules random cluster generation of the HiSeq and MiSeq flowcells, the X
can hybridize to the complementary oligos on the flow cell, thus Ten flowcells contain a pre-formatted grid of nano-wells, which
forming a bridge structure (Fig. 1A; steps 3–5). each can produce
 one sequence polony. This allows for optimized cluster densities and

Two flowcells in parallel 16 SMRT cells

in combination with faster scanning protocols, specifically tailored

High output mode

software and new sequencing chemistry this system can produce
~600 Gb of data in a single day or ~1.8 terabases within three days,

Ion318 Chip IonP1 chip

which is enough for 5 and 15 whole human genomes, respectively.

Remarks
Output per run 3.2. Ion torrent technology

For the PGM/Proton sequence platforms the sequence

templates are generated on a bead or sphere via emulsion PCR
(emPCR) [5,6]. An oil–water emulsion is created to partition small
reaction vesicles that each ideally contains one sphere, one library
molecule and all the re- agents needed for amplification. Two
# reads per run

primers that are complementary to the sequence library adapters

are present, but one is only present in solution while the other is
bound to the sphere. This serves to select for the library molecules
with both an A and a B adapter while excluding those molecules with
two A or B adapters from loading on the beads dur- ing emPCR (Fig.
Read length (bp)

2B). In addition, this ensures a uniform orientation of the sequence

library molecules on the sphere. During the emPCR steps, indi- vidual
library molecules get amplified to millions of identical copies that are
bound to the beads to allow ultimate detection of the signal.
Although one emPCR reaction can generate billions of templated
spheres, some aspects inherent to the emPCR method, in addition to
700

the general biases during PCR amplification, prevent optimal output.

Due to the double Poisson distribution behavior, it is impossible to
Run time

achieve optimal loading of one library molecule into all individual vesi-
cles. In fact 1/3 of the vesicles will have the one molecule to one vesicle
ratio, the remaining 2/3 will be either without a molecule or have more
Run types

than one. In addition, breaking of the emulsion and recovery of the

spheres are inefficient even with the latest automated systems. In the
final step spheres containing amplified DNA are selected in an enrich-
end Single end

ment step from empty spheres and the loaded spheres are deposited
into the sequencing chip.
The Ion torrent chip consists of a flow compartment and solid state
Detection

pH sensor micro-arrayed wells that are manufactured using

processes built on standard CMOS technology. The detection of the
end Single

incorporated bases during sequencing is not based on imaging of

fluorescent signals but on the release of an H+ during extension of
release

each nucleotide. The release of H+ is detected as a change in the

end Single

pH within the sensor wells. Due to the lack of the time consuming
SingleProton

imaging a sequencing run can be completed within 4 h. Since there

is no detectable difference for H+ released from an A, C, G or T
platforms. detection Pyrophosphate detection

bases, the individual dNTPs are applied in multiple cycles of

consecutive order. If upon delivery of a dNTP no change in pH is
detected in a specific well, that nucleotide is not present in the
template at the next available position. Alternatively, if a change in
System Ligation Synthesis Synthesis

pH is detected, that base is in the template. In contrast to the

Illumina's SBS method the dNTPs used are not blocked and when
the template contains a series of a nucleotide after each other (a
homo- polymer stretch), the entire stretch of identical bases will be
Table 1

extended, leading to an accordingly stronger pH change which is

Pyrophosphate

directly propor- tional to the number of identical bases incorporated.

Relative to a single ‘A’ a stretch of ‘AA’ will give a 2 fold increase in
the pH, while an ‘AAA’ template will yield a 1.5 fold (3/2) increase
Synthesis

in pH relative to ‘AA’ and for 6 vs. 5 identical bases this relative

Summary of the output per sequencing technology

increase is just 1.2 fold. This decrease of the relative increase of the
GS Junior

change in pH as the homopoly- mer length increases reduces the

Synthesis

probability by which a homopolymer region is called correctly.

The dNTPs are added in a predefined flow order. At the first release
XL+

of the system, this order was a repetitive T–A–C–G sequence. As

Synthesis
FLX Titanium

with Illumina sequencing, not all of the template molecules on a

templated sphere get extended in perfect synchrony. On average
Pacific biosciences Helicos
Synthesis

LifeTechnologies

0.5–1% of the molecules deviate from the flow either because they
Illumina/solexa

lag behind due to improper extension or they advance ahead due to

Roche GS

carry over of dNTPs from a previous cycle. In order to minimize

this de-phasing, the flow order was changed to a more sophisticated
sequence that incorporated A–T–A catch-up type flows. This scheme
allows incomplete extension of
 Fig. 2. Template generation via bridge amplification (A) or emPCR (B).

the A nucleotide to catch up after the T base. Although this does come at a
those of the above mentioned sequencers. First of all, the technology
cost of decreased overall read length, the overall quality of the read does
works with single molecule detection, i.e., the optics used are sensitive
improve. Still, the quality of the reads gradually decreases towards the
enough to detect incorporation of one fluorescently labeled nucleotide.
ends of the reads. By taking into account the flow order, it is possible to
Consequently, template preparation does not require any amplification
make flow-aware base caller algorithms and flow-space aware aligner
steps, and the prepared library molecule is the sequencing template. Li-
software and variant detection tools that take the actual flow order into
brary preparation is similar to shotgun libraries for the other platforms,
account when processing the data in order to generate higher accuracy
i.e., fragmentation of the gDNA to the required size, here multi-kb size,
data [7,8]. The present error rate for substitutions is ~ 0.1% [8] which
followed by end repair and either A/T overhang or direct blunt adapter
is very similar to that of the Illumina systems. The main point of criticism
ligation. The main difference is that the adapters have a hairpin struc-
the system endures is the homo-polymer errors. Despite many improve-
ture (SMRT loop adapters) so that after ligation the dsDNA fragments
ments the 5-mer homo-polymer error rate is still at 3.5% [8].
will have become circular (Fig. 1B). Pre-preparation of the sequencing
Since the initial release of the Ion torrent platform, this technology
template consists of annealing a sequencing primer to the ssDNA region
has evolved at a very rapid pace. The output specs of the first Ion-
of the SMRT loop adapters, followed by binding of the DNA polymerase
314 chip were a mere 10 Mb. Through increasing the total surface
to form the active polymerization complex. A combination of insert size
area of the chips and the sensor well density, all on the 350 nm
and read length will determine whether a short molecule is read several
CMOS technology, in addition to increasing the average read length
times (CCS or consensus circular sequencing) or a long molecule only
from 100 up to 400 bp, the newest Ion-318 chips produce ~ 1 Gb.
once.
For the Ion Proton System 110 nm CMOS technology was used to
The standard PacBio DNA library preparation starting amounts can
manufacture the Proton-I chips. The diameter of the spheres and
be as high as 1–5 μg total genomic DNA or 0.5–1.0 μg sheared and
the sensor wells decreased which allowed the number of wells to
size selected DNA fragments for 10 kb libraries. These numbers are
increase to ~ 165 million per chip. The Proton-I chips currently
unex- pectedly high for a system that reads single DNA molecules.
yield 60–80 million reads per run, reaching 10 Gb. This is enough to
These high input requirements may limit the use of the PacBio
sequence two human exomes at ~50× coverage. The announced
system for low input applications such as ChIP-Seq or single-cell
Proton-II chip will have 4× the number of sensor wells, with an
genomics. The bottleneck lies at the stringent XP bead clean-up
expected output of ~ 32 Gb per chip promising to generate a whole
steps and Exo III and VII treatments during library preparation to
human genome at ~ 10× coverage, still within the 4 h run time. This
exclude short and/or non-circular fragments from the final library
output puts it at par with a paired-end run on single HiSeq2000 lane.
pool. A lot of sample is lost during these steps, but not taking these
Life Technologies have developed an alternative method to generate
measures which would lead to suboptimal output of the runs.
polonies called Wildfire [9]. The process generates clusters on a Alternative methods have been explored that bypass these stringent
solid surface using isothermal amplification without denaturation or library preparation steps and immediately proceed to sequencing the
amplifi- cation cycles. Although initially designed for the Solid 5500 unprocessed DNA template molecules either by specifically primed or
system, it is likely that this method could be applied to the Ion random hexamer primed sequencing on plas- mid DNA. Although the
torrent semicon- ductor sequencing as well. This may involve output of the SMRT cells was far below standard specs, Coupland et
spheres as an intermediate carrier or clusters may be generated al. were able to sequence the entire M13mp18 ge- nome at average
directly into the sensor wells. A Proton-III chip has been 56× coverage using as little as 3.1 ng input DNA [10]. These
announced that will double the number of wells to 1.2 billion, experiments demonstrated that it is possible to directly sequence
leading to an expected output of ~ 64 Gb per run. small single- and double-stranded DNA genomes without the need
With these output levels, the Ion Proton will become a competitor for any DNA hungry library preparation steps.
to the current Illumina HiSeq systems. The sequencing reaction takes place at the bottom of the ~ 150,000
zero-mode waveguide (ZMW) wells [11] on a SMRT cell. These ZMW
3.3. Pacific biosciences technology are small reaction wells that each ideally contains one complex consisting
of template molecule, sequencing primer and DNA polymerase bound to
The principles underlying the pacific biosciences single molecule the bottom of the ZMW [12]. Unlike the Illumina and Life Technologies
real-time (SMRT) sequencing technology are quite different from
platforms, PacBio does not rely on interrupted cycles of extension and im-
the sequencing reaction. In the C3 chemistry, photo-protected
aging to read the template strand. Instead, the fluorescent signals of the
nucleo- tide analogs are used which shield the polymerase from
extended nucleotides are recorded in real time at 75 frames per second
damage and consequently, half of the reads are over 8–10 kb in
for the individual ZMWs. This is achieved by a powerful optical system
length when using the P5 enzyme (Fig. 3). With the current max.
that illuminates the individual ZMWs with red and green laser beamlets
movie length of 180 min, read lengths of over 40,000 bases have
from the bottom of the SMRT cell and a parallel confocal recording system
been reported. With these specifications the output per SMRT cell
to detect the signal from the fluorescent nucleotides [13]. The width of
reaches ~ 400 Mb for the PacBio RSII.
the ZMWs is chosen in relation to the laser wavelength such that the
One much debated aspect of the PacBio data is the high single pass
light cannot pass through the ZMWs but a zeptoliter sized illumination
error rate of 10–15%, the majority of which are insertion/deletion errors
zone is formed at the bottom of the ZMWs where the active polymerase
with only a small fraction of miscalls. It is important to keep in mind
complex is bound.
that in contrast to the other sequencing technologies, the errors in the
As with the Illumina platform, the four nucleotide species are
PacBio reads are randomly distributed and do not occur more
labeled with a different fluorescent label. However, a crucial
frequently towards the end of the reads. This property can be used to
difference is that the label for the PacBio system is terminally
create con- sensus calls from information of multiple reads covering
phospholinked nucleotides, meaning the label is cleaved off during
each reference position. Consensus accuracy can reach over 99.999%
strand extension. In addition, the nucleotides do not contain a
by using the PacBio Quiver software [17]. Although this does require
terminator group allowing continuous extension of the growing
a coverage of
DNA copy. When a nucleotide complementary to the template is
~ 40× per base, these levels can easily be reached with the output
bound in position by the polymerase within the illu- mination zone
of a few SMRT cells for small (bacterial) genomes.
of the ZMW, the identity of the nucleotide is recorded by its
Despite the relatively low output of the system per SMRT cell, the
fluorescent label. During extension the label is cleaved off, diffuses
PacBio long read data, absence of GC bias and insight into the kinetic
outside the illumination zone, and the complex is ready for the next
extension. In essence, the system records a movie of the activity of state of the polymerase during sequencing present a niche of specific
the polymerase during a rolling circle amplification of the template. applications for this system that cannot be covered by any of the other
The po- lymerase used for sequencing in the PacBio system is a currently available sequence platforms.
modified version of phi29 [14] which although exhibiting reduced 3–
5 exonuclease activ- ity, still has many of the properties of the 4. Future sequencing technology
original phi29 like high processivity of several hundred kilobases,
low error rate of ~ 10e− 5 [15], no GC bias and strand displacement The advancements made on sequencing technology over the last
properties [16]. years have been impressive. However, the ultimate sequencing plat-
Through several advancements at the technological, bioinformatics form would work on single DNA or RNA molecules without any
and chemistry level, the average output per SMRT cell has (pre-) amplification, without use of optical steps, reads of Mb to Gb
increased considerably since the release of the system early 2011. in length, no GC bias, high read accuracy and would be flexible
The output has mainly increased though achieving longer average enough to generate as many sequence reads as are necessary for the
read lengths, starting from the ~ 1 kb at the time of release, while specific research ques- tion at hand. In addition, it should be both
the number of reads passing filter remained ~ 50–60 K per SMRT cheap to acquire and run, easy to operate, have short run times and
cell. Although the maximal achievable read lengths that can be simple or no library pre- preparation steps. Needless to say, this
obtained with this technol- ogy are directly related to the length of sequencing platform does not exist, yet. In the next section we will
the sequencing time, not all polymerase complexes reach identical discuss one emerging sequencing platform which may have the
read lengths. The main cause for this is photo-damage of the phi29 potential to make the next step towards these ultimate sequencers.
polymerase which terminates

Fig. 3. Total read length distribution for PacBio reads obtained with the P5 enzyme in combination with the C3 chemistry. Blue bars (left y-axis) represent the number of reads
and the black line (right y-axis) represents the total amount of data from reads longer than the read length on the x-axis. The data presented is from 8 SMRT cells run on the same
library. The total number of bases and reads is indicated with the average per SMRT cell in parentheses.
4.1. Oxford nanopore sequencing Finally transcripts

Expected characteristics for the nanopore sequencers are single

molecule, amplification free, base detection without labels, long
reads, low GC bias and scalable in data output. The basic principle
behind the technology is tunneling of (polymer)molecules through a
pore that sep- arates two compartments. Physical presence of the
molecule passing through the pore causes a characteristic temporary
change in the poten- tial between the two compartments which
allows for identification of the specific molecule [18]. Two version of
nanopore DNA sequencing are being developed, i.e., using the
natural pore forming protein alpha- hemolysin [19] or manufactured
solid state pores [20,21].
Oxford nanopore technologies (ONT) is one of the companies work-
ing on building nano-pore sequencing devices. Although ONT has put
their focus on nucleic acid sequencing, in principle this technology
could be applied to any (bio-)polymer as long as the molecules yield
distinguishable changes in the current between the compartments. In
Q4 2012 Oxford nanopore announced the early access release of their
MinION system. This is a palm sized sequencing device that facilitates
real-time analysis of single molecules such as DNA and RNA. However,
specifics on read length, accuracy and run times are difficult to obtain.

5. NGS applications

Below we will briefly describe a range of NGS applications. Each

application requires a minimal amount of reads of a pre-defined length
in order to obtain a dataset which allows the researcher to draw reliable
conclusions. Specifications differ for each application but in Table 2
we give a rough indication of the minimal dataset required. Still, de-
pending on the specific research question, the number of reads and/or
read length may differ.

5.1. Expression analysis

The development of micro-array technology revolutionized bio-

medical research, for the first time allowing overall characterization
(genome-wide) of gene expression. Yet the technology had several
flaws limiting sensitivity and specificity, that are overcome by
sequenc- ing. A first application of NGS was in gene expression
profiling. While detection of low expressed genes using micro-arrays
is limited by back- ground noise, sensitivity of sequence based
studies is predominantly limited by the depth of sequencing.
Recent publications show that RNA-based gene expression can be
measured in many ways. Initially, cost-directed, SAGE-like
approaches were popular [22], but dropping costs now drives
analysis towards full RNA sequencing (RNAseq) [23], requiring
much deeper sequencing yet through analysis of the entire
transcript revealing the complete picture incl. differential splicing
and allelic expression. A combination of SNP-array based GWAS
studies and NGS-based RNAseq is now used to study gene activity
per allele and detect variants that affect transcription both in cis
and trans (eQTLs) [24]. NGS has also been extensively used to study
RNA structure in detail and specific methods were developed to
characterize e.g. tran- scription initiation using the 5′-start (cap site)
[25] or the 3′-end of tran- scripts [26].
Small RNAs were re-discovered and large studies were
performed
measuring e.g. miRNA expression in great detail [27,28]. Enhancers
were recently found to initiate RNA polymerase II transcription,
produc- ing so-called eRNAs. Through the presence of balanced
bidirectional capped transcripts (from CAGE analysis), the FANTOM5
consortium was able to obtain a genome-wide atlas of active enhancers
across human cell types and tissues [29].
A recent addition is ribosome profiling [30] where not all RNA is
isolated but only that RNA which at the time of sampling is being
trans- lated into protein. Besides giving a picture much closer to
protein levels the technology can also be used to annotate the genome
(translation ini- tiation site, termination site and reading frame used).
reveal whether they contain the so-called untranslated open unstudied.
reading frames (uORF), in yeast often found in stress-related genes.

5.2. ChIP-seq

Another application boosted by NGS technology was the study of

protein binding sites in genomic DNA, especially that of transcription
factor binding sites based on Chromatin ImmunoPrecipitation
(ChIP) [31]. Initially ChIP-enriched sequences were analyzed using
array technology but since array content was limited choices had to be
made re- garding the sequences to analyze. In general promoter regions
of genes and CpG-rich regions were selected. Since it was often difficult
to enrich the sequences of interest to high purity, the signals obtained
were often rather weak. The advantage of sequencing the ChIP-products
enriched is that all sequences bound are identified and that the number
of sequence read can be used to determine the sensitivity; even when
enrichment is weak, increasing the number of reads can help to obtain
a good signal. Many studies have been performed using ChIP-seq and
DNase I hyper- sensitive site (DHS) mapping of which those of the
ENCODE [32] and FANTOM5 [29] projects are most impressive,
revealing genome wide profiles and binding sites for a range of DNA
binding proteins.

5.3. Methylation

Like ChIP-seq, the power of NGS gave a boost to the study of

(genome- wide) DNA methylation. Irrespective of the technology
used to isolate the methylated sequences, e.g., methylated DNA
immunoprecipitation- sequencing (MeDIPseq), methylated CpG
island recovery assay (MIRA- Seq) or whole-genome shotgun
bisulphite sequencing (WGSBS) (reviewed in Liard et al [33]),
NGS-analysis clearly reveals all sequences enriched. Besides
enriching methylated DNA sequences, chemical tricks can be used to
identify methylated nucleotides. Sequencing both untreat- ed and
bisulphite-treated DNA will highlight the C-nucleotides that are
methylated and not chemically converted resulting in a T when se-
quenced. Although powerful, the lower complexity of the
bisulphite treated DNA (most C's converted to a T) will cause some
problems during analysis (determining the origin of the reads
obtained). Another compli- cating factor is that, since methylation is
usually not 100%, a significant sequence depth will be required to
get meaningful results. Single molecule sequencing
technologies, like that of pacific biosciences (see above), are
attractive alternatives especially since they are able to detect all
DNA modifications present and not only methylation of C-
residues. Many NGS methylation studies have been presented
among which those of the ENCODE consortium [32]. An elegant
smaller study is that by Herb et al. [34] in honeybees where they
show that sub- stantial differences exist in the methylation
pattern in the brain be- tween nurses and foragers but not
between a queen and the worker castes. Interestingly, the
difference between nurses and foragers was reversible.

5.4. De novo genome sequencing

A main application of NGS technology is the complete

characteriza- tion of the entire genome of a particular species.
After tackling first the main model organisms (yeast, Escherichia
coli, Drosophila, Arabidopsis, mouse) and the human genome, an
ever increasing number of genome sequences is currently being
performed. An important player here is BGI (Beijing Genomics
Institute) with statements like “Does it taste good, we'll sequence
it”, “Does it look cute, we'll sequence it” and their “1000 Plant and
Animal Reference Genomes Project” and Ten Thousand Microbial
Genomes Project. Considerable effort has also been invested in
the analysis of genomes from endangered or even extinct species,
like the panda [35], mammoth [36] and early humans [37].
Together these projects produce an enormous amount of data
that, besides for some evolutionary studies, remains largely
Table 2
Recommendation for data requirements for a selection of NGS applications.

Application # reads/sample Run type # read length (bp) Remark

Transcriptome analysis
Tag based (SAGE/CAGE) N 10 million Single end 20–50
SmallRNA N 10 million Single end 20–50
mRNA Seq N 30 million Paired-end N50 Efficient exclusion of rRNA derived sequences
increases
the resolution of the transcripts of interest
Ribosome profiling N 20 million Single end 20–50
ChIP-Seq N 20 million Single or Paired-end ≥50 Specificity of the ChIP enzyme determines the # reads
needed. Low specificity ~ more background = more
reads needed
De novo sequencing 30× genome coverage, long single-end reads and Paired-end As long as possible Ideal PacBio long reads. Or combination of paired-end,
preferably more. mate-pair and PacBio.

Meta-genomics
Tag based (ITS, 16S) N100,000 Paired-end, long single-end reads As long as possible Complexity of the specific biosphere determines both
the primer pairs and/or #reads per sample. Longer
reads allow for better differentiation between related
species
Shotgun N 100 million Paired-end, long single reads As long as possible Complexity of the specific biosphere determines the
library insert size and/or #reads per sample.

Methylation analysis
Whole genome N 400 million Paired-end ≥100 Ideal situation: all PacBio long reads on
native/ unmodified shotgun libraries.
Enrichment strategies N 50 million Paired-end ≥100
Infections N 25 million Single or Paired-end ≥100 ~2% of cell-free DNA from plasma is of non-human
origin
Non-invasive prenatal testing N 10–20 million Single-end N50 Trisomy detection from cell-free fetal DNA in maternal
plasma

Disease gene identification diagnostics

Whole genome 1 billion Paired-end ≥100 30× average coverage
Exome (50 Mb) N 60 million Paired-end ≥100 50× average coverage, N 75% on target

5.5. Metagenomics
RNA sequencing one gets an overview of “what's happening” [44].
The technology facilitates the study of the consequences of environ-
A complex variant of de novo genome sequencing is “sequence it
mental changes as well as way to determine the cause of
all”, metagenomics. Using brute force sequencing, simply reading all
disturbances. Enormous progress has been made in a medical
DNA sequences present in a sample, metagenomics is a way to make
setting. Studying the human microbiome gave a range of surprising
an inventory of what is present in a sample, of what is living where. A
findings, incl. its enor- mous complexity containing 10 × more cells
simple but effective application of this is trying to detect the cause of
than the human body and 1000 × more genes. The microbiome of
an infectious disease. Simply analyzing all DNA from control versus
the human gut has been studied in great detail [45] including its
infected (diseased) individuals will reveal the “extra” DNA which most
relation with phenotypes like obesity [46]. Microbiomics is hot and
likely derives from the infectious agent. The approach was used success-
it will undoubtedly bring new insights in the interplay between
fully to identify e.g. colony collapse disorder killing honeybees [38] but
human health and the bugs living on and in within us.
also to identify the cause of diseases that killed thousands of humans in
the past, inclusive the black death [39].
Metagenomics can be performed by a sequence it all approach or
5.6. Non-invasive prenatal testing
by focusing on specific uniformly conserved sequences like e.g.
ribosomal RNA genes only. The latter approach has two main
To obtain DNA from a fetus, prenatal diagnosis generally
advantages; 1) the complexity of the data obtained is much smaller,
involves the costly and risky sampling of either chorionic villi or
and 2) more sequences can be assigned to a specific organism or a
amniotic fluid. It is long known that DNA of the fetus can be found
group of related organisms. The latter facilitates some semi-
in maternal blood (cell free serum), yet it has low abundance and
quantitative analysis which is much more difficult when analyzing
low quality and it is not easy to discriminate fetal from maternal
all sequences mixed from many organ- isms with largely varying
DNA. These characteristics prevented wide-spread implementation
genomesizes. Metagenomics focussing on rDNA genes has been
of prenatal tests performed on maternal blood. However, the
used to study many different things, incl. e.g. the effect of the 2004
enormous power of NGS technology seems particularly attractive
tsunami on microbial ecologies in marine, brackish, freshwater and
for non-invasive prenatal testing (NIPT). To detect trisomies, in
terrestrial communities in Thailand [40]. Targeting the chloroplast
particular trisomy-21 or Down's syndrome, a very simple but
trnL gene was successfully used to study airborne pollen and its
effective brute-force method was developed: sequence, map, and
relation with hay-fever [41].
count. DNA isolated from maternal serum is sequenced, reads are
A unique feature of metagenomics approaches is that one does
mapped to the human genome and counted per chromosome.
not need to culture the organisms that one wants to study. The
When 5–10 million reads are mapped, trisomies will reveal
most im- pressive result of first studies that read all DNA sequences
themselves by giving a significantly too high number of reads
was that up to ten times more organisms were encountered than
mapping to a partic- ular chromosome [47]. A recent study showed
seen previously. One can now study organisms that no one is able
that when genome se- quencing of both parents, genome-wide
to culture and/or that no one has ever seen. Based on DNA
maternal haplotyping and deep sequencing of maternal plasma
sequencing one gets an idea of the complexity and constitution of
DNA are combined even the ge- nome sequence of an 18.5 weeks
entire ecosystems [42,43]. Using
human fetus can be determined [48].
5.7. Disease gene identification
studies, low accuracy (N 85%) single-molecule long-read sequences
can be sufficient to make a significant difference. De novo genome
A combination of genome-wide association studies (GWAS) and
assembly can be improved considerably when long kilobase-sized
specific targeting by sequence capture of the genomic regions
reads are avail- able that span gaps from short-read paired-end
detected is now used extensively trying to identify the variants that
sequencing. The single- molecule technologies are amplification free
functionally link the DNA with the phenotype. Similarly, genome
and thereby not hampered by PCR-based artifacts like uneven
sequencing can be used as a tool to characterize genetic variation in
amplification and GC-bias. Conse- quently, they give a much more
a specific population, determine haplotype structure and use this
uniform coverage and span GC-rich regions. In addition they may
knowledge to impute alleles and boost the outcome of GWAS
span repeat structures or duplications that cannot be resolved using
analysis [GoNL consortium, 2014, in press].
short read sequencing. English et al. have used PacBio data for
One of the most impressive applications of NGS lies in the field of
upgrading existing draft genome assemblies derived from Illumina
human genetics and disease gene identification. In the past larger fam-
sequencing data by looking for reads that extend into or cover gaps
ilies were an absolute requirement for a successful approach. Without
in the assembly [56]. A paper by Loomis et al. showed that the
being able to first map a disease gene to a specific position and then
system has no problems sequencing long regions of 100% GC
zooming in on the genes in that region, the human genome was simply
content of CGG trinucleotide repeat expansions [57].
too big and analysis too costly. Some successes were obtained using
Amplification-free methods facilitate the analysis of DNA modifica-
candidate disease gene approaches but generally these only worked
tions, deriving from either cellular processes like methylation [58]
when for a specific disease a new gene was discovered making similar
and/or from damage (irradiation, chemicals, etc) [59]. DNA
genes or neighboring genes in a certain pathway obvious candidates.
modifica- tions present on the template molecule affect the DNA
NGS studies were much more successful, even when only one or a few
polymerase activity to a certain extent, i.e., the time needed for
cases are available [49]. Parent–child trio analysis turned out to be
incorporation of a nucleotide at a particular site. The identity of
very effective to reveal dominant de novo diseases [50], while recessive
modifications can be inferred by analysis of the kinetic state of the
diseases can be revealed when several unrelated cases are available or
polymerase. This works well for modifications that have a large
when clearly damaging variants are present [51]. The latter successes
effect on the polymerase activity, e.g., N6-methyladenine (m6A)
could already be obtained with exome sequencing, i.e. a method to
and N4-methylcytosine (m4C) [60,61]. The 5-methylcytosine (m5C)
zoom in on the 1–2% protein coding sequences of the human genome
modification has a weaker signal but given enough coverage can still
only. Needless to say that, when cost drops further, full genome
be inferred from the data [61]. How- ever, conversion of 5-
sequencing will be used to detect also deleterious variants that are not
methylcytosine to 5-carboxylcytosine through the Ten-eleven
in the protein coding regions. Early steps towards the ultimate applica-
Translocation Gene Protein 1 (Tet1) results in a greater disturbance
tion, genome-based medicine/personalized medicine have been set
in the signal, thus making it easier to detect even at lower coverage
by the UK and Saudi Arabia which last year both announced projects
[62].
to sequence the genome of 100,000 individuals.
Other applications of long-read sequencing are RNA structure analy-
sis [63] and studies to unravel complex repeat structures (e.g.
5.8. Human disease and health
segmental duplications in the human genome) and large segments of
repetitive DNA.
Thus far studies have been mostly performed on the level of cell
cultures, whole tissues or sorted cell populations. Although the yield
6. Online supplement
per cell, 30%–70% of all RNA or DNA present, can still be improved,
re- cent NGS developments have now made genome-wide single cell
6.1. Solid sequencing
anal- ysis feasible. Individual cells turn out to be quite different
showing extensive genomic and transcriptomic heterogeneity in both
The Solid systems read DNA by an intricate sequencing-by-
normal development and disease [52]. This turns out to be especially
ligation scheme [64]. After positioning the templated beads on the
true for cancer tissue being a complex mixture of many different cell
flow-cell, the first step is hybridization of a primer complementary
popula- tions each carrying a range of genomic rearrangements driving
to the com- plete adapter followed by hybridization of octamer
its unre- stricted growth. Dissecting these using (very) deep
probes. The first two nucleotides of these probes represent 16
sequencing of the cancer genome/transcriptome as a whole or from
dinucleotide combina- tions, bases 3–5 are degenerate and bases 6–
single cell analysis should give us a tool to identify the so-called driver
8, also degenerate, contain a fluorescent label for identification of
mutations. These will be different in different tumors and instrumental
bases 1 and 2. Four different di- nucleotide combinations are
to direct treat- ment and prescribe the best (set of) drugs to be used,
labeled with the same fluorescent tag. Use of 4 different fluorescent
personalized cancer treatment. Similarly NGS approaches will be used
groups allows for labeling all 16 dinucleo- tide tags. When a probe
to study drug resistance, identify their mechanism and provide
anneals adjacent to the adapter primer strands are ligated and
strategies to combat resistance [53]. Coordinated by the International
information on the first two bases is recorded from the fluorescent
Cancer Genome Consortium (ICGC) a large project is ongoing trying to
signal at bases 6–8. These last three bases are removed and new
resolve the geno- mic changes present in many forms of cancers by
octomer probes are allowed to hybridize and ligate, providing
analyzing 50 cancer/ normal tissue pairs [54]. In due time NGS information on bases 6 and 7, i.e., position +5 relative to the
developments will start to impact our daily life. While it will initially be previous cycle, of the template. Depending on the required read
used as a molecular micro- scope to diagnose disease, ultimately it will length, 5–7 of such cycles are performed after which the entire
also be used to monitor our personal health. Our genome sequence will synthesized strand is removed from the template. A new primer
be read once, but e.g. blood- derived RNA analysis, completed with with a −1 nt shift in end po- sition is hybridized and octomer probes
proteomics and metabolomics measurements, will be used on a regular are allowed to hybridize and li- gate in 5–7 cycles as described above,
basis to study the status of our body [55], the Whole-body-BIOscan. but now probing nucleotides 0–1, 5–6, and 10–11 etc. of the
template. In total 5 of these re-priming cycles are performed. The
5.9. Single molecule & long read sequencing template sequence is subsequently decoded from the color labels
from the two ligation events per base [65].
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taining few read errors, are not essential for all applications. For specific generate paired-end reads. However, due to limitations of
sequencing by ligation scheme the maximal read length is limited to
75 bases for read 1 and 35 bases for read 2. Although the total
number of reads generated per Solid run is comparable to HiSeq,
the total output in Gb per run is only half of that for HiSeq due to
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