Whole Genome Amplification Methods And Protocols

by Thomas Kroneis

Author Thomas Kroneis Isbn 978 1493929894 File size 4 6 MB Year 2015 Pages 704 Language English File format PDF Category Biology This volume mirrors the holistic feature of whole genome amplification WGA technology by combining reviews detailed basic methods and advanced sample workflows The first part of the book covers an overview of the development of WGA techniques throughout recent years including general considerations on bias in WGA possible sample pre enrichment strategies and how

Publisher :

Author : Thomas Kroneis

ISBN : 978 1493929894

Year : 2015

Language: English

File Size : 4.6 MB

Category : Biology



Preface
It is the nature of (lab) techniques that they steadily improve. The development of highresolution analysis such as comparative genome hybridization or the need of multiple analyses from minute amounts of template—down to the single-cell level—entails improved
sample preparation. Hence, amplification of template DNA is necessary to meet the requirements of state-of-the-art analysis. Although not representing an endpoint in analysis, equal
and unbiased amplification of DNA is a conditio sine qua non for downstream methods and
so whole genome amplification (WGA) methods were developed, adapted, and optimized
in parallel and with regard to the lab techniques that allowed for increasingly powerful
analyses. Apart from methods longing for a certain amount of starting template, WGA is
also necessary in case of multiple analyses to be done from low-template or single-cell
samples. This may not only allow for repeated analyses of one and the same sample but also
for combining diverse strategies such as targeted and screening approaches. This volume on
whole genome amplification is meant to mirror the holistic feature of WGA technology by
combining reviews, detailed basic methods, and advanced sample workflows. In the first
chapters the interested reader will gain an overview of the development of WGA techniques
throughout the recent years. Furthermore, general considerations on bias in WGA and how
to run a single-cell lab are given and possible sample pre-enrichment strategies are introduced. The second part focuses on major WGA methods and—most important—also protocols allowing assessing the WGA product quality. Covering most chapters, the final part
contains advanced protocols that go hand in hand with WGA. These chapters address issues
such as sample preparation using laser microdissection, WGA from partially degraded DNA
(formalin-fixed paraffin-embedded samples), circulating tumor cells, or even ancient samples. Following the techniques described here will most likely result in successful whole
genome amplification even though some of the described methods (e.g., laser microdissection) require some attention and improve with practice. Thus, this volume shall enable the
newcomer to get started and yield results within short time and serve old hands as rich
source of detailed information and inspiration.
Graz, Austria

Thomas Kroneis

v

Chapter 1
Principles of Whole-Genome Amplification
Zbigniew Tadeusz Czyz, Stefan Kirsch, and Bernhard Polzer
Abstract
Modern molecular biology relies on large amounts of high-quality genomic DNA. However, in a number
of clinical or biological applications this requirement cannot be met, as starting material is either limited
(e.g., preimplantation genetic diagnosis (PGD) or analysis of minimal residual cancer) or of insufficient
quality (e.g., formalin-fixed paraffin-embedded tissue samples or forensics). As a consequence, in order to
obtain sufficient amounts of material to analyze these demanding samples by state-of-the-art modern
molecular assays, genomic DNA has to be amplified. This chapter summarizes available technologies for
whole-genome amplification (WGA), bridging the last 25 years from the first developments to currently
applied methods. We will especially elaborate on research application, as well as inherent advantages and
limitations of various WGA technologies.
Key words Whole-genome amplification, PCR-based amplification, Ligation-mediated amplification,
Multiple displacement amplification

1

Challenges of Analyzing Minimal Quantities of Genomic DNA
For most high-throughput assays in molecular biology large amounts
of high-quality genomic DNA (gDNA) are needed as starting
material. However, depending on the source of the sample, its
inherent characteristics, and the spectrum of downstream analyses,
this requirement simply cannot be met. In some applications, e.g.,
prenatal genetic diagnostics (PDG) or minimal residual cancer, the
amount of starting material is extremely limited and often restricted
to only one individual cell, which corresponds to approximately
7 pg of gDNA [1]. Additionally, in some instances, as in forensics,
paleobiology or when processing formalin-fixed paraffin-embedded
tissue (FFPE) specimens, the sample processing procedures and/
or storage may diminish the quality and quantity of the available
DNA. In these cases direct analysis of the sample’s gDNA is technically challenging and enables assessment of only limited amount of
genetic markers.

Thomas Kroneis (ed.), Whole Genome Amplification: Methods and Protocols, Methods in Molecular Biology, vol. 1347,
DOI 10.1007/978-1-4939-2990-0_1, © Springer Science+Business Media New York 2015

1

2

Zbigniew Tadeusz Czyz et al.

To enable comprehensive analysis of such demanding samples
by modern molecular assays, gDNA has to be amplified. The
amplification procedure has to (1) ensure high genomic coverage;
that is, as much as possible of all 3 × 109 nucleotides comprising the
human genome have to be amplified; (2) maintain the inherent
sequence composition; that is, avoid artificial loss of one or even
both gene copies (maternal and paternal) without introducing artificial sequence variation; and (3) allow reliable quantification of
copy number variation; that is, all regions of the genome have to
be amplified homogeneously.
During the last decades, several methods for whole-genome
amplification (WGA) have been developed. Most of them rely on
the principle of polymerase chain reaction (PCR), a powerful technique allowing exponential amplification of the DNA using thermostable DNA polymerases and short oligonucleotide primers [2].
In its original form, PCR was designed to amplify specific DNA
loci with limited amplicon length due to the processivity of the
polymerase. For example, Taq DNA polymerase which is typically
used in PCR is amenable to generate amplicons of approximately
1000 base pair in length. Thus, to amplify whole genomes three
basic principles have been applied: (1) increasing the amount of
priming events, (2) reducing the complexity of the genome prior
to amplification (i.e., by fragmentation in smaller fragments), or
(3) utilizing alternative enzymes with higher processivity.

2

First Attempts to Amplify Whole Genomes by PCR-Based Technologies
The first approach to amplify a genome used non-degenerated
primers targeting the most conserved regions of repetitive Alu
motifs within the genome [3]. This method, called interspersed
repetitive sequence (IRS) PCR, allows amplification of fragments
directly adjacent to Alu elements. Although Alu elements are abundantly present within the human genome, their distribution within
the genome is not uniform [4], which results in a bias towards
amplification of regions enriched for Alu sequences [5].
Additionally, Alu sequences are not frequent for some other species
(e.g., mouse), decreasing the applicability of the method for single
cells in studies involving animal models [5]. IRS PCR was primarily used for generation of probe libraries specific for designated
regions of the human genome from either mixed DNA sources,
i.e., human/rodent somatic cell hybrids [6–8], or microdissected
human chromosomes [9].
One way to assure a more uniform distribution of the priming
events across the genome is partial or complete randomization of
primer sequences. Degenerate oligonucleotide primed (DOP)
PCR is based on the assumption that random primers anneal
uniformly across the genome [10]. The method utilizes primers

Principles of Whole-Genome Amplification

5

enzymatic treatment, i.e., Omniplex™/GenomePlex™ technology
by Rubicon Genomics [36, 37]. The method is less dependent on
the quality of the starting material, as following the fragmentation
and fill-in reaction PCR adaptors can be ligated even to partially
degraded template, e.g., in FFPE tissue specimens. Moreover, due
to the use of non-degenerated primers this approach ensures constant
priming efficiency, which results in high DNA yields. Nevertheless,
the priming pattern remains erratic and unreproducible as in PEPand DOP-PCR.
Thus far, LM-PCR-based GenomePlex™ technology has been
used in a variety of applications. The method enabled analysis of as
little as 5 ng of FFPE-derived DNA material [38] or as few as 2000
microdissected cells by aCGH [39]. Moreover, GenomePlex™ technology was successfully applied for aCGH-based analysis of single
disseminated cancer cells [40–42] and enriched populations of circulating tumor cells [43, 44] and single human blastomeres [45].
Here, however, due to high-level technical noise, designated algorithms had to be developed to allow evaluation of single-cell aCGH
data sets [41]. More recently, GenomePlex™ technology was used
for low-coverage high-throughput sequencing of single-cell nuclei
sorted by FACS [46]. Based on profiles of copy number changes,
this approach allowed to investigate the population structure and
evolution of tumorigenic clones in individual tumor specimens [46].
Massive parallel sequencing was also used to sequence a panel of
cancer-related genes in single-cell GenomePlex™ WGA products
generated from colorectal cancer circulating tumor cells, facilitating discovery of discrepancies in mutation spectrum between primary tumor, metastasis, and corresponding tumor cells in
circulation of the same cancer patients [47].
Another fully deterministic WGA technology utilizing the
LM-PCR approach is the single-cell comparative genomic hybridization (SCOMP) published by Klein and colleagues [48]. In this
approach the fragmented representation of the genome is achieved
by the use of restriction endonuclease MseI, whose 4-base pairlong restriction motif is distributed with an average spacing of 126
base pair across the human genome (based on the hg19 build of
the human genome)—an optimal size for the subsequent PCRbased amplification. Still, this approach may be less favorable for
templates with more infrequent distribution of MseI restriction
sites. Following the restriction digestion PCR-adaptor sequences
are ligated to the MseI representation of the genome, assuring a
deterministic, reproducible priming pattern and high genomic
coverage of WGA. Furthermore, the designated design of the
PCR adaptors used in SCOMP minimized the risk of their multimerization and undesirable mispriming within the sampled
genome, thereby improving the efficiency of the PCR. Collectively,
the unique design of SCOMP makes it particularly advantageous
for the downstream assays, wherein reproducible coverage and

6

Zbigniew Tadeusz Czyz et al.

composition of the input material are essential for the analysis
(i.e., genotyping and targeted sequence analyses). This aspect is
particularly important for the diagnostic assays used in the clinic.
SCOMP was successfully applied to amplify single-cell DNA of
disseminated and circulating tumor cells allowing a number of locusspecific analyses (i.e., direct Sanger sequencing-, RFLP-, and STRbased detection of LOHs) [48–50]. Importantly, comprehensive
representation of the single-cell DNA by SCOMP was demonstrated by metaphase CGH allowing detection of copy number
alterations (CNAs) in single tumor cells in numerous studies [48,
49, 51–54]. Moreover, SCOMP facilitated successful metaphase
CGH-based analysis of single human blastomeres, allowing detection of unbalanced translocations and mosaicism within individual
embryos [55]. More recently, products of SCOMP were analyzed
using a BAC clone-based [56] and oligonucleotide aCGH platforms [57, 58] providing high-quality data and allowing detection
of CNAs as small as 53 kb in size. Moreover, applicability of
SCOMP was also demonstrated for dissected FFPE tissue sections
providing high-quality results of both metaphase and array-based
CGH [59, 60]. In both of these studies SCOMP outperformed
DOP-PCR allowing more accurate and unbiased analysis of copy
number changes. The unique deterministic design and power of
the method in the analysis of clinical material (e.g., disseminated
cancer cells and FFPE tissue specimens) recently led to commercialization of the principle method as Ampli1™ WGA kit (Silicon
Biosystems SpA, Bologna, Italy).

4

High Processivity by MDA-Based WGA
Strand displacement amplification (SDA) or multiple displacement
amplification (MDA) [61] is based on rolling circle amplification—
a replication mechanism naturally occurring in the λ and various
other bacteriophages [62]. The method was initially adapted to
amplify circular DNA templates [63] and later also used for WGA
of single cells [12]. MDA utilizes enzymes as the highly processive
Phi29 or Bst DNA polymerases [12, 64] with proofreading activity
[65]. In principle, exonuclease-resistant, random hexamers bind to
denatured DNA followed by an isothermal amplification [12, 66].
As a consequence of the strong strand displacement activity of the
applied polymerase, generated fragments become available for secondary priming events leading to the generation of a network of
hyperbranched structures and thus multiple overlapping copies of
the starting material. High processivity of the Phi29 DNA polymerase results in generation of relatively large amplicons (>10 kb)
[12], facilitating high genomic coverage of single-cell genomes
[67]. Notably, however, utilization of MDA leads to considerable
sequence representation bias [68], and a tendency to generate

Principles of Whole-Genome Amplification

7

chimeric DNA rearrangements in the amplified DNA [69].
This results in a significant rate of allelic dropout (ADO) [70] or
preferential amplification (PA) particularly affecting highly polymorphic sequences [71]. These effects are further pronounced in
samples with low DNA quantity [64, 72] and especially with fragmented template DNA [73, 74]. This reasoning does not support
the application of MDA on clinical samples (as, e.g., circulating
tumor cells), for which fixation and transport logistics may lead to
considerable degradation of high-molecular DNA. For these types
of samples, PCR-based WGA approaches were suggested as the
better alternative than MDA [75].
Despite the mentioned shortcomings, MDA technology has
been applied in numerous studies on single-cell DNA, i.e., for
genotyping of short tandem repeats [76, 77], assessment of
copy number changes by CGH [5] or aCGH [64, 78, 79], and
more recently whole-exome or whole-genome sequencing [80–
83]. Furthermore, DNA yields after MDA-based amplification
are sufficient to allow multiple downstream analysis with the
same single-cell sample [84]. As MDA-based WGA is an easyto-use and efficient approach to generate large quantities of
genomic DNA from small sample sizes it has been commercialized (e.g., Qiagen’s REPLI-g product family and GenomiPhi by
GE Healthcare) and is widely used to generate a long-lasting
source of DNA for downstream genetic analyses of small sample
sizes.

5

WGA Approaches Combining MDA and PCR
In an effort to synergize the advantages and negate the disadvantages of both MDA- and PCR-based WGA, the company Rubicon
Genomics developed PicoPlex™, the first technology to combine
both WGA principles [85]. Apart from the patent holder Rubicon
Genomics, this WGA technology is also vended by the New England
Biolabs Inc. (Single Cell WGA kit), BlueGnome (SurePLEX™),
and Perkin Elmer (EasyAmp™).
In the PicoPlex™ WGA protocol genomic DNA is initially
amplified in an MDA-based process utilizing a set of four non-selfcomplementary primers. These so-called self-inert primers are
composed of base pair combinations that do not participate in the
Watson-Crick base pairing, i.e., A–C, A–G, T–C, and T–G. Through
this intervention formation of primer dimers is precluded, which
has a strong positive impact on the efficiency of the reaction. Selfinert primers are composed of two sections: degenerated sequence
at the 3′-end responsible for frequent priming in the genome and
the fixed sequence at the 5′-end. During the initial MDA-based
pre-amplification step, fixed sequences are incorporated to the end
of each amplicon. In a second step these molecules are amplified by

Principles of Whole-Genome Amplification

9

detection of aneuploidy and single-nucleotide variants. In a recent
study on CTCs of lung cancer patients, single-cell genomes were
analyzed by exome and whole-genome sequencing after
CellSearch® detection and MALBAC [98]. Although putative
“druggable” copy number variations and sequence variations could
be discovered, high allelic dropout at the single-cell level could be
observed [98], questioning the reliable use of the method on fixed
single CTCs from cancer patients. Recently, MALBAC has been
made commercially available by Yikon Genomics (Beijing, China).

6

Conclusion
The increasing number of publications and commercialized
molecular methods to analyze genomes of single cells, as reviewed
in this chapter, depicts the increased interest in studies on cellular
heterogeneity. In the future, the link between inherent genomic
changes of individual cells with their functional state would certainly improve our understanding on clonal evolution and cellular
adaptation. One possibility to achieve this goal is the analysis of
genome and transcriptome of the same individual cell as demonstrated by the group of Christoph Klein [99]. Correlation of results
obtained by this approach with the cellular phenotype of individual
cells (as, e.g., by multicolor immunostaining) would provide a
basis to better understand cellular heterogeneity and its implications in biology and medicine.

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Preface
It is the nature of (lab) techniques that they steadily improve. The development of highresolution analysis such as comparative genome hybridization or the need of multiple analyses from minute amounts of template—down to the single-cell level—entails improved
sample preparation. Hence, amplification of template DNA is necessary to meet the requirements of state-of-the-art analysis. Although not representing an endpoint in analysis, equal
and unbiased amplification of DNA is a conditio sine qua non for downstream methods and
so whole genome amplification (WGA) methods were developed, adapted, and optimized
in parallel and with regard to the lab techniques that allowed for increasingly powerful
analyses. Apart from methods longing for a certain amount of starting template, WGA is
also necessary in case of multiple analyses to be done from low-template or single-cell
samples. This may not only allow for repeated analyses of one and the same sample but also
for combining diverse strategies such as targeted and screening approaches. This volume on
whole genome amplification is meant to mirror the holistic feature of WGA technology by
combining reviews, detailed basic methods, and advanced sample workflows. In the first
chapters the interested reader will gain an overview of the development of WGA techniques
throughout the recent years. Furthermore, general considerations on bias in WGA and how
to run a single-cell lab are given and possible sample pre-enrichment strategies are introduced. The second part focuses on major WGA methods and—most important—also protocols allowing assessing the WGA product quality. Covering most chapters, the final part
contains advanced protocols that go hand in hand with WGA. These chapters address issues
such as sample preparation using laser microdissection, WGA from partially degraded DNA
(formalin-fixed paraffin-embedded samples), circulating tumor cells, or even ancient samples. Following the techniques described here will most likely result in successful whole
genome amplification even though some of the described methods (e.g., laser microdissection) require some attention and improve with practice. Thus, this volume shall enable the
newcomer to get started and yield results within short time and serve old hands as rich
source of detailed information and inspiration.
Graz, Austria

Thomas Kroneis

v

Chapter 1
Principles of Whole-Genome Amplification
Zbigniew Tadeusz Czyz, Stefan Kirsch, and Bernhard Polzer
Abstract
Modern molecular biology relies on large amounts of high-quality genomic DNA. However, in a number
of clinical or biological applications this requirement cannot be met, as starting material is either limited
(e.g., preimplantation genetic diagnosis (PGD) or analysis of minimal residual cancer) or of insufficient
quality (e.g., formalin-fixed paraffin-embedded tissue samples or forensics). As a consequence, in order to
obtain sufficient amounts of material to analyze these demanding samples by state-of-the-art modern
molecular assays, genomic DNA has to be amplified. This chapter summarizes available technologies for
whole-genome amplification (WGA), bridging the last 25 years from the first developments to currently
applied methods. We will especially elaborate on research application, as well as inherent advantages and
limitations of various WGA technologies.
Key words Whole-genome amplification, PCR-based amplification, Ligation-mediated amplification,
Multiple displacement amplification

1

Challenges of Analyzing Minimal Quantities of Genomic DNA
For most high-throughput assays in molecular biology large amounts
of high-quality genomic DNA (gDNA) are needed as starting
material. However, depending on the source of the sample, its
inherent characteristics, and the spectrum of downstream analyses,
this requirement simply cannot be met. In some applications, e.g.,
prenatal genetic diagnostics (PDG) or minimal residual cancer, the
amount of starting material is extremely limited and often restricted
to only one individual cell, which corresponds to approximately
7 pg of gDNA [1]. Additionally, in some instances, as in forensics,
paleobiology or when processing formalin-fixed paraffin-embedded
tissue (FFPE) specimens, the sample processing procedures and/
or storage may diminish the quality and quantity of the available
DNA. In these cases direct analysis of the sample’s gDNA is technically challenging and enables assessment of only limited amount of
genetic markers.

Thomas Kroneis (ed.), Whole Genome Amplification: Methods and Protocols, Methods in Molecular Biology, vol. 1347,
DOI 10.1007/978-1-4939-2990-0_1, © Springer Science+Business Media New York 2015

1

2

Zbigniew Tadeusz Czyz et al.

To enable comprehensive analysis of such demanding samples
by modern molecular assays, gDNA has to be amplified. The
amplification procedure has to (1) ensure high genomic coverage;
that is, as much as possible of all 3 × 109 nucleotides comprising the
human genome have to be amplified; (2) maintain the inherent
sequence composition; that is, avoid artificial loss of one or even
both gene copies (maternal and paternal) without introducing artificial sequence variation; and (3) allow reliable quantification of
copy number variation; that is, all regions of the genome have to
be amplified homogeneously.
During the last decades, several methods for whole-genome
amplification (WGA) have been developed. Most of them rely on
the principle of polymerase chain reaction (PCR), a powerful technique allowing exponential amplification of the DNA using thermostable DNA polymerases and short oligonucleotide primers [2].
In its original form, PCR was designed to amplify specific DNA
loci with limited amplicon length due to the processivity of the
polymerase. For example, Taq DNA polymerase which is typically
used in PCR is amenable to generate amplicons of approximately
1000 base pair in length. Thus, to amplify whole genomes three
basic principles have been applied: (1) increasing the amount of
priming events, (2) reducing the complexity of the genome prior
to amplification (i.e., by fragmentation in smaller fragments), or
(3) utilizing alternative enzymes with higher processivity.

2

First Attempts to Amplify Whole Genomes by PCR-Based Technologies
The first approach to amplify a genome used non-degenerated
primers targeting the most conserved regions of repetitive Alu
motifs within the genome [3]. This method, called interspersed
repetitive sequence (IRS) PCR, allows amplification of fragments
directly adjacent to Alu elements. Although Alu elements are abundantly present within the human genome, their distribution within
the genome is not uniform [4], which results in a bias towards
amplification of regions enriched for Alu sequences [5].
Additionally, Alu sequences are not frequent for some other species
(e.g., mouse), decreasing the applicability of the method for single
cells in studies involving animal models [5]. IRS PCR was primarily used for generation of probe libraries specific for designated
regions of the human genome from either mixed DNA sources,
i.e., human/rodent somatic cell hybrids [6–8], or microdissected
human chromosomes [9].
One way to assure a more uniform distribution of the priming
events across the genome is partial or complete randomization of
primer sequences. Degenerate oligonucleotide primed (DOP)
PCR is based on the assumption that random primers anneal
uniformly across the genome [10]. The method utilizes primers

Principles of Whole-Genome Amplification

5

enzymatic treatment, i.e., Omniplex™/GenomePlex™ technology
by Rubicon Genomics [36, 37]. The method is less dependent on
the quality of the starting material, as following the fragmentation
and fill-in reaction PCR adaptors can be ligated even to partially
degraded template, e.g., in FFPE tissue specimens. Moreover, due
to the use of non-degenerated primers this approach ensures constant
priming efficiency, which results in high DNA yields. Nevertheless,
the priming pattern remains erratic and unreproducible as in PEPand DOP-PCR.
Thus far, LM-PCR-based GenomePlex™ technology has been
used in a variety of applications. The method enabled analysis of as
little as 5 ng of FFPE-derived DNA material [38] or as few as 2000
microdissected cells by aCGH [39]. Moreover, GenomePlex™ technology was successfully applied for aCGH-based analysis of single
disseminated cancer cells [40–42] and enriched populations of circulating tumor cells [43, 44] and single human blastomeres [45].
Here, however, due to high-level technical noise, designated algorithms had to be developed to allow evaluation of single-cell aCGH
data sets [41]. More recently, GenomePlex™ technology was used
for low-coverage high-throughput sequencing of single-cell nuclei
sorted by FACS [46]. Based on profiles of copy number changes,
this approach allowed to investigate the population structure and
evolution of tumorigenic clones in individual tumor specimens [46].
Massive parallel sequencing was also used to sequence a panel of
cancer-related genes in single-cell GenomePlex™ WGA products
generated from colorectal cancer circulating tumor cells, facilitating discovery of discrepancies in mutation spectrum between primary tumor, metastasis, and corresponding tumor cells in
circulation of the same cancer patients [47].
Another fully deterministic WGA technology utilizing the
LM-PCR approach is the single-cell comparative genomic hybridization (SCOMP) published by Klein and colleagues [48]. In this
approach the fragmented representation of the genome is achieved
by the use of restriction endonuclease MseI, whose 4-base pairlong restriction motif is distributed with an average spacing of 126
base pair across the human genome (based on the hg19 build of
the human genome)—an optimal size for the subsequent PCRbased amplification. Still, this approach may be less favorable for
templates with more infrequent distribution of MseI restriction
sites. Following the restriction digestion PCR-adaptor sequences
are ligated to the MseI representation of the genome, assuring a
deterministic, reproducible priming pattern and high genomic
coverage of WGA. Furthermore, the designated design of the
PCR adaptors used in SCOMP minimized the risk of their multimerization and undesirable mispriming within the sampled
genome, thereby improving the efficiency of the PCR. Collectively,
the unique design of SCOMP makes it particularly advantageous
for the downstream assays, wherein reproducible coverage and

6

Zbigniew Tadeusz Czyz et al.

composition of the input material are essential for the analysis
(i.e., genotyping and targeted sequence analyses). This aspect is
particularly important for the diagnostic assays used in the clinic.
SCOMP was successfully applied to amplify single-cell DNA of
disseminated and circulating tumor cells allowing a number of locusspecific analyses (i.e., direct Sanger sequencing-, RFLP-, and STRbased detection of LOHs) [48–50]. Importantly, comprehensive
representation of the single-cell DNA by SCOMP was demonstrated by metaphase CGH allowing detection of copy number
alterations (CNAs) in single tumor cells in numerous studies [48,
49, 51–54]. Moreover, SCOMP facilitated successful metaphase
CGH-based analysis of single human blastomeres, allowing detection of unbalanced translocations and mosaicism within individual
embryos [55]. More recently, products of SCOMP were analyzed
using a BAC clone-based [56] and oligonucleotide aCGH platforms [57, 58] providing high-quality data and allowing detection
of CNAs as small as 53 kb in size. Moreover, applicability of
SCOMP was also demonstrated for dissected FFPE tissue sections
providing high-quality results of both metaphase and array-based
CGH [59, 60]. In both of these studies SCOMP outperformed
DOP-PCR allowing more accurate and unbiased analysis of copy
number changes. The unique deterministic design and power of
the method in the analysis of clinical material (e.g., disseminated
cancer cells and FFPE tissue specimens) recently led to commercialization of the principle method as Ampli1™ WGA kit (Silicon
Biosystems SpA, Bologna, Italy).

4

High Processivity by MDA-Based WGA
Strand displacement amplification (SDA) or multiple displacement
amplification (MDA) [61] is based on rolling circle amplification—
a replication mechanism naturally occurring in the λ and various
other bacteriophages [62]. The method was initially adapted to
amplify circular DNA templates [63] and later also used for WGA
of single cells [12]. MDA utilizes enzymes as the highly processive
Phi29 or Bst DNA polymerases [12, 64] with proofreading activity
[65]. In principle, exonuclease-resistant, random hexamers bind to
denatured DNA followed by an isothermal amplification [12, 66].
As a consequence of the strong strand displacement activity of the
applied polymerase, generated fragments become available for secondary priming events leading to the generation of a network of
hyperbranched structures and thus multiple overlapping copies of
the starting material. High processivity of the Phi29 DNA polymerase results in generation of relatively large amplicons (>10 kb)
[12], facilitating high genomic coverage of single-cell genomes
[67]. Notably, however, utilization of MDA leads to considerable
sequence representation bias [68], and a tendency to generate

Principles of Whole-Genome Amplification

7

chimeric DNA rearrangements in the amplified DNA [69].
This results in a significant rate of allelic dropout (ADO) [70] or
preferential amplification (PA) particularly affecting highly polymorphic sequences [71]. These effects are further pronounced in
samples with low DNA quantity [64, 72] and especially with fragmented template DNA [73, 74]. This reasoning does not support
the application of MDA on clinical samples (as, e.g., circulating
tumor cells), for which fixation and transport logistics may lead to
considerable degradation of high-molecular DNA. For these types
of samples, PCR-based WGA approaches were suggested as the
better alternative than MDA [75].
Despite the mentioned shortcomings, MDA technology has
been applied in numerous studies on single-cell DNA, i.e., for
genotyping of short tandem repeats [76, 77], assessment of
copy number changes by CGH [5] or aCGH [64, 78, 79], and
more recently whole-exome or whole-genome sequencing [80–
83]. Furthermore, DNA yields after MDA-based amplification
are sufficient to allow multiple downstream analysis with the
same single-cell sample [84]. As MDA-based WGA is an easyto-use and efficient approach to generate large quantities of
genomic DNA from small sample sizes it has been commercialized (e.g., Qiagen’s REPLI-g product family and GenomiPhi by
GE Healthcare) and is widely used to generate a long-lasting
source of DNA for downstream genetic analyses of small sample
sizes.

5

WGA Approaches Combining MDA and PCR
In an effort to synergize the advantages and negate the disadvantages of both MDA- and PCR-based WGA, the company Rubicon
Genomics developed PicoPlex™, the first technology to combine
both WGA principles [85]. Apart from the patent holder Rubicon
Genomics, this WGA technology is also vended by the New England
Biolabs Inc. (Single Cell WGA kit), BlueGnome (SurePLEX™),
and Perkin Elmer (EasyAmp™).
In the PicoPlex™ WGA protocol genomic DNA is initially
amplified in an MDA-based process utilizing a set of four non-selfcomplementary primers. These so-called self-inert primers are
composed of base pair combinations that do not participate in the
Watson-Crick base pairing, i.e., A–C, A–G, T–C, and T–G. Through
this intervention formation of primer dimers is precluded, which
has a strong positive impact on the efficiency of the reaction. Selfinert primers are composed of two sections: degenerated sequence
at the 3′-end responsible for frequent priming in the genome and
the fixed sequence at the 5′-end. During the initial MDA-based
pre-amplification step, fixed sequences are incorporated to the end
of each amplicon. In a second step these molecules are amplified by

Principles of Whole-Genome Amplification

9

detection of aneuploidy and single-nucleotide variants. In a recent
study on CTCs of lung cancer patients, single-cell genomes were
analyzed by exome and whole-genome sequencing after
CellSearch® detection and MALBAC [98]. Although putative
“druggable” copy number variations and sequence variations could
be discovered, high allelic dropout at the single-cell level could be
observed [98], questioning the reliable use of the method on fixed
single CTCs from cancer patients. Recently, MALBAC has been
made commercially available by Yikon Genomics (Beijing, China).

6

Conclusion
The increasing number of publications and commercialized
molecular methods to analyze genomes of single cells, as reviewed
in this chapter, depicts the increased interest in studies on cellular
heterogeneity. In the future, the link between inherent genomic
changes of individual cells with their functional state would certainly improve our understanding on clonal evolution and cellular
adaptation. One possibility to achieve this goal is the analysis of
genome and transcriptome of the same individual cell as demonstrated by the group of Christoph Klein [99]. Correlation of results
obtained by this approach with the cellular phenotype of individual
cells (as, e.g., by multicolor immunostaining) would provide a
basis to better understand cellular heterogeneity and its implications in biology and medicine.

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