Padlock Probe-Based Assays for Molecular Diagnostics Anja Mezger

Padlock Probe-Based Assays for
Molecular Diagnostics
Anja Mezger
©Anja Mezger, Stockholm University 2015
ISBN 978-91-7649-155-3 pp1-53
Printed in Sweden by Holmbergs, Malmö 2015
Distributor: Department of Biochemistry and Biophysics,
Stockholm University
The possibilities are limited only
by our imagination and determination,
and not by the physics.
Mike Duke
To my family,
for believing in me
List of Publications
This thesis is based on the following papers:
Mezger A, Öhrmalm C, Herthnek D, Blomberg J, Nilsson M. Detection of
rotavirus using padlock probes and rolling circle amplification. PLoS One 9,
e111874 (2014).
Mezger A, Gullberg E, Göransson J, Zorzet A, Herthnek D, Tano E, Nilsson
M*, Andersson DI*. A general method for rapid determination of antibiotic
susceptibility and species in bacterial infections. J Clin Microbiol 53, 425-432
(2015).
Mezger A, Allen S, Cavelier L, Hultén M, Nilsson M. Elimination of maternal
DNA for accurate non-invasive prenatal testing: a pilot study. Submitted.
Mignardi M*, Mezger A*, Larsson C and Nilsson M. Oligonucleotide gap-fill
ligation for mutation detection and sequencing in situ. Submitted – under review.
* These authors contributed equally.
Reprints were made with permission from the publishers.
Related work by the author
Mezger A*, Kühnemund M*, Nilsson M, Herthnek D. Highly specific DNA
detection employing ligation on suspension bead array readout. New
Biotechnology (2015). [Epub ahead of print].
Østerberg FW, Rizzi G, Donolato M, Bejhed RS, Mezger A, Strömberg M,
Nilsson M, Strømme M, Svedlindh P, Hansen MF. On-chip detection of rolling
circle amplified DNA molecules from Bacillus globigii spores and Vibrio
cholerae. Small 10, 2877-2882 (2014).
Gomez de la Torre TZ*, Ke R*, Mezger A, Svedlindh P, Strømme M, Nilsson
M. Sensitive detection of spores using volume-amplified magnetic nanobeads.
Small 8, 2174-2177 (2012).
Gomez de la Torre TZ, Mezger A, Herthnek D, Johansson C, Svedlindh P,
Nilsson M, Strømme M. Detection of rolling circle amplified DNA molecules
using probe-tagged magnetic nanobeads in a portable AC susceptometer.
Biosens Bioelectron 29, 195-199 (2011).
* These authors contributed equally.
Contents
Introduction ............................................................................................ 9
In vitro nucleic acid detection ............................................................... 10
Polymerization-based technologies ......................................................................10
Ligation-based technologies ................................................................................12
Sequencing-based technologies ...........................................................................14
In situ nucleic acid detection................................................................. 17
Fluorescent in situ hybridization ..........................................................................17
Methods for signal amplification in situ .................................................................18
In situ sequencing .............................................................................................20
Diagnostic methods used in bacteriology and virology .......................... 21
Bacterial identification by culture and biochemical methods ....................................21
Microscopy for rapid microbial diagnosis ...............................................................22
Immunoassays ..................................................................................................23
PCR-based methods for pathogen identification .....................................................23
Mass spectrometry ............................................................................................24
Methods for antibiotic susceptibility testing ...........................................................25
Summary of diagnostic methods .........................................................................27
Methods for prenatal diagnosis of chromosomal aneuploidies .............. 28
Traditional prenatal testing .................................................................................28
Non-invasive prenatal testing ..............................................................................29
Molecular Diagnostics in Oncology ........................................................ 32
Present investigations ........................................................................... 34
Detection of rotavirus using padlock probes and rolling circle amplification ...............34
A general method for rapid determination of antibiotic susceptibility and species in
bacterial infections ............................................................................................35
Elimination of maternal DNA for accurate non-invasive prenatal testing: a pilot study 36
Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ ...........37
Populärvetenskaplig sammanfattning på svenska ................................. 40
Acknowledgments ................................................................................. 41
References ............................................................................................ 44
Abbreviations
ACTB
ASP
AST
bDNA
C2CA
cff-DNA
CLIA
CVS
EGFR
EIA
ELISA
EM
FISH
FRET
HER2
HIV
ISH
KRAS
LAMP
LNA
MALDI
MELAS
MIC
MLPA
MPS
MS
MSAFP
NGS
NIPD
NIPT
OLA
PAPP-A
PCR
PNA
QF-PCR
qPCR
RCA
RCP
RT-PCR
SDA
smFISH
SNV
TB
TOF
UTI
WHO
β-hCG
Actin beta
Antibiotic susceptibility profile
Antibiotic susceptibility testing
Branched DNA
Circle-to-circle amplification
Cell-free fetal DNA
Clinical Laboratory Improvement Amendments
Chorionic villus sampling
Epidermal growth factor receptor
Enzyme immunoassay
Enzyme-linked immunosorbent assay
Electron microscopy
Fluorescent in situ hybridization
Fluorescence resonance energy transfer
Human epidermal growth factor receptor 2
Human immunodeficiency virus
In situ hybridization
Kirsten rat sarcoma viral oncogene homolog
Loop-mediated isothermal amplification
Locked nucleic acid
Matrix-assisted laser desorption/ionization
Mitochondrial encephalomyopathy, lactic
acidosis, and stroke-like episodes
Minimum inhibitory concentration
Multiplex ligation-dependent probe amplification
Massively parallel sequencing
Mass spectrometry
Maternal serum alpha-fetoprotein
Next-generation sequencing
Non-invasive prenatal diagnosis
Non-invasive prenatal testing
Oligonucleotide ligation assay
Pregnancy-associated plasma protein-A
Polymerase chain reaction
Peptide nucleic acid
Quantitative fluorescent PCR
Quantitative PCR
Rolling circle amplification
Rolling circle product
Reverse-transcription PCR
Strand displacement amplification
Single-molecule FISH
Single nucleotide variant
Tuberculosis
Time of flight
Urinary tract infection
World Health Organization
β-human chorionic gonadotropin
Introduction
Rapid and reliable identification of the factors underlying a disease is essential
for correct treatment and ultimately for the patient’s health. The optimal test
should excel in several parameters in order to guarantee accurate and affordable
diagnosis. Clinical and analytical sensitivity must be high, as well as specificity
and precision. Especially in infectious disease diagnosis, an assay must be rapid
in order to minimize the time until adequate treatment can be initiated.
Additionally, cost, required technical skills and infrastructure must be kept to a
minimum to allow wide application, also in low resource settings. A plethora of
diagnostic tools already exists, but none of them excels in all stated aspects.
Although it should be noted, that the importance of each parameter varies from
field to field. Time, for example, is not as important in oncology and prenatal
testing as it is in infectious disease diagnostics.
The ability of delivering effective care depends on accurate diagnosis.
Molecular assays have the advantage of providing additional information on the
nucleic acid or protein level, which can be used to maximize the clinical benefit.
Knowledge of the nucleic acid content allows not only rapid diagnostics, but
also targeted therapies. The work presented in my thesis focused on the
development of nucleic acid-based methods for molecular diagnostics. The aim
was to develop several assays that have the potential to be applied in clinical
practice as they overcome certain limitations of already existing methods. The
covered areas include viral detection, antibiotic resistance profiling, noninvasive prenatal diagnosis (NIPD) and in situ mutation detection,
demonstrating the wide applicability of padlock probes for clinical use.
In this thesis, I will first give an overview of molecular technologies used for
nucleic acid detection, which are the basis of a range of diagnostic tests. Then, I
will describe nucleic acid and protein-based technologies that are commonly
applied in clinical diagnostics. The advantages of nucleic acid-based diagnostics
in the fields of infectious disease diagnostics, prenatal testing and in oncology
will be discussed in the respective subsection. In the last part, I will describe and
discuss the developed assays that this thesis is composited of and give a future
outlook.
9
In vitro nucleic acid detection
A number of methods are currently available for nucleic acid detection aiming at
identifying a specific nucleic acid sequence. This goal can be reached by
techniques that are mainly based on polymerization, ligation, sequencing or a
combination of these. I will give an overview of some of the most prominent
techniques on which many assays, currently used in the field of molecular
diagnostics, are based.
Polymerization-based technologies
Polymerization can be used to specifically detect and amplify a target sequence
as polymerases possess the enzymatic ability to copy the sequence content of a
template strand. The most widely used polymerization-based amplification
technique is the polymerase chain reaction (PCR). Invented 30 years ago it
allows the exponential amplification of a target sequence 1-3. The target DNA
sequence is amplified by cyclically heat denaturing the DNA double strand,
annealing the two primers and extending them by polymerization. The primers
hybridize to complementary sequences of the target strand and thus, flanking the
target site. Thermal cycling is required since the temperature must be repeatedly
altered between the denaturation, extension and annealing steps. The
development of quantitative PCR (qPCR) made accurate quantification of the
target DNA possible. In real-time qPCR, the amplification process is
continuously monitored by measuring the increase in fluorescence whereby the
fluorescence intensity is directly proportional to the target DNA concentration
and can be used for DNA quantification4, 5. Dyes, such as SYBR green I that
preferentially stain double stranded DNA or alternatively, fluorescent resonance
energy transfer (FRET) probes can be used. FRET probes are short
oligonucleotide probes labeled with a fluorophore/quencher pair. The most
commonly used FRET probes are TaqMan probes. Upon binding of the probe
and subsequent hydrolysis by the 5’-3’ exonuclease activity of the Taq
polymerase the fluorophore is released and due to the lack of proximity it is no
longer quenched4, 6, 7. qPCR can accurately detect a two-fold difference in DNA
concentration8. Absolute quantification can be achieved by the inclusion of an
internal standard9 or by the use of a standard curve that is constructed by
amplification of known amounts of target nucleic acids 10. Additionally, qPCR
offers a lower risk of cross-contamination as a closed system from sample to
readout can be used. Furthermore, the turnaround time is significantly
decreased, since quantification does not require any post-PCR manipulation.
Digital PCR achieves an even higher quantitative accuracy whereby the target
DNA is diluted and compartmentalized with a concentration of less than one
10
copy per compartment and individually amplified11. The number of positive
wells is counted after amplification reflecting the number of initial target
molecules present. Digital PCR allows the analysis of rare mutations 11-13, which
has been difficult with conventional PCR in which bulk DNA, compared to
single copies, is analyzed. Increasing the number of compartments leads to an
increase in sensitivity as a larger fraction of the sample is analyzed 11, 14. The
introduction of droplet microfluidics allows the analysis of a large number of
compartments in a cost-effective manner without the need for larger sample
volumes15.
All PCR-based methods require precise temperature control. Isothermal
amplification methods, on the other hand, do not rely on thermal cycling and
thus implementation into microfluidic chips and use in low resource settings is
facilitated. Several polymerization-based isothermal amplification methods have
been developed in the last decades. Two isothermal amplification techniques,
both used in diagnosis of infectious diseases16-19, are described in greater detail
below, followed by a description of rolling circle amplification (RCA).
Strand displacement amplification (SDA) uses primers containing a recognition
site for a nicking enzyme and a target complementary part. After initial
extension of the primers, the nicking enzyme creates a free 3’ end, which is
extended by an exonuclease-deficient polymerase displacing the downstream
strand. Exponential amplification of the target sequence can be achieved by
using two primers targeting strands of opposite polarities20. The target site must
not include the recognition site of the enzyme used as it would be digested by
the nicking enzyme during the amplification process. Another exponential
isothermal amplification method that has been used for detection of
microorganisms is the loop-mediated isothermal amplification (LAMP)
method18, 19. LAMP requires several primers and a polymerase possessing
strand-displacement activity21. The amplification product contains stem-loop
structures, which will initiate further amplification. A disadvantage of LAMP is
the complicated primer design, targeting six different genomic regions using
four primers21. The likelihood of primer-primer interactions becomes larger with
the increase in the number of primers and thus, makes multiplexing difficult 22.
The work in my thesis is based on RCA, which is an efficient and well-exploited
isothermal method to amplify short DNA circles, including padlock probes
(described in more detail below)23-25. A short primer hybridized to the circle
initiates polymerization. φ29 DNA polymerase possesses several characteristics
that make it an excellent choice for RCA. It is a highly processive enzyme and
does not require accessory proteins26. Furthermore, the strand displacement
activity enables a continuous amplification of the circularized padlock probe26
and the 3’ to 5’ exonuclease activity allows to efficiently use the DNA target as
a primer by digesting overhanging nucleotides (target-primed RCA)27.
11
Common for all mentioned methods is the fact that specificity solely depends on
primer hybridization. An increase in specificity can be achieved by using the
intrinsic properties of ligases to distinguish single base mismatches.
Ligation-based technologies
In nature, ligation is used during replication and as a repair mechanism to seal
single-strand breaks in duplex DNA or to repair double-strand breaks28-31.
Ligases catalyze the formation of a phosphodiester bond between the 3’
hydroxyl group of one DNA strand and the 5’ phosphate group of the other
DNA strand30, 31. The ligation of single-strand breaks is templated by the
complementary strand and mismatches at the ligation site inhibit strand sealing
to some extent with the highest discriminatory power at the 3’ end of the nick 3235
. Several DNA detection and amplification technologies are based on the
above described properties of ligases. The oligonucleotide ligation assay (OLA)
was one of the first technologies that utilized the specificity of DNA ligases 36.
Although not a nucleic acid amplification method, I will shortly describe OLA
as several other methods are based on its principle. The first description of the
method used T4 DNA ligase to ligate two adjacent complementary
oligonucleotides and thereby discriminating single nucleotide mismatches36. A
ligation product is only formed if the two oligonucleotides are perfectly basepaired at the ligation site. By using a thermostable ligase and thermal cycling the
ligation product can be linearly amplified (termed ligase detection reaction) 37.
The ligation product is first heat-denatured from its target sequence and by
subsequent lowering of the temperature two new short oligonucleotides can
hybridize and be ligated and thus be linearly amplified 37. Addition of a second
set of oligonucleotides, complementary to the first one, results in exponential
amplification (termed ligase chain reaction)37.
Another example of a ligation-based amplification method, which has been
commercialized for mutation detection in various genetic disorders (MRCHolland), is the multiplex ligation-dependent probe amplification (MLPA)38. In
MLPA, two probes, built up of identical end sequences for primer binding and a
target complementary part that, like in OLA, hybridize to adjacent target sites 39.
Spacer sequences of different length between the primer binding and the target
site allow multiplex detection and amplification39. Upon ligation the probes are
amplified by PCR and separated by capillary electrophoresis39.
Padlock probes, on which the work in this thesis is based on, are a further
development of OLA. Padlock probes are linear oligonucleotides with two target
complementary arms that are linked via a backbone sequence (Figure 1)40. The
backbone can contain sequences used for detection or recognition sites for
restriction enzymes. Upon hybridization the two ends are enzymatically joined
12
by ligation forming a topologically locked circle40. Like in OLA specificity
relies on hybridization and on the fidelity of the ligase32, 36, 40. Padlock probes are
locally amplified by RCA, by a factor of 1,000 per hour25, and collapse
spontaneously into µm-sized coiled structures25, 41. These amplification products,
termed rolling circle products (RCPs) can either be directly detected25 or further
amplified by, for example, circle-to-circle amplification (C2CA)42. RCPs can be
detected in numerous ways, such as fluorescence based microscopy yielding a
digital assay43 or by colorimetric methods44. C2CA increases sensitivity and
allows detection of target sequences in a wide dynamic range42, 43. To initiate a
second round of RCA, the RCPs must first be monomerized. A short
oligonucleotide, containing an enzymatic restriction site, is hybridized to its
complementary sequence in the RCP and digested upon addition of a restriction
enzyme. These short monomers are re-ligated to form circles, which in turn can
be amplified by RCA42. The use of padlock probes compared to the
amplification techniques described above offers several advantages: (i) single
nucleotide resolution can be achieved due to the high fidelity of the ligase 45, 46,
(ii) padlock probes can be highly multiplexed without the need of extensive
optimization allowing the simultaneous detection and amplification of more than
10,000 target sites47, (iii) the first round of RCA products are topologically
linked to their target sequence40 allowing washing steps and yielding spatial
information if applied in situ. The latter will be discussed in a later section( in
situ RCA and sequencing).
13
Figure 1. Schematic illustration of padlock probes and rolling circle amplification. A) Illustration of a padlock
probe. The two target complementary arms are linked by a backbone sequence, which can contain different
sequence elements such as a restriction site (in blue) and a site for hybridization of a detection oligonucleotide
(in green). B) A ligase (in pink) seals the nick of the two juxtaposed padlock probe arms. C) The circular
molecule is amplified by φ29 DNA polymerase (in yellow) creating a concatemer of multiple copies of a
sequence with opposite polarity to the padlock probe. The resulting rolling circle product can be monomerized
by enzymatic digestion after hybridization of a restriction oligonucleotide to the restriction site (not shown). D)
The monomers are re-ligated, templated by undigested restriction oligonucleotides. E) Ligated circles are
amplified in a second amplification step. The amplified products can be detected in an optical imaging system by
hybridization of short fluorescently labeled oligonucleotides (not shown).
Sequencing-based technologies
DNA sequencing technologies have been implemented into diagnostics, as the
sequence content may give valuable information about disease and thus, allow
administration of effective treatment. Application areas include mutation
detection in cancer and hereditary diseases, prenatal testing and microbiology
14
where sequencing is mainly used for epidemiological purposes. With the
introduction of next-generation sequencers, sequencing costs have dropped
significantly and this trend is expected to continue48, therefore sequencing might
become the diagnostic tool.
In 1977, one of the first sequencing methods was developed: Sanger
sequencing49. For the next three decades, until the next-generation sequencing
(NGS) technologies arose, Sanger sequencing was practically the only
technology used in any sequencing project50. In Sanger sequencing, the four
nucleotides are mixed together with their dideoxynucleotide analogs, which can
be incorporated into a DNA sequence but cannot be elongated, creating a
mixture of sequence fragments of different lengths49. The original DNA
sequence can be derived after separating the resulting fragments according to
their length49. Sanger sequencing excels at read length but suffers high costs.
Although offering long read lengths, this sequencing method is tedious and
unsuitable for high throughput. Substantial progress was made with the
introduction of an automated DNA sequencer using fluorescently labeled
primers and thus, eliminating the need of X-ray film development51. Further
development of the sequencing chemistry included the introduction of
fluorescently labeled dideoxynucleotides permitting the synthesis of
fluorescence-tagged fragments in one reaction52. The introduction of capillary
array electrophoresis for separation and detection of sequencing products
substantially increased throughput53 and thereby significantly contributed to the
completion of the human genome project54.
In 2005, the breakthrough came with the introduction of two novel NGS
technologies: the 454 sequencing-by-synthesis technology55 and the multiplex
polony sequencing technology, based on sequencing-by-ligation56. The 454
technology uses a modified version of the pyrosequencing protocol to sequence
the template strand which is coupled to beads in picolitre-sized reaction wells55.
Signal detection is based on the detection of pyrophosphate, released during
nucleotide incorporation57. Polony sequencing, developed by Shendure et al., on
which the sequencing by oligonucleotide ligation and detection (SOLiD)
technology is based, uses beads that bind the amplification products generated
by emulsion PCR56. Fluorescently labeled degenerated probes are hybridized to
the amplification products and ligated to an anchor primer (Figure 2). Multiple
cycles of hybridization, ligation, detection and cleavage are performed whereby
the read length is determined by the number of cycles 56. Besides the two NGS
technologies mentioned above several alternative technologies have been
developed in the last decade. Illumina uses single-nucleotide addition of
reversible dye terminators, each nucleotide being identified by a specific
fluorescent label58. Ion Torrent, on the other hand, does not use fluorescence to
determine the sequence content, but measures the pH change caused by the
release of hydrogen ions during nucleotide incorporation 59. The described NGS
15
technologies rely all on the same basic principles. First, a sequence library of
fragments with covalently attached adaptors is constructed to form clusters of
the original templates. Second, the sequencing is massively parallel, consisting
of a nucleotide addition, detection and a cleaving/washing step. As a
consequence, NGS technologies allow higher throughput at a fraction of the cost
required for Sanger sequencing. The downside is the shorter read length, which
makes it more difficult to uniquely map the reads to a reference genome. The
read length is determined by the signal-to-noise ratio, averaging 50-150 bp for
most NGS compared to 800-900 bp achieved with Sanger sequencing60.
Figure 2. Schematic illustration of sequencing-by-ligation chemistry. An anchor primer is hybridized to its
target sequence and ligated to fluorescently labeled, degenerated nonamers. After image acquisition the ligation
product is stripped off and the cycle is repeated.
Sequencing of individual molecules, opposed to sequencing clusters, has the
advantage of eliminating the bias produced during library preparation and the
possibility of detecting DNA modifications, such as methylation. The difficulty,
though, is signal detection, as the sensor needs to be sensitive enough to detect
the signal changes from a single molecule. Pacific Biosciences, commercialized
in 2010, uses a technology where library fragments are bound to a single DNA
polymerase molecule, which is then deposited on the chip surface61. The
polymerase has been engineered to allow incorporation of fluorescently labeled
nucleotides and the polymerization rate has been decreased in order to detect
single incorporated nucleotides61. In 2012, sequencing of DNA methylations has
been achieved using the technology developed by Pacific Biosciences62, 63. An
alternative to the above described single molecule NGS technology is the use of
nanopores. Nanopores function as ion channels and measure the change in ion
flux when a single DNA molecule is threaded through the pore 64. The four
nucleotides can be distinguished due to their unique electrical signature 65.
However, a major challenge remains to slow down the translocation rate as
commercial detectors lack sufficient temporal resolution causing a high error
rate66. Nevertheless, nanopore sequencing has the potential to become a routine
analytical tool in the future.
16
In situ nucleic acid detection
In vitro methods can accurately quantify the simultaneous presence or absence
of different target sequences, but lack spatial information. Bulk measurements
cannot give information about gene expression patterns in heterogeneous tissues
as they mask the information by yielding an average expression profile. In situ
methods, however, allow the analysis of spatially-resolved gene expression
patterns in heterogeneous tissue. Such level of resolution is needed for example
in the study of the transcriptome in cancer tissue to direct treatment, as bulk
measurements can mask the existence of a minor population such as cancer stem
cells. Other application areas include the study of naturally heterogeneous
tissues such as brain tissue. In the following sections I will give a brief overview
of different technologies used to achieve spatially-resolved transcriptome
analysis. Common to most in situ techniques are the following steps: tissue
fixation and permeabilization followed by hybridization of probes. Signal
detection and/or amplification vary between the techniques described.
Fluorescent in situ hybridization
With the development of in situ hybridization (ISH) in 1969 it was possible to
study nucleic acids directly in cells. Gall and Pardue demonstrated the detection
of ribosomal DNA by hybridization of radiolabeled RNA67. Advancements in
fluorescence microscopy have replaced radiolabeled probes with fluorescentlylabeled probes. In the original fluorescent in situ hybridization (FISH) protocol,
a fluorochrome is covalently bound to the RNA probe allowing DNA detection
by hybridization and subsequent fluorescent detection68. Although the first FISH
protocols were less sensitive than autoradiography it had significant advantages:
increased spatial resolution and increased speed, making results available in one
day68. mRNA detection, using biotinylated nucleotides in the probe sequence
and either antibodies or fluorescently-labeled avidin for detection, was
demonstrated by Singer et al. in 198269.
Despite the potential of increased spatial resolution of FISH, compared to ISH,
which used radiolabeled probes, early protocols allowed only qualitative
information and not absolute counting of target sequences 68. The low signal-tonoise ratio of long probes, partly caused by quenching of adjacent dyes, which
are randomly distributed along the probes, does not allow absolute
quantification70. Improvements in probe design and imaging technology made
absolute quantitation with high spatial resolution possible. Instead of long,
randomly labeled probes, Femino et al. used several, adjacent to each other, 50base pair long probes that were labeled at predefined positions with five
fluorophores per probe and a GC content of ~50%. These improvements permit
17
counting of diffraction-limited spots, each arising from one single transcript71.
Multiply labeled probes have been used for analysis of transcript distribution in
yeast64, 65 and mammalian cells72, and for detection of transcription sites in
tissue73. Differences in spot intensity due to incomplete hybridization or due to
only partially fluorescent probes impede the differentiation between true signals
and nonspecifically bound probes. Further optimizations, yielding higher
specificity and sensitivity, included construction of a probe library whereby each
probe is 17-22 nucleotides long and labeled with a single fluorophore74. This
approach has been applied, amongst others, to simultaneously detect multiple
transcripts in mammalian single cells74, for detection of fusion transcripts75 and
for the detection of stem-cell markers in tissue76. Inclusion of modified
nucleotides, such as peptide nucleic acids (PNA) and locked nucleic acids
(LNA) increases probe binding stability and thus, probe lengths can be
shortened77, 78. Using these modifications, short transcripts such as micoRNA
molecules can be specifically detected, although signal amplification is
necessary79, 80.
The multiplexibility of single-molecule FISH (smFISH) is limited by the
number of fluorophores that can be spectrally distinguished. These limitations
have been bypassed by either using combinatorial fluorescent barcodes or by
sequential hybridization81, 82.
Methods for signal amplification in situ
Traditional smFISH, due to low signal strength, requires high-magnification
objectives resulting in long acquisition times. The acquired signal can be
improved either by target or by signal amplification. In situ PCR and reversetranscription PCR (RT-PCR) have been demonstrated for target amplification,
but due to diffusion of amplification products this method is less quantitative83,
84
. For signal amplification, either the signal, as in branched DNA (bDNA) or
the probe sequence, as in in situ RCA, can be amplified to obtain higher
sensitivity.
As in FISH, signal detection using bDNA relies solely on hybridization. Instead
of directly detecting mRNA molecules by hybridization of short fluorescentlylabeled probes to the target sequence a gene-specific probe, to which a preamplifier binds, is hybridized85. Multiple amplifier probes hybridize to the preamplifier probe onto which labeled detection probes hybridize 85. The above
described mechanism of bDNA results in a local increase in fluorescence
without the need of target amplification. bDNA has been used for in situ
detection of viral DNA and mRNA in various cell types and in tissue85, 86.
Another approach to obtain signal amplification combines immunoRCA with
ISH. A labeled probe is hybridized to its target sequence and detected by
18
immunohistochemistry combined with RCA87. The bound antibody carries a
primer, which upon complementary binding of circular DNA initiates RCA and
amplifies the signal which is detectable by hybridization of fluorescent probes87.
RCA is an isothermal amplification mechanism and the target molecules are
tethered to their amplification product, thus yielding bright diffraction limited
spots. Therefore, it is an ideal signal amplification method for in situ analyses,
which require high spatial resolution.
The specificity of in situ detection can be improved if detection of target
molecules does not depend solely on hybridization, but also on enzymatic
ligation. Padlock probes, amplified by RCA, have been used to detect DNA as
well as mRNA in situ41, 88, 89. Larsson et al. demonstrated the detection of mRNA
molecules by first reverse transcribing the mRNA to cDNA. mRNA is then
digested by RNase H followed by hybridization and ligation of the padlock
probe to the cDNA target sequence (Figure 3)89. Examples of application areas
include the differentiation of gene isoforms, detection of somatic mutations in
tissue and detection of viral RNA89-91.
Figure 3. In situ rolling circle amplification. A) cDNA is synthesized from mRNA (in black) using
LNA modified primers (in grey). B) The mRNA is degraded by RNaseH and (C) a complementary
padlock probe hybridizes to its target sequence on the cDNA. D) The nick is sealed by a highly
specific ligase (in pink). E) Ligated padlock probes are locally amplified by a highly processive DNA
polymerase (in yellow). Fluorescently labeled oligonucleotides (orange stars) are hybridized to the
rolling circle product (RCP) and (F) visualized in a fluorescence microscope (RCPs in orange, nuclei
in blue).
19
In situ sequencing
Sequencing of the amplification products can be used to overcome the limitation
of the low multiplexity of traditional FISH, which is defined by the fluorophores
that can be spectrally resolved. In 2013, Ke et al. demonstrated targeted in situ
sequencing of RCA products allowing multiplex detection of mRNA transcripts.
Either an integrated barcode in the backbone of the padlock probe or a short
target site, filled by polymerization to circularize the padlock probe, can be
sequenced92. Drmanac et al. applied the sequencing-by-ligation technology to
sequence RCA products generated in vitro93. In situ sequencing is based on the
sample principle. An anchor primer is hybridized to the RCA products and a
sequencing library, consisting of random hexamers with one fixed position, is
ligated to it92. Each base is encoded by one fluorophore. The simultaneous
sequencing of 31 probes, partly based on the commercially available
OncotypeDx Breast Cancer assay, has been shown in breast cancer tissue
sections92.
A similar, but untargeted, approach has been described by Lee et al. Random
primers containing a sequencing adaptor are used for cDNA synthesis. After
RNA digestion and circularization of single-stranded cDNA, primers,
complementary to the sequencing adaptor, are used to prime the RCA. The RCA
products are covalently linked to cellular proteins and sequenced using the
SOLiD chemistry achieving a read length of 27 nucleotides. Partition
sequencing, using pre-extended anchor primers with mismatches at the ligation
site, is used to decrease signal density in order to discern single amplification
products. The authors demonstrated the simultaneous sequencing of thousands
of genes and their cellular localization94.
20
Diagnostic
methods
used
bacteriology and virology
in
Numerous methods are available that are commonly used for the diagnosis of
diseases of viral or bacterial origin. To give an overview of this broad field some
of the most widely used methods are described below. Requirements on methods
used in clinical practice differ regarding automation, sample handling and time
from methods used solely in a research setting. Cost-benefit analysis is
important in decision-making whether or not new methods should be
implemented into routine diagnostics. For a new method to be used for clinical
diagnosis it must not only be more sensitive or more multiplexed than currently
used methods, but time saving, labor and costs are equally important. Thus,
some methods have been used in routine diagnostics with little change for more
than 100 years albeit more sensitive but at the same time more expensive
methods exist.
Bacterial
methods
identification
by
culture
and
biochemical
For many bacterial infections culture, either on solid or in liquid medium, is
considered the gold standard in microbiology with it beginning already in the
19th century. Robert Koch demonstrated in 1881 the use of solid media for
bacterial culture, which can further be used for the isolation of pure cultures 95.
This laid the basis for medical diagnosis of bacterial infections as pure cultures
are essential for correct pathogen identification and determination of antibiotic
susceptibility. Most bacterial culturable pathogens form colonies on solid media
in 24-48 hours, but slow growing bacteria such as Mycobacterium tuberculosis
require longer culture time due to long generation time. Escherichia coli, the
most common pathogen in urinary tract infections (UTI)96, for example, has a
generation time of about 20 min97 whereas M. tuberculosis has a generation time
between 18 and 24 hours98. Bacterial growth on agar plates allows phenotypic
identification by morphology and odor. A single bacterium can give rise to a
colony consisting of thousands of clones99 and thus, microbial viability can be
examined. The plate count method, counting the number of colonies grown on
an agar plate, allows quantitative analysis of a sample. With its simplicity and
high sensitivity of 10 bacteria per ml100 it is highly suitable for clinical
diagnostics. Despite protocol optimizations it is estimated that less than 2% of
all bacteria can be grown in culture99. Non-culturable bacteria, such as the
syphilis causing bacterium Treponema pallidum, cannot be detected by culture
21
but must be detected by other means such as microscopic examination or by
molecular methods101.
To further identify or confirm identification by morphology of a single culture,
several biochemical tests can be performed. A number of commercially
available systems for bacterial identification exist where biochemical tests are
incorporated into a strip format allowing simple inoculation and identification of
clinically relevant pathogens with a specificity of around 90%102. Besides
limited specificity, the downside of these tests is the long turnaround time as
pure cultures are needed and the test itself requires an overnight incubation. In
recent years, automated systems such as the Vitek2 (BioMérieux) and the
Phoenix system (Becton Dickinson) have been widely used in large clinical
laboratories and replaced the above mentioned strip-based tests as they allow
automation and highly multiplexed analysis in a short period of time. For
optimal specificity and sensitivity a pre-culture is still needed103.
Cultures combined with biochemical tests are commonly used in clinical
microbiology laboratories due to the high sensitivity, specificity and ease of use,
but the long turnaround time impedes fast diagnosis. The below described
methods are more rapid and have partly replaced identification solely based on
culture and biochemical tests, but usually have other drawbacks such as lower
sensitivity or higher costs.
Microscopy for rapid microbial diagnosis
Light microscopy combined with different staining methods can be used for
bacterial identification. Smear microscopy for diagnosis of tuberculosis (TB)
was developed more than 100 years ago104 allowing diagnosis of the most
infectious cases (>5,000 – 10,000 bacilli per ml sputum)105. Due to its
simplicity, low demand on equipment and low cost per sample it is still the most
commonly used method, particularly in low resource settings. However, the
biggest drawback of smear microscopy is the low sensitivity, which is 50% of
culture106, and the inability to establish drug resistance profiles.
Electron microscopy (EM) has been widely used for diagnosis in virology107.
The high resolution of EM, due to the short wavelength of electrons, enables
imaging of viral particles108. Only a simple negative staining, to increase
contrast between specimen and background, is required before visualization
using EM109. As the staining procedure does not involve any pathogen specific
reagents, EM allows viral classification up to the family level without prior
knowledge of the infectious agent. A wide range of samples can be used for EM:
vesicle fluids, body excretions and biopsies. Sensitivity levels for EM are
around 105 particles per ml without the use of enrichment techniques 107 and time
from sample to readout can be as little as 15 min107. Despite its rapidity and low
22
cost per sample EM requires highly skilled personal and highly expensive
equipment and thus, is not suitable for screening purposes. The emergence of
nucleic acid-based methods has greatly replaced EM in viral diagnostics, but it
is still partly used for urgent diagnosis.
Immunoassays
Like microscopy, immunoassays have been widely replaced by nucleic acid
detection methods for clinical diagnosis, but are still used in certain instances.
Since the development of the enzyme-linked immunosorbent assay (ELISA)110
and the enzyme immunoassay (EIA)111 in 1971 numerous assays based on these
principles have been used in clinical diagnostics. Briefly, ELISA is based on the
detection of an antigen by an enzyme-linked antibody. Upon binding and
subsequent washes to remove unbound antibodies, a chemical substrate for
detection is added110. Application areas include the detection of the human
immunodeficiency virus (HIV)112 and detection of bacteria belonging to the
genus Borrelia, causing Lyme disease113. Sensitivity and specificity can be
extremely high with >98% reported for some commercial HIV assays 114, but
might be insufficient for other applications115.
Latex agglutination assay is another type of immunoassay that is used for
detection of pathogens, e.g., for rotavirus A detection116. Latex beads are
sensitized with antigens forming visible aggregates upon antibody binding 117.
The same principle can be used to detect antigens by immobilizing the
corresponding antibody onto the beads. These assays are rapid and simple
without requiring expensive equipment or extensive training, but their
specificity and sensitivity depend heavily on the antibodies used. Furthermore,
only conserved epitopes can be targeted as antigenic drift can cause falsenegative results.
The drawbacks of immunoassays, such as low sensitivity, are partially addressed
by nucleic acid amplification-based testing methods, which have entered into
routine diagnostic settings.
PCR-based methods for pathogen identification
Quantitative PCR is increasingly being used for routine diagnostics of infectious
diseases as it offers several advantages over more traditional methods: fast
turnaround time, high specificity and high sensitivity. Microbial pathogens can
be directly detected in clinical specimens rendering culture unnecessary. In
bacteriology, diagnostic applications include panbacterial PCR118, species
specific PCR119, 120 and antibiotic susceptibility testing (AST)121-123. Although
PCR-based methods are used for detection of fast growing bacteria, culture
23
remains the reference diagnostic method in many cases. Quantitative PCR is
particularly suited for the diagnosis of slow growing or difficult to culture
bacteria, such as Mycobacteria120 or Chlamydia trachomatis119, 124, and for lifethreatening conditions where rapid and correct treatment is essential as it
significantly reduces morbidity125.
In contrast to bacteriology, PCR-based methods are widely used in virology as it
offers several advantages over traditional methods like virus isolation in cell
lines126, 127. Cell culture for viral detection, which was regarded as the gold
standard for several decades, is slow and requires extensive technical expertise.
The introduction of qPCR to routine virology omitted the need of culture in
most instances offering rapid and sensitive diagnosis126. Although considered as
the “new” gold standard there is a risk that PCR-based methods will fail to
detect highly variable viruses. As reported by Ripa et al., genetic drift can cause
failure in primer hybridization and thus, unsuccessful amplification 128.
Therefore, constant surveillance of circulating strains is necessary.
Mass spectrometry
Mass-spectrometry (MS) plays a growing role in clinical microbiology
laboratories as it is a powerful tool for identification of microbial biomarkers 129.
MS measures the mass in relation to charge (m/z). The substrate to be analyzed
is first ionized in vacuum and then separated in a mass analyzer containing an
electro-magnetic field. Separation is based on the m/z ratio of the ionized
particles and the ratio output is measured by a detector. A widely used massspectrometer in clinical microbiology laboratories is the time-of-flight (TOF)
mass-spectrometer, which offers sensitive, parallel ion detection with a very
high mass range. The flight time of each particle to the detector is recorded
whereby heavier particles have a longer travel time130, 131. Different ionization
techniques have been developed to enable the study of biological samples. One
commonly used technique is matrix-assisted laser desorption/ionization
(MALDI). In MALDI, the sample is first mixed with an appropriate matrix
followed by short laser pulses to desorb and ionize the sample for analysis in a
mass-spectrometer132. The matrix thereby absorbs the radiation and prevents
fragmentation of large molecules133. Thus, MS coupled to MALDI allows
analysis of complex biological matrices, as there is little or no fragmentation of
biomolecules during the ionization stage.
MALDI-TOF MS has entered clinical microbiology laboratories allowing
simple, rapid, and high-throughput analysis of patient samples for the presence
of pathogens. The spectrograms obtained are compared to a database for
pathogen identification. Already in 1975, Anhalt and Fenselau demonstrated the
use of MS for identification of bacteria134. Since then there have been a number
24
of reports on the use of MALDI-TOF for reliable identification of bacterial
species135-137 but all of these studies required bacterial isolates. Seng et al.
observed an accuracy of 95.4% compared to conventional phenotypic
identification establishing its usability in routine diagnostics 135. In order to have
an even greater impact on clinical diagnosis, identification of pathogens directly
from body fluids is desirable. Direct identification has been shown on blood
culture samples but several washing and centrifugation steps were required 138140
. Overall, the specificity in these studies ranged between 75.8 and 80.4% and
required culture before analysis138-140. Thus, further development such as
database updates and optimization of sample preparation protocols are needed
for accurate analysis of body fluids. As sensitivity of MALDI-TOF MS is low,
only body fluids containing a high bacterial load e.g., urine samples or blood
cultures can be analyzed.
Methods for antibiotic susceptibility testing
Antibiotic susceptibility testing is an important part of clinical microbiological
diagnosis as effective antibiotics determine treatment success. The most
commonly used methods rely on phenotypic analysis such as disk based
diffusion assays and microdilution assays. PCR is commonly used for genotypic
testing in research settings, but is not widely implemented in diagnostic
laboratories with an exception being drug susceptibility testing for TB.
The classic approach to AST is based on the determination of the minimum
inhibitory concentration (MIC), which is the lowest concentration of an
antibiotic that inhibits growth. In 1971, an international collaborative study on
antibiotic sensitivity testing tried to standardize testing procedures for
determination of MICs and recommended agar dilution and disk diffusion
assays141, which are still commonly used in routine clinical microbiology
laboratories. The agar dilution method uses a series of agar plates with different
concentrations of antibiotics to determine the MIC for visible microbial
growth142. A similar method, called broth dilution method, uses liquid medium
instead of agar plates. The disk based diffusion assay has the advantage that it is
technically quite simple and easy to perform by low-skilled users. Furthermore,
several antimicrobial substances can be tested on the same plate. Antibioticimpregnated disks are placed on an agar plate, inoculated with the strain to be
tested and incubated overnight. Zones of no growth are measured and zone
diameters are compared to published values to classify the tested strain as
susceptible or resistant143. The agar dilution method is considered to be the
standard method and therefore, other methods need to be correlated to it142. The
above described methods for AST require a pure culture as a starting point plus
at least one overnight culture. There is an obvious need for more rapid methods
25
that can readily be applied in the clinics. I will describe some of the most
promising and widely used technologies below.
Instead of phenotypic determination of antibiotic resistances, qPCR can be used
for genotypic analysis detecting the presence of resistance genes or point
mutations causing resistance. There have been several reports in the last two
decades describing the use of qPCR for AST144-147. One widely used qPCR
system for drug resistance detection in TB is the GeneXpert MTB/RIF test
(Cepheid), endorsed by the World Health Organization (WHO) in 2010 148. This
system has the advantage of a fully integrated sample processing from nucleic
acid isolation to amplification and detection149 and thus limiting crosscontamination and hands-on time. Furthermore it has a significantly improved
turnaround time of two hours120 compared to conventional culture based testing,
which can take weeks to months in the case of M. tuberculosis98. Although much
faster than the methods described above, the major disadvantage of PCR-based
tests is that novel, previously unknown resistance genes will be missed as they
will not be covered by the PCR primers. In addition, the presence of a resistance
gene does not automatically imply phenotypical resistance150. Furthermore,
multiplexing and thus, targeting all relevant mutations requires extensive
optimizations.
Recently, MALDI-TOF MS has been used to phenotypically detect resistances
towards β-lactams151-155. Detection by MS for these classes of antibiotics is
possible by monitoring the mass peak for the antibiotic and their corresponding
hydrolyzed product154. Absence of the peak of the hydrolyzed product
corresponds to susceptible bacteria, as they do not synthesize the enzymes able
to degrade β-lactams. The main limitation of this technique is the requirement of
a fresh culture, usually grown overnight156. In order to be considered a general
method for AST, the detection of resistance mechanisms where the antibiotic
uptake is reduced or the drug is pumped out of the cell and the detection of
multi-resistant bacteria must be further developed as current methods are either
too labor-intensive and expensive or are unable to accurately quantify antibiotic
concentrations156.
Several methods for ASTs have been developed using microfluidic
technologies157-159. Cira et al. and Choi et al. used a microfluidic device that
visually monitors the growth of bacteria in the presence of antibiotics 158, 159.
Microfluidic approaches offer the advantage that several antibiotics and
concentrations can be tested in parallel with a very low number of bacteria
required but they most often lack species identification and thus additional
methods are needed.
26
Summary of diagnostic methods
Below is a brief summary of the above described methods (Table 1). As can
be seen in the table each method has its advantages and disadvantages and
thus, must be carefully chosen for each diagnostic question and depending
on available resources.
Table 1. Comparison of widely used diagnostic methods in bacteriology and virology
Method
Time
(approx.)
Pros
Cons
Refs
Culture (bacteria)*
Overnight
Sensitivity (10
bacteria per ml)
AST is possible
Trained personnel for
identification required
Only 2% of all bacteria are
culturable
99, 100
Light microscopy
(bacteria)
Few hours
Applicable to low
resource settings
Short time
Sensitivity (5,000-10,000
bacilli/ml for M. tuberculosis)
No AST possible
105
Electron
microscopy (virus)
15 min-1
hour
Short time
No prior knowledge
of virus needed
Sensitivity (105 particles/ml)
Low throughput
Skill-based
High equipment cost
107
Immunoassay
(virus)
30 min-1
hour
Short time
Simplicity
Suitable for
automation
Specificity depends on available
antibodies
Antigenic drift can cause falsenegatives
Low multiplexity
160
qPCR (virus)
2-3 hours
Sensitivity
Specificity
High-throughput
Suitable for
automation
Antigenic drift can cause falsenegatives
Low multiplexity
Trained personal required
Cost per sample
128,
160
Short time
Sensitivity (subculture of 105
Accuracy
bacteria is required)
Simplicity
High equipment costs
High-throughput
Certain bacterial species are
No prior knowledge
difficult to distinguish
of bacteria needed
*
The pathogenic agent in brackets denotes the pathogen that the method is mainly used for
MALDI-TOF MS
(bacteria)
10 min
135,
161,
162
27
Methods for prenatal diagnosis of
chromosomal aneuploidies
Trisomies are the most common chromosomal anomalies in humans, mainly
caused by non-disjunction during meiosis. Trisomy 21, known as Down
syndrome, is the most common one163, occurring in 1 in 800 live births164,
making up 30-40% of all chromosomal abnormalities 163. The second and third
most prevalent trisomies are trisomy 18 (Edwards syndrome), followed by
trisomy 13 (Patau syndrome). As chromosomal aneuploidies have severe effects
on the offsprings’ health, several prenatal diagnostic and screening methods
have been developed to aid in prenatal counseling.
Molecular, non-invasive, diagnostics offer several advantages compared to more
traditional, invasive techniques. The biggest advantage of accurate non-invasive
techniques is to render invasive procedures unnecessary, which are associated
with a certain risk for procedure-related pregnancy loss165, 166. In addition to the
information gained regarding chromosomal aberrations, targeting nucleic acids
with single nucleotide resolution can yield information about other potential
health defects, e.g., mutations causing cystic fibrosis.
Traditional prenatal testing
Traditionally, prenatal screening methods rely on the measurement of several
biochemical markers in maternal blood and on ultrasonography. For definite
diagnosis invasive procedures are needed. Already, in 1984 an association
between low maternal serum alpha-fetoprotein (MSAFP) levels and
chromosomal abnormalities was reported167. An increase in nuchal translucency
was first correlated with chromosomal defects in 1992168. Several options for
prenatal screening for fetal chromosomal abnormalities to risk-stratify patients
are available, e.g., measurement of nuchal translucency thickness and
concentration of maternal serum biochemical markers such as the pregnancyassociated plasma protein-A (PAPP-A), maternal serum free β-human chorionic
gonadotropin (β-hCG), inhibin A, unconjugated estriol and MSAFP169. Upon
presentation for prenatal screening in the first trimester combined first and
second trimester screening should be offered as it has the highest detection rate
of chromosomal aneuploidies170. The quadruple test alone, consisting of protein
concentration determination of MSAFP, hCG, unconjugated estriol and inhibin
A has a detection rate for Down syndrome of about 80%170. By combining this
first trimester screening with measurement of nuchal translucency and
measurement of PAPP-A detection rates for Down syndrome of up to 96% can
be achieved170.
28
Upon receiving a high risk score genetic counseling in combination with a first
trimester chorionic villus sampling (CVS) (at about 9-11 weeks of gestation) or
a second trimester amniocentesis (at about 15 weeks of gestation) should be
offered169. Traditionally, chromosome diagnosis was done by karyotyping.
Condensed chromosomes, present in the metaphase, are stained by Giemsa
banding and analyzed in a microscope171. Since amniotic fluid does not contain
dividing cells a cell culture must be performed first and thus, delays diagnosis.
Cell culture is also used to confirm diagnosis on cells obtained directly by
CVS172. Molecular methods for prenatal diagnostics such as FISH and
quantitative fluorescent PCR (QF-PCR) provide a faster turnaround time.
Although faster than karyotyping, FISH is labor intensive and requires intact
cells to accurately detect chromosomal aberrations173. QF-PCR on the other
hand requires the presence of polymorphisms such as short tandem repeats and
is less informative than karyograms172. Despite newly developed techniques
chromosome karyotyping via Giemsa banding remains the gold standard for
detection of aneuploidies from invasive samples with a false negative rate of
below 1%174. Although conventional chromosome analysis has a very high
detection rate, small but significant risks are associated with the necessary
invasive procedures. Procedure-related pregnancy loss rates after secondtrimester amniocentesis has been estimated to be about 1 in 370-600165, 166 and
similar loss rates are estimated for CVS166. Furthermore, the long turnaround
time of traditional techniques render non-invasive prenatal testing (NIPT), an
attractive alternative. In addition, waiting times for screening results between the
first trimester and second trimester screening can cause psychological burden on
the patient.
Non-invasive prenatal testing
Fetal cells have been recovered from maternal plasma for the first time already
in 1969175 and the feasibility of using these to detect Down’s syndrome has been
demonstrated176, 177. Nonetheless, they have not been extensively used in
prenatal diagnostics due to their rarity, limiting robust detection178. In 1997, Lo
et al. discovered cell-free fetal DNA (cffDNA) in maternal plasma and serum179.
The presence of large amounts of background maternal DNA has posed
challenges to the development of accurate NIPD technologies. It has been shown
that cffDNA concentration correlates with gestational age180. The small fraction
of fetal DNA, up to 10 % in gestational week 10181, in maternal blood makes it
challenging to detect chromosomal aneuploidies using cffDNA as its sequence
content is nearly identical to maternal DNA. Evidence exists that the majority of
cffDNA is derived from apoptotic cells as it is enriched for nucleosome-bound
fragments182 with a size peak at <200-300 bp, shorter than cell-free maternal
DNA182-184. cffDNA has a relatively short half-life of less than two hours and is
cleared rapidly from circulation after delivery174, 175. Hence, it is well suited for
29
prenatal diagnosis as no fetal DNA from former pregnancies will be present at
the point of testing. Fetal progenitor cells, on the other hand, have been detected
in maternal blood even 27 years postpartum185. Several different non-invasive
methods using cffDNA have been developed in the last couple of years with the
majority being sequencing-based. Below I describe approaches that are based on
differential methylation to distinguish fetal from maternal DNA, approaches that
use massively parallel sequencing (MPS), and approaches using digital PCR. I
will then give a short overview of commercially available assays for NIPD
detecting chromosomal aberrations.
The methylation pattern between placental-derived and maternal-derived DNA
differs and several markers have been discovered that can potentially be used in
NIPD186, 187. Several methods have been reported using methylation markers for
NIPD of trisomies188-190. Approaches used include bisulfite conversion and
sequencing of PDE9A, a gene on chromosome 21 that is completely methylated
in blood but unmethylated in placenta188, fetal DNA enrichment using
methylation-specific antibodies combined with qPCR189 and bisulfite conversion
combined with methylation specific PCR190. These studies based on differential
methylation patterns had significant drawbacks such as low sensitivity 188, 190,
high false-positive rates188, 190 or showed low reproducibility191 leading to poor
diagnostic performance.
Fan et al. and Chiu et al. demonstrated the use of MPS to precisely enumerate
DNA fragments in a locus-independent fashion and thereby detecting fetal
aneuploidies in maternal blood without any further enrichment of fetal DNA 182,
192
. The large number of counted sequences allowed robust diagnosis of fetal
trisomy 21, detected by the overrepresentation of the corresponding sequence
tags182, 192. Chromosomes with a high GC content showed a large variation in
sequence counts and thus, might have limited detection sensitivity. Chromosome
13, 18 and 21 showed low variance making them suitable for NIPD using DNA
sequencing182.
Digital PCR on the other hand is technically challenging as it requires thousands
of PCR reactions to correctly detect trisomy 21 in samples having low fractional
DNA concentrations193, 194. Thus, for accurate diagnosis additional enrichment of
fetal DNA sequences from maternal plasma is required.
In the last couple of years several tests for NIPD have been commercialized but
have not yet been implemented into routine diagnostics. Most commercially
available tests for NIPD rely on next-generation sequencing as sequencingbased technologies proved to be advantageous in terms of sensitivity, precision
and robustness195. The US-based companies, Verinata and Sequenom, use nontargeted MPS technologies whereas Natera uses targeted SNP-based sequencing
and until recently Ariosa’s technology was based on chromosome selective
30
sequencing196-199. In 2014, Ariosa demonstrated that their chromosome targeted
approach in combination with microarrays yields more accurate results than
when combined with sequencing technologies200. Costs for a single test range
approximately between $800 and $2,700199 contributing to the fact that NIPT is
not yet implemented for population-wide prenatal screening.
31
Molecular Diagnostics in Oncology
Cancer is a disease characterized by a large degree of heterogeneity, exhibiting
intra- as well as inter-tumor heterogeneity201-203. Underlying causes of the
observed heterogeneity include numerous somatic mutations, such as point
mutations, copy number variations and chromosomal rearrangements202, 204, 205.
These molecular signatures characterize a cancer and can offer valuable
information about the best treatment course. However, detailed characterization
of these signatures is not possible by the widely used TNM staging system,
which stands for tumor, node and metastasis. The tumor is examined based on
size, the proximity to lymph nodes and the presence of metastasis. Molecular
methods, on the other hand, can give information about the mutational landscape
of a tumor which can be used to estimate the likelihood of recurrence in breast
cancer patients206 or response to therapy206. Additionally, information on the
origin of the tumor207 can be gained from these signatures. Evidence exists that
target-based treatment strategies are only effective in a subpopulation of cancer
patients and thus, knowledge of the mutational status of a tumor is essential for
effective targeted therapy208, 209. Predictive biomarkers include mutations in
cancer driver genes, such as in the epidermal growth factor receptor (EGFR) in
lung cancer, changes in methylation, and changes in expression levels as
observed for the estrogen receptor in breast cancer208.
About a decade ago a number of molecular tests for gene expression profiling in
cancer emerged on the market. Many of these tests for cancer diagnostics are
based on PCR or microarrays. Sequencing-based technologies are often only
used as a discovery tool or for confirmatory purposes, but are expected to play
an increasing role in cancer diagnostics in the near future. Perou et al.
demonstrated a large heterogeneity in expression pattern in breast cancer 210.
Based on the observed heterogeneity several assays have been developed for
risk stratification of patients206, 211-214. Genomic Health’s Oncotype Dx, a
multigene breast cancer assay, has been incorporated into clinical guidelines of
the American Society of Oncology215. The Oncotype Dx assay determines the
risk for recurrence and response to therapy in breast cancer by examining 21
genes in a RT-PCR-based assay206.
Most molecular tests used in the clinical diagnosis of cancer do not have single
cell resolution as nucleic acids are analyzed in bulk measurements. Thus, spatial
information and detailed information on heterogeneity are lost. Sanger
sequencing has been used for mutation detection in the Kirsten rat sarcoma viral
oncogene homolog (KRAS). Although, in samples containing less than 10%
tumor cells a significantly lower mutation rate was reported and thus, requiring
manual microdissection for tumor enrichment216. Newly developed assays,
32
mostly PCR-based, have reported improved sensitivities of as low as 1% mutant
alleles217. But nonetheless, these molecular methods must often be accompanied
by histopathology to guarantee accurate diagnosis. In situ techniques are able to
combine molecular testing and histopathology into one test and thus, could
eliminate the requirement of several assays.
One example of in situ techniques for cancer diagnosis is the use of FISH for
gene expression analysis in breast cancer patients, as different patterns are
associated with different clinical outcomes218. Overexpression of the human
epidermal growth factor receptor 2 (HER2) is commonly measured since it is
positively correlated with response to trastuzumab, a monoclonal antibody
binding to HER2 and thus inhibiting proliferation219. As described earlier, FISH
is an excellent method for low-plex gene expression analysis in situ. However, it
does not offer single nucleotide resolution needed for the analysis of point
mutations that might significantly affect treatment success.
Padlock probes, on the other hand, are a reliable tool for detection of point
mutations in situ. The potential of padlock probes for clinical application has
been demonstrated by mutational analysis of the most common sample
preparations used in diagnostic routine: fresh frozen tissue, formalin-fixed,
paraffin-embedded tissue, tissue microarrays and tumor touch imprints. A
mutation detection rate of 1% makes the padlock probe technology a potential
tool for clinical diagnosis of tumor samples90.
33
Present investigations
Detection of rotavirus using padlock probes and rolling
circle amplification
In this work, we applied padlock probes to detect a highly variable double
stranded RNA virus in clinical samples.
Rotavirus is a highly contagious virus, causing diarrhea. By the age of five
nearly every child has experienced at least one episode of rotavirus infection 220.
Rotavirus is the number one pathogen recorded for hospitalizations due to
gastrointestinal diseases221 and contributes to a high number of deaths, mainly in
developing countries222. Thus, the financial burden of rotavirus infections is high
and simple and accurate diagnostic methods are needed to control its outbreak,
and for surveillance purposes. Diagnosis is usually achieved by a latex
agglutination assay. Although the latex agglutination assay is rapid and easy to
perform, it lacks sensitivity223. RT-PCR, which is mainly used in research
settings, overcomes this limitation, but due to the highly variable nature of
rotavirus, strain variants can be easily missed if not covered by the primers.
In this study we designed a pool of padlock probes covering ~95% of the
published rotavirus A sequences in the NCBI database. The pool consisted of six
padlock probe mixes whereby four of these included degenerated bases creating
a total number of 58 unique padlock probes. As padlock probes are easily
multiplexed and amplified independently of each other, new strain variants can
be included in the assay by a simple addition of new padlock probes to the
already existing pool. RNA was extracted from fecal samples and transcribed
into cDNA using degenerated primers able to cover a large part of rotavirus
sequences. RNA/cDNA hybrids were heat denatured and the cDNA was
subsequently captured on magnetic beads. Padlock probes were hybridized and
ligated at temperatures significantly higher than the melting temperature of each
probe arm to ensure specific ligation. Unbound probes were washed away as
they inhibit the subsequent amplification reactions. Ligated and circularized
padlock probes were amplified by RCA. To increase the amplification factor,
RCPs were monomerized by restriction enzyme digestion and a second round of
RCA was carried out. Fluorescently labeled RCPs were detected in a
microfluidic setup using an optical imaging system.
With the setup described, we achieved a sensitivity of 1,000 synthetic targets
equivalent to the cDNA. To evaluate the efficiency of a multiplexed assay,
different numbers of probes were added and amplified. A decrease in signal with
an increase in probe number was not observed. Thus, inhibition of competing
34
probes could be ruled out. Twenty-two clinical samples were tested with our
assay, including two rotavirus negative samples. Seventeen of these samples
were diagnosed as positive and confirmed by a PCR-based assay using agarose
gels as a readout format except for one sample that resulted positive only by the
padlock assay. The remaining three rotavirus positive samples were neither
detected by our assay nor did they yield a specific band on the gel. This might
be explained by failed cDNA synthesis due to low RNA quality or due to
mismatched primers.
To summarize, we demonstrated a highly variation tolerant assay for detection
of viral RNA using padlock probes and RCA. The whole assay from cDNA
synthesis to readout can be completed within three hours. Our assay has
advantages over RT-PCR as it can be highly multiplexed to cover a wide range
of strain variant without loss in efficiency.
A general method for rapid determination of antibiotic
susceptibility and species in bacterial infections
In this work, we established a rapid assay for simultaneous species identification
and AST directly in clinical samples.
Extensive use and over-prescription of antibiotics has led to an increase in
selective pressure on bacteria and contributed to the spread of antibiotic
resistances224. Resistance mechanisms develop rapidly and no novel class of
antibiotics has been discovered in the last 25 years. The WHO recently
published a report underlining the alarming rate at which pathogens become
resistant and thus, impact our ability to treat common infectious diseases 225. In a
post-antibiotic era, previously curable diseases might turn to be deadly once
again. As traditional identification of bacterial species and determination of
antibiotic susceptibilities is slow, antibiotic treatment is often empirical and
thus, ineffective drugs might be prescribed. Therefore, rapid AST is essential to
maximize optimal use of antibiotics.
We merged the traditional culture based diagnostics with molecular methods for
rapid identification and quantitation to achieve AST and demonstrated its use for
diagnosis of UTI. We designed padlock probes for the common urinary
pathogens, E. coli, Pseudomonas aeruginosa and Proteus mirabilis, targeting
the 16S rRNA gene. Samples were split into several vials and cultured in the
presence and absence of antibiotics for a short period of time. Subsequently
bacteria were lysed by simple addition of sodium hydroxide and heat. The
targeted DNA sequences were captured on magnetic beads followed by a quick
wash to remove the sample matrix. The species specific padlock probes were
hybridized and ligated in order to be amplified by C2CA. For detection, an
35
optical imaging system was used allowing digital quantification of RCPs.
Comparison of counts of antibiotic containing samples with the sample lacking
antibiotics established the antibiotic resistance profile (ASP) of the targeted
bacterial species.
We first investigated the time necessary to detect bacterial growth. Using our
padlock probe-based method growth could already be detected after 30 min of
culture whereas a viable count did not detect any growth before 90 min of
culture. Second, we tested the specificity of the padlock probes by parallel
incubation with the three target bacterial strains. No unspecific signal was
observed for non-matching padlock probes. To establish an ASP for E. coli for
ciprofloxacin and trimethoprim, three different strains were tested. As expected,
resistant strains showed growth in the presence of the antibiotic they were
resistant to. Only minor growth was observed in the presence of the antibiotic
for which the tested bacterial strain lacked resistance mechanisms. A first set of
32 patient samples was used as a training set to establish cut-off values for
determination of antibiotic resistances in E. coli. The developed algorithm was
verified by a blind prospective study comprising 56 urinary samples. Species
identification for E. coli was correct in 55 out of the 56 analyzed samples with
the 56th sample having fewer bacteria than required to be considered a UTI 226.
No false-positives were observed and resistance profiles were correctly
identified for all samples containing E. coli above the threshold for UTI.
Furthermore, all results were in concordance with the routine testing performed
at a clinical laboratory.
In conclusion, we have established an assay that is not limited to UTI, but can be
adapted for AST in other infections, such as TB or sepsis. The number of
antibiotics tested can be increased by simply adding an extra vial per antibiotic.
Additional pathogens can be targeted by either a parallel or multiplexed
approach using a multicolor detection system rendering this assay suitable for
general bacterial identification and AST.
Elimination of maternal DNA for accurate non-invasive
prenatal testing: a pilot study
In this work, we have established an assay for detection of trisomy 21 and 18
that uses differential methylation for enrichment of fetal DNA and padlock
probes amplified by RCA for precise quantitation.
Traditional prenatal diagnosis for chromosomal abnormalities rely on screening
methods, such as measurement of biochemical markers and nuchal
translucency169. If high-risk scores are obtained, invasive methods can be used to
confirm diagnosis169. Invasive methods are however associated with a certain
36
risk for procedure-related abortions165, 166. The discovery of cff-DNA179 has
initiated intensive research to develop precise and reliable tests for NIPD with
the goal to render invasive tests unnecessary. In the last couple of years
numerous methods, mainly sequenced-based, have been developed195. A
drawback of these methods are the laborious nature and high cost. We therefore
propose a padlock probe-based assay achieving precise enumeration of target
molecules by RCA and fluorescent detection.
Target sites, that are hypermethylated in placental-derived DNA and
hypomethylated in maternal DNA were chosen186, 187. All sites included the
recognition sequence for HpaII, a methylation sensitive restriction enzyme.
Before probe hybridization and ligation, samples were incubated with HpaII
cleaving unmethylated maternal-derived DNA and thus, inhibiting probe
ligation and subsequent amplification. Ligated padlock probes were amplified
and detected using a confocal microscope or an automated, commercially
available two-color imaging system. A ratio of signals received from the two
chromosomes was calculated and used for determination of trisomy.
An excellent sensitivity of between 30 and 300 genomic equivalents was
achieved with the above described assay. We observed a decrease in variation
with an increase of counted objects, yielding a coefficient of variation of below
5% for 3,000 genomic equivalents. Female DNA, reflecting maternal DNA, was
used to determine the enrichment factor obtained by HpaII digestion. A threeand five-fold enrichment for chromosome 21 and chromosome 18, respectively,
was obtained by using HpaII. Fourteen CVS samples, including four trisomy 21
samples and four samples with non-targeted chromosomal abnormalities, were
analyzed. The use of a cut-off value allowed correct identification of all trisomy
21 samples. When CVS samples were spiked into female DNA, a fetal fraction
of 30-40%, was required for accurate diagnosis.
To conclude, we have developed an assay that allows correct identification of
trisomy 21 and 18 in invasive samples based on precise quantification of RCPs.
With slight adaptations, such as increasing the number of differentially
methylated target sites, it should be possible to increase sensitivity and thus,
precision for robust and accurate NIPT.
Oligonucleotide gap-fill ligation for mutation detection
and sequencing in situ
In this work, we developed a novel approach for in situ mutation detection using
gap-fill ligation. Specificity, in this assay, relies on the requirement of two
ligation events to occur in order to circularize a padlock gap probe. Furthermore,
the generated RCPs can serve as a substrate for in situ sequencing.
37
The high specificity of ligases can be utilized to distinguish single nucleotide
differences45, 46. Oligonucleotide gap-fill ligation combined with padlock gap
probes and RCA can be used to visualize SNVs or point mutations in situ.
Padlock gap probes are similar to conventional padlock probes, but differ at the
ligation site. Instead of directly ligating the two target complementary arms, a
short gap oligonucleotide is ligated to the padlock arms. Hence, two ligation
reactions instead of one are necessary to circularize the padlock gap probe. The
use of short gap oligonucleotides ensures specificity and offers a simple way to
distinguish between mutant and wild-type sequences.
We designed padlock gap probes, spanning a gap of six nucleotides, and a pool
of the corresponding gap oligonucleotides to target the mitochondrial A3243G
mutation causative of mitochondrial encephalomyopathy, lactic acidosis, and
stroke-like episodes syndrome (MELAS)227 and an expressed SNV in the ACTB
gene. Furthermore, we targeted a mutational hotspot in the EFGR gene
indicative for treatment response. Two requirements are necessary to obtain a
signal with our approach. The targeted mutation or SNV must be present and the
respective gap probe needs to be phosphorylated at the 5’ end in order to be
ligated. Thus, absence or presence of a signal was used to discriminate closely
related sequences. We evaluated the assay for efficiency and specificity using a
pool of gap probes. The specificity of the gap-fill ligation was further validated
by in situ sequencing.
A mutant cell line for the A3243G mutation and a wild-type cell line were tested
with the gap-fill approach. A total of seven gap probes were used and signal was
only detected in the wild-type cell line if the fully complementary gap probe was
phosphorylated. Phosphorylation of the mutant gap probe did not yield any
signal on the wild-type cell line, but on the cell line carrying the targeted
mutation. We estimated an efficiency of ~80% for the padlock gap probes
compared to conventional padlock probes. We further showed the applicability
of our approach to mRNA by specific detection of an SNV in mouse and human
fibroblasts. Addition of the gap probes, all being phosphorylated, generates
substrates for in situ sequencing making this assay suitable for simultaneous
multiplex mutation detection. We successfully genotyped the SNV in ACTB in
human and mouse fibroblast cells using gap-fill ligation combined with in situ
sequencing. Furthermore, we detected a clinically relevant point mutation in the
EGFR gene in lung cancer tissues. Specificity of the gap-fill ligation was
confirmed by in situ sequencing.
This assay design has the advantage of offering a simpler and cheaper design for
multiplexed mutation detection compared to the use of a pool of padlock probes
targeting several mutations in a short stretch of nucleotide sequence. The double
ligation ensures high specificity with minimal loss of efficiency. Potential
application areas include screening for mutations in mutational hotspot areas.
38
Concluding remarks and future
perspective
In this thesis, I have demonstrated the use of padlock probes combined with
RCA for a wide spectrum of diagnostic purposes. Although the novel methods
described in this thesis are not yet ready for implementation into routine
diagnostics, they certainly have the potential for application in clinical
diagnostics especially if they can be automated and thus, minimize manual
labor. Automation, labor time and cost are important factors one must consider
before employing new techniques in a clinical setting. Several efforts have been
undertaken, by our group and others, in order to automate padlock-based RCA
protocols, both in vitro and in situ228-231. However, they still require further
development to allow sample processing in a completely automated fashion.
This can, for example, be done by implementing the whole protocol on a
pipetting robot or by designing a dedicated microfluidic chip for each assay.
Besides automation, additional parameters must be fulfilled until a laboratory
test can be offered for clinical diagnosis. In the US, laboratory-developed tests
that provide information on diagnosis and treatment options must adhere to the
Clinical Laboratory Improvement Amendments (CLIA)232. Similar guidelines
exist in other countries. CLIA requires the verification and validation of the
accuracy and reliability of a diagnostic test to ensure the validity of clinical test
results232, 233. Thus, the assay developments described in this thesis are still at the
very beginning on the potential road to a clinical diagnostic laboratory.
In summary, I have described the development of padlock probe-based assays
for use in molecular diagnostics in four different application areas. The
application areas, described in this thesis, are quite diverse, demonstrating the
wide applicability of padlock probes and rolling-circle amplification in the
diagnostic field. Depending on the diagnostic question padlock probes might be
an interesting alternative to already existing methods as they have advantageous
properties in regard to specificity and multiplexity, and additionally offer local
target amplification and digital quantification. If used in the right context,
padlock probes combined with RCA can generate powerful diagnostic tools
overcoming limitations of techniques currently employed in diagnostics.
39
Populärvetenskaplig
sammanfattning på svenska
Hur framgångsrik en medicinsk behandling blir beror ofta på tillgången till
exakta och tillförlitliga diagnostiska analyser för att vägleda läkare i val av
behandlingsmetoder. Ett optimalt test utmärker sig med avseende på specificitet
och känslighet. Beroende på användningsområdet kan snabbhet, låg kostnad och
enkelhet vara lika viktiga kriterier. För infektionsdiagnostik är analystiden
avgörande eftersom tid till påbörjad behandling oftast är kritisk för
tillfrisknande. Inom andra områden är tidsaspekten av mindre betydelse, till
exempel inom icke-invasiv fosterdiagnostik, en metod med vilken man testar för
fosterskador och ärftliga sjukdomar. Inom fosterdiagnostiken är specificitet och
känslighet de viktigaste parametrarna eftersom en feldiagnos kan få stora
konsekvenser.
I denna avhandling beskriver jag utvecklingen av fyra olika metoder avsedda för
molekylär diagnostik, alla baserade på så kallade ”padlock prober” (molekylära
hänglås) och rullande cirkel amplifiering. Trots att de utvecklade metoderna är
avsedda för olika tillämpningsområden, såsom diagnostik av infektiösa
sjukdomar, fosterdiagnostik och onkologi, har de gemensamt att de övervinner
vissa begränsningar som nu tillgängliga diagnostiska metoder har. Denna
avhandling innehåller två nya tester som riktar sig mot smittämnen: ett test för
att specifikt detektera rotavirus, ett mycket variabelt dubbelsträngat RNA-virus
som orsakar bland annat diarré. Det andra testet utgör ett nytt analysformat för
antibiotikaresistensbestämning vilket är snabbt och generellt tillämpbart för
olika patogener. Vidare beskriver jag utvecklingen av en metod för att anrika
fostrets DNA ur moderns blod, som använder DNA metylering som markör för
fostrets DNA. Detta DNA används för att exakt mäta kromosomförhållanden
och därmed upptäcka trisomi 21 och 18, vilket innebär en extra kopia av en viss
kromosom hos fostret. Den fjärde metoden som beskrivs i denna avhandling
använder speciella hänglåsprober för att upptäcka diagnostiskt relevanta
punktmutationer med hög specificitet direkt i celler och tumörvävnad.
De presenterade metoderna har potential att användas inom klinisk diagnostik,
efter automatisering av protokollen samt studier som validerar och verifierar
resultaten. Dessutom visar dessa metoder på den breda tillämpbarheten av
hänglåsprober som genom sina egenskaper avseende specificitet och
multiplexitet, är utmärkta verktyg för specifik detektion av nukleinsyra i provrör
och i celler.
40
Acknowledgments
This thesis would not have been possible without the contribution and support of
many others and I would like to express my sincere gratitude to everybody
involved in one way or another:
My supervisor Mats for accepting me as a PhD student and introducing me to
the world of padlock probes and RCA. Thank you for all your inspiration,
encouragement and endless optimism, never doubting that I will finish in time!
Thank you for believing in me! I really feel lucky I was able to pursue my PhD
studies in your group.
I spent the first two years of my PhD studies in Uppsala and would like to thank
Ulf Landegren, Ola, and Masood for creating such a nice and inspiring
research environment! It has been a great place to work in.
Dan Andersson, thank you for all your enthusiasm and for always making me
feel welcome in your lab! It was great getting the chance to work with you on
the UTI project!
I also would like to thank my collaborators who have contributed to the work
presented in this thesis: David, Christina and Jonas, for working together on
the rotavirus project. Erik, Anna, Eva, David and Jenny for the wonderful
collaboration on the UTI project. Erik, thanks for teaching me everything I
needed to know about microbiology, for coming in at the weekend to open the
door for me and sharing your lab space with me (sorry for the mess…)! Thank
you Eva for introducing me to routine clinical diagnostics and Jenny for always
having good advice on anything related to RCA. Maj, Lucia, and Stephanie,
for the valuable collaboration on the trisomy project. Thank you Maj for all
your input and comments. You are a truly inspiring scientist! Marco, Xiaoyan,
Chatarina, Linnea and Johan, thank you for the great collaboration on the gapfill project!
To the present and past members of the Nilsson lab for creating such a nice
place to work in and for all the fun in and outside the lab: Marco, thank you for
everything! For never getting tired of listening to my complaints, for always
trying to cheer me up, for the shopping trips, for the good advice I received
during the years and for everything else which I would neither have the time nor
the space to mention here! I don’t know how I would have survived without
you! ;-) Anna, thank you for being such a great friend and colleague! For all the
laughter, for sharing the highs and lows of life and work, for the glasses of wine
and for offering me samples for my diagnostic projects ! Iván, thanks for
being the number one resource in any chemistry-related question, for the best
41
popcorn of all times and for bringing me, together with all my stuff, safely
home! And of course, for being super enthusiastic about the boat! You will be a
great marinero! Annika, thanks for taking such good care of the lab, for the
great road trip and for always lending an ear for all and everyone! David, thank
you for introducing me to the lab and teaching me everything about C2CA and
for the fruitful collaborations! Malte and Elin L, thanks for the great
snowboarding trip to Järvsö! It was really fun! Malte, thanks for all the great
talks, for sharing lab bench and for being enthusiastic about our projects (at least
most of the time )! Elin L, for sharing a room at the conference, for the nice
conversations and for organizing the lab! Tom, thanks for the yummy pirogi, the
delicious hazelnut vodka and for taking good care of our very demanding
Aquila! Thomas, thanks for the nice chats, the recommendations on restaurants
and pubs and with Iván and Malte for supplying the lab with home-brewed
beer! Camilla, thanks for supervising with me my first student and all the help
on the magnetic projects! Xiaoyan, thanks for your help on the gap-fill project
and sorry for stressing you out about it! Thanks, Di for being such a nice desk
mate, for the interesting talks on science and keeping me updated in the field of
NIPT! Pavan, thanks for the great moving-in party and the nice Indian food!
Jessica, thanks for the nice chats at lunch and in the lab! Tagrid, thank you for
your encouragements and for always having a smile on your face! Rongqin,
thank you for sharing your knowledge! You always had an answer to any RCA
related question! Amel, thanks for the nice banana cupcakes and conversations
over lunch! Lotte and Elin F, thank you for the great time in Barcelona and for
bringing me to see Flamenco! Chenglin and Sibel it is great you have joined
this group. Thanks to Mustapha, Eva and all other students for contributing to
the nice atmosphere we have in the lab!
For the Uppsala people: Ola, Karin, Björn, Linda, Johan V, and CarlMagnus, thank you all for the great swimming sessions! Karin, thanks a lot for
the BBQ, Eurovision song contest and Christmas fika! It was always fun and
delicious! Björn, thanks for all the great talks in the lab and on the way home to
Flogsta! Carla thank you for great company in and outside the lab! Thanks for
the great Indian dinner at your place! Thanks Gucci, for the fikas, the glasses of
wine and chats! Elin E, thank you for your help with administrative stuff and of
course for the nice climbing sessions! Christina, thanks for keeping the lab so
organized and for always knowing where I could find stuff! Liza, for sharing
office space! Tonge, your good mood is contagious! Thanks! Agata and
Caroline, together with Karin, Linda and Elin L, thanks for the great time in
Berlin! It was a lot of fun! Junhong, thanks for the common courses we took
and organizing the Rudbeck masquerade together! Johan O, thanks for all the
computer help! Erik, thanks for all administrative work making it even easy to
fill out EU time sheets! Rasel, for always being happy and for fun talks!
Rachel, for being a great roommate at the retreat! Spyros, for your
42
encouragements and talks! Monica, Lena, Maria, Ida, Irene, Gaëlle, Felipe,
Andries, Lei, Anne-Li, Axel, Johanna and Joakim, thanks for great
discussions and making Rudbeck/BMC such a nice place!
For the people at SciLife: Thanks Tim, Elin B and Burcu for the nice
conversations over a beer, during collaborations or fika! Tim, thanks for the
tennis matches!
For Q-linea: Janne, thank you for your help with the Aquila in person and
electronically from far away. Thanks for fixing it every single time and giving
us an introduction about its inner life! Jenny, Anna and Jonas, thank you for
your help with the old blob counter and always welcoming us at Q-linea!
For my friends near and far: Anja, Franzi, Marlen, and Kasia, thank you for
the great friendship and support you have given me throughout the years! For
everything we experienced in the last couple of years, the talks, laughter, travels,
dinners and much more. It feels good to know that I can always count on you!
You made Sweden a much brighter place! Thanks Anja, Franzi and Marlen for
always offering me a place to stay in Uppsala! Merci, Kasia for hosting us in
Paris! It was great fun! Rachel, thanks for all the fun we had in Uppsala and
thank you for proof reading this thesis! Tina, thanks for being such a good
friend for all these years! For always lending me an ear and giving me support
when I needed it! For sharing the good and bad moments in life and always
having advice concerning work, life, just everything! No matter where you
lived, your door was always open for me! Thanks! Anja S, thanks for
encouragements, support and inspiration! Thank you for all the visits during the
last couple of years! Each one was unique and great! Anna, Alex K, and Anke,
vielen Dank für Eure Freundschaft! Ich bin mir sicher, dass egal wo es mich hin
verschlägt, ich mich auf Euch immer verlassen kann!
Micke, tack för allt vi har upplevt tillsammans under de gångna sex åren. Du har
alltid funnits där för mig när jag behövt dig som mest! Ord kan inte tillräckligt
beskriva hur tacksam jag är för det.
For my family: Mama, Papa, Oma, Opa und Omi, ich möchte mich von
Herzen bei Euch bedanken! Ihr habt mir mein Studium ermöglicht und ich
verdanke es Euch, dass ich bis hierhin gekommen bin. Nie habt ihr meine
Entscheidung oder mein Vermögen zu promovieren angezweifelt und ich konnte
Eurer Unterstützung immer sicher sein! Vielen Dank! Jens und Lars, ich kann
mich glücklich schätzen Euch als Brüder zu haben! Egal ob ich Hilfe mit
Übersetzungen, formellen Schreiben oder mit Weihnachtsgeschenken benötigte,
ich konnte immer auf Euch zählen. Danke!
43
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