Single cell whole genome amplification on-chip for
forensic purposes
Loes Steller 11814268 MA Forensic Science Literature thesis (5 EC) 14-12-2019 Supervisor: dr. B.B. Bruijns & dr. J.C. Knotter Examiner: prof. dr. A.D. Kloosterman Word count: 9348 (excl references and abstract)Abstract
DNA analysis via short tandem repeats (STRs) is a valuable tool for human identification of biological crime traces. However, crime traces with a limited amount of DNA are often not sufficient for STR analysis. In this review, a novel approach using single cell whole genome amplification (WGA) on a microfluidic chip is proposed, with the aim to improve the success rate of DNA analysis in forensic investigations. WGA functions as an extra amplification step to increase the amount of DNA that is available for STR-PCR, which could improve the success rate of obtaining a DNA profile and allow mixture deconvolution already at the amplification stage. Via performing single cell WGA on-chip in enclosed nanoscale volumes, time, cost and chance of contamination could potentially be reduced compared to tube-based WGA. Moreover, the portable nature of microfluidic chips might enable direct analysis at the crime scene. This review aims to provide an overview of available single cell WGA methods, including kits for polymerase chain reaction-based, multiple displacement amplification-based and hybrid methods for single cell WGA. The performance of each method analyzed with STR analysis will be discussed, in order to determine whether these methods could be useful in forensic investigations. Furthermore, available microfluidic chips for single cell WGA reported in literature will be described.Table of contents
Abstract I
Table of contents II
1 Introduction 1
1.1 STR analysis challenging due to limited DNA samples 1
1.2 Forensic uses of microfluidic devices 1
1.3 Whole Genome Amplification on-chip 2
2 Whole genome amplification 4
2.1 PCR-based WGA 5
2.2 MDA-based WGA 8
2. 3 Hybrid methods 9
3 On-chip whole genome amplification 13
3.1 MDA on-chip 14
3.2 MALBAC on-chip 19
4 Conclusions and recommendations 21
4.1 WGA preceding STR profiling increases success rate 21
4.2 On-chip WGA reduces contamination and reaction volume 22
6 Supplementary material 27
6.1 Search strategy 27
1 Introduction
1.1 STR analysis challenging due to limited DNA samples
Donor identification of biological crime traces is often crucial for linking perpetrators, victims, locations and/or items, thereby reconstructing what happened during the crime. The main method used for human identification of biological crime traces is deoxyribonucleic acid (DNA) profiling via the analysis of short tandem repeats (STRs). 1 STRs are repeats of a sequence of 2-13 nucleotides (nt) that are present in non-coding regions of genomic DNA. The number of repeats present at each STR allele differs between individuals, and could be used to distinguish individuals. Nowadays, various multiplex polymerase chain reaction (PCR) kits for the amplification of a set of STR loci can be used to establish a DNA profile from a crime sample. Statistical methods to determine the discriminatory power of the established DNA profiles, including random match probability calculations and likelihood ratio estimations, have been developed for interpretation. Together, these developments have resulted in STR analysis becoming a valuable tool for human identification in forensic casework.2
However, often only a minimal amount of DNA or partly degraded DNA is found at crime scenes and STR analysis using these crime traces remains challenging. For example, a study by Mapes et al. (2016) has shown that from half of the 2260 traces from forensic casework that were sent in for DNA profiling no DNA profile could be established.1 From 13% of the other half of the analyzed crime traces a DNA profile could be established, but these DNA profiles did not meet the criteria for storage in a DNA database. A comparative study with regard to the nature of the items from which samples were taken and the DNA concentration of each sample revealed that the success rate of DNA profiling is highly dependent on the DNA concentration of the sample. It does not depend on the nature of the item from which the sample was taken. 3 For example, for
bloodstains and cigarette buds high probabilities for succcessful DNA profiling and high mean DNA concentrations were reported. In contrast, items with with low probabilities for successful DNA profiling, such as tie-wraps, also have low mean DNA concentrations. 3 Thus, forensic investigations
could benefit from the development of novel approaches that increase the success rate of DNA profiling from traces containing a limited amount of DNA or degraded DNA.
1.2 Forensic uses of microfluidic devices
Recent developments in the field of microfluidics have highlighted the potential of a lab-on-a-chip (LOC) or microfluidic device as novel method for DNA profiling using samples containing a limited amount of DNA and mixed DNA samples. 4 Microfluidic devices are widely applied in the field of
(medical) biology. For example, they have been developed for single-cell analysis and drug screening using circulating tumor cells to determine treatment strategies, or single-cell analysis of blastocyst cells for preimplantation genetic screening. 5 Less well studied is the potential of microfluidic devices
in the field of forensic science. Microfluidic devices contain enclosed microchannels, which reduce the chance of contamination. Furthermore, potentially some of the steps that are necessary for DNA profiling can be combined, thereby reducing the time that is needed for analysis as well as the chance of contamination. Finally, reactions can be performed in small volumes, which allows fast heating, cooling and mixing of the sample, resulting in a short reaction time. Taken together, microfluidic devices could become a valuable tool in the field of forensic science.4,6
Multiple steps could be distinguished from sampling at the crime scene to genetic analysis and improvements in each step could potentially increase the chance of obtaining a DNA profile. Bruijns (2019) reviewed available microfluidic methods for each step of DNA analysis- i.e. sampling, sample work-up, amplification, detection and secure storage. 4 Most commercial and research
microfluidic devices that have been developed could be used to perform one of the steps of DNA analysis. For example, a ceramic collection device for sample collection at the crime scene could be used as an alternative to cotton swabs7 and Brown and Audet (2008) reported microfluidic chips that
were developed for optical, electrical and chemical lysis of single cells. 8 In addition, chips integrating
several steps of DNA profiling have been developed.
For example, the BioChipSet cassette (NetBio), which is commercially available as ANDE Rapid DNA, and the RapidHIT (Applied Biosystems) integrate DNA purification, STR-PCR, electrophoretic separation and detection in one chip. 9 Both chips generate STR profile of reference
buccal swabs and RapidHiT can also generate STR profiles from other human samples. Despite the emerging interests with regard to microfluidics in forensic science, the applicability of microfluidic chips for DNA analysis in forensic investigations remains largely unknown. The aim of this review is to explore the applicability of a microfluidic approach for single cell whole genome amplification (WGA) as a means to improve the success rate of DNA profiling in forensic investigations.
1.3 Whole Genome Amplification on-chip
The conventional method for DNA amplification in forensic investigations is STR-PCR. Here, a set of STRs is amplified via PCR and the resulting fragments are separated based on length - i.e. the number of repeats - via (capillary) electrophoresis to establish a DNA profile. However, when dealing with limited and mixed DNA samples, this method does not always suffice. 3 It could be difficult to
determine which alleles originate from each donor and allele drop-in and drop-out could occur. An alternative method to obtain genetic information from these samples is to increase the number of cycles for amplification. However, an unwanted effect of increasing the number of amplification cycles is that it also increases the chance of obtaining a false DNA profile due to artifacts, including the amplification of contaminant DNA. Another alternative method is to obtain a mitochondrial DNA (mtDNA) profile via mtDNA profiling, as single cells can contain hundreds of mtDNA copies. A drawback of this method is the low discriminatory power of mtDNA profiles, due to its maternal inheritance. 10 A proposed approach in order to improve the success rate of DNA profiling using limited and mixed samples is single cell WGA and subsequently standard STR-PCR. Through preamplification of the whole genome enough DNA could be obtained to perform STR-PCR and create a DNA profile. 10 Such a method would be valuable for analyzing crime traces where the amount of available sample is limited, as only a single cell would be required to be able to establish a DNA profile. In addition, single cell analysis would allow mixture deconvolution. This could be valuable in cases where a crime trace contains mixed DNA from the victim and one or more suspects, such as sexual assault cases. However, a drawback of single cell WGA is its time-consuming nature and the high number of manual steps, which could result in contamination. In general, the protocol of various WGA kits that are available takes 3-9 hours and involves multiple rounds of manually adding a buffer or reaction mix to the sample, followed by thermal cycling. 11 The development of a novel method for single cell
WGA using a microfluidic device could potentially be less time-consuming, more automated and less prone to contamination compared to tube-based WGA. Moreover, when coupled to off- or on-chip
STR profiling, the success rate of STR profiling using limited and mixed DNA samples could be increased.
The aim of this report is to compare the available methods for single cell WGA in order to determine whether they could potentially be performed on-chip. The principle of single cell WGA and give examples of several PCR-based, multiple displacement amplification (MDA)-based and hybrid WGA methods that have been used for the amplification of single cells and/or degraded DNA are discussed in chapter 2. 5,12 Chapter 3 provides an overview of available microfluidic chips for
single cell WGA and evaluate their potential use together with subsequent off-chip STR-PCR in a forensic setting.
2 Whole genome amplification
One human diploid cell contains ~6 picograms (pg) of DNA, whereas 100 pg of input DNA is needed for STR analysis. 3,4 As a result, DNA obtained from crime traces consisting of only a few cells is not
sufficient for conventional STR-PCR. In order to obtain enough DNA from these limited samples for subsequent STR-PCR, an extra amplification step is desired. Furthermore, it would be valuable to amplify single cells from mixed crime traces separately prior to STR-PCR. This would allow mixture deconvolution already at the amplification stage, instead of during interpretation of DNA profiles. Here, we propose single cell WGA as a means to improve the success rate of STR profiling using crime traces with degraded DNA or a limited amount of DNA.
WGA is a method for the amplification of an entire genome, using only a small quantity of DNA. For example, the DNA present in one single cell ( ~6 pg) could be amplified to generate µg quantities of DNA. 13 Various methods for WGA have been described in literature and these methods
could be performed in a PCR tube or in a microfluidic device. The methods can be divided into three categories: PCR-based methods, multiple displacement amplification (MDA) based methods and hybrid methods (table 1). Hybrid methods aim to combine some advantages and overcome disadvantages of PCR- and MDA-based methods. For example, PCR- and MDA-based methods use exponential amplification, resulting in amplification bias, an unwanted effect that is reduced in hybrid methods via using a combination of quasi-linear and exponential amplification.
A number of studies investigated the use of WGA with the aim to increase the success rate of STR analysis for forensic purposes. 14-16 Studies that did not use single cells but a higher amount of
DNA as input fall outside the scope of this review. Single cell WGA is widely applied in a medical setting, as it has been proven to be a valuable tool to amplify DNA derived from cells that exist in small numbers. For example, single cell WGA is used to amplify circulating tumor cells in order to identify cancer-associated mutations in these cells and adjust therapy according to this information.17,18 This example demonstrates the applicability of WGA for intact cells that have not been exposed to environmentally challenging conditions or chemical substances. In contrast, few in-depth studies have addressed the use of single cell WGA in a forensic setting where DNA quality is often impaired and one sample could contain DNA from multiple donors. 12,19 This chapter contains an overview of available WGA kits that have been used for the amplification of single cells and/or degraded DNA samples. 5,12 Furthermore, in order to determine which methods could potentially be performed on-chip in a forensic context, the results of a number of comparative studies in which the performance of different WGA kits was compared is discussed.
Table 1. Overview of single cell WGA methods.
WGA: whole genome amplification; DOP-PCR: degenerate oligonucleotide polymerase chain reaction; LA-PCR: ligated adaptor PCR; MDA: multiple displacement amplification
Kit name WGA technique Company Duration DNA input DNA yield Refs
DOP-PCR DOP-PCR NA ~2.5 h 15 pg 300 ng 20,21
iDOP-PCR DOP-PCR NA ~1.5 h 15 pg 1 μg 20
DOPlify DOP-PCR PerkinElmer ~3 h 1 cell >1.25 μg 22 Ampli1 WGA kit LA-PCR Silicon
Biosystems
~7 h 1 cell 2-4 μg 28
Genomeplex single cell WGA4 kit
LA-PCR Sigma Aldrich ~4 h 1 cell 5-10 µg 29
REPLI-g single cell kit MDA Qiagen ~9 h 1 cell 25–40 µg 32
TruePrime single cell WGA kit
MDA Sygnis ~3 h 1 cell 3-10 μg 23
Illustra single cell GenomiPhi DNA amplification kit
MDA GE Healthcare ~3 h 1 cell 4-7 μg 24
PicoPLEX WGA kit Displacement DOP-PCR Rubicon Genomics ~2.5 h 1 cell 2-5 μg 39 MALBAC single cell WGA kit Multiple annealing and looping based amplification cycles Yikon Genomics ~4 h 1 cell 2-5 μg 25
2.1 PCR-based WGA
2.1.1 degenerate oligonucleotide primed PCR
A well-established method for PCR-based WGA is called degenerate oligonucleotide primed PCR (DOP-PCR) (figure 1). Here, primers consisting of six random nucleotides flanked by a fixed sequence at the 3’ and 5’ ends are added to the DNA sample to be amplified. 26 During the first 5-8
amplification cycles, a low annealing temperature allows binding of the fixed 3’ end and the random primer sequence to complementary DNA sequences. After primer binding, a raise in temperature
(75–80 °C) allows strand extension via Taq polymerase with a rate of ~150 nucleotides (nt) per second. This enzyme does not have 3’ —> 5’ exonuclease proofreading, resulting in an error-rate of approximately 1 in 9000 nt and amplification of fragments up to 1500 basepairs (bp). 20,27 During the
subsequent cycles of amplification (>25), the annealing temperature is raised, resulting in mostly non-random primer binding, and thus amplification of the amplicons with complete complementarity to the primer. 5 A disadvantage of DOP-PCR is its inability to cover the whole
genome due to the short amplicon length. Furthermore, uneven exponential amplification results in amplification bias; sequences difficult to amplify will be under amplified or not covered at all whereas sequences that are easy to amplify will be over amplified. 5 With 15 pg DNA input, a DNA yield of 300 ng can be obtained, which would be sufficient for STR profiling. 21 Maciejewska et al. (2013) investigated the use of DOP-PCR for forensic purposes and observed a DNA yield of 5620x and 2032x when using an input of 1 ng non-degraded or degraded DNA, respectively. 12 For degraded DNA, this yield exceeds the yield obtained with MDA (section 2.2), which could be due to the need of smaller templates for DOP-PCR. Figure 1. Principle of degenerate oligonucleotide primed PCR.
Degenerate primers consist of a random hexamer sequence, flanked by a fixed 5’ and 3’ sequence. Semi-random binding of degenerate primers and extension takes place during the first 5-8 amplification cycles. During the exponential amplification step, the annealing temperature is raised, resulting in non-random primer binding and extension.
Over the years, multiple variants of DOP-PCR have been developed. 20,22,28 Here, modified
primers and other DNA polymerases with better proofreading capabilities are used in order to improve the genome coverage and to reduce the amount of required input DNA. For example, for iDOP-PCR a DNA input of 15 pg is sufficient to obtain genome coverage of 60% and DOPlify (PerkinElmer) can be performed with single cells. 20,29
2.1.3 PCR with ligated adaptors
The DOP-PCR and DOP-PCR variant methods described above all use random primers for amplification, contributing to amplification bias. Multiple PCR-based methods have been developed with the aim to reduce amplification bias via genomic fragmentation followed by ligation of a
Degenerate primer design Fixed 5’Random hexamer Fixed 3’
Degenerate primer binding and extension
Exponential amplification 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
universal primer binding sequence to each fragment (figure 2). Two kits in which this method is used are Ampli1 (Silicon Biosystems) and GenomePlex WGA4 (Sigma Aldrich).
The principle of Ampli1 is based on enzymatic digestion of the complete genome with Msel, which cleaves TTAA sequences in the DNA. After digestion, an adaptor sequence is ligated to the 3’ and 5’ ends of each fragment. This sequence is complementary to the universal primer that is added to the sample during amplification. 30 WGA using one universal primer results in a more uniform
amplification compared to random primers. GenomePlex WGA4 differs from Ampli1 in the DNA fragmentation step, where Ampli1 uses an enzyme for DNA fragmentation and GenomePlex WGA4 uses heat-induced DNA fragmentation. 31 Here, the isolated DNA is heated to 99 °C for four min. This
method could be preferred over enzymatic fragmentation, as it does not lead to biased amplification of amplicons containing the sequence recognized by the restriction enzyme.31 Figure 2. Principle of Ampli1 and GenomePlex WGA4 kits for PCR with ligated adapters. Random fragmentation of genomic DNA is performed via digestion with the Msel restriction enzyme (Ampli1) or via heat (GenomePlex WGA4). Upon fragmentation, a universal adapter sequence is ligated to the 3’ and 5’ ends of the genomic DNA fragments. This sequence is complementary to a universal primer that will be added during amplification.
Biezuner et al. (2017) compared the performance of seven single cell WGA kits, including Ampli1 and GenomePlex WGA4. 13 The performance of each kit was evaluated via determining
genome coverage and error rate of microsatellite loci. Of all analyzed kits, Ampli1 showed the highest genome coverage, with an average of 1095.5 amplicons. In contrast, GenomePlex WGA4 showed the lowest genome coverage, with an average of <200 amplicons. The error rate of GenomePlex WGA4 is lower compared to Ampli1, but does not excel the error rate of MDA-based methods. 13 Maciejewska et al. (2013) investigated the use of GenomePlex WGA4 for forensic
purposes and observed an increase in DNA yield of 153x and 75x when using an input of 1 ng non-degraded or degraded DNA, respectively. 12 Compared to the other investigated methods, which did not include Ampli1, this is a low amplification efficiency. ATATTAAT A T ATATTA ATAT TATA
Msel digestion Heat-induced
Random genomic DNA fragmentation
Ampli1 GenomePlex WGA4
genomic DNA fragments
Adapter sequence ligation
Amplification Universal primer binding
In conclusion, PCR-based WGA methods that rely on DNA fragmentation, adaptor ligation and amplification with a universal primer show reduced amplification bias compared to DOP-PCR. Since the DNA is fragmented prior to the amplification step, the sample does not need to contain intact DNA. Therefore, Ampli1 and GenomePlex WGA4 might be suitable for crime samples with degraded DNA, such as old samples from cold cases. 12,32
2.2 MDA-based WGA
A difference between PCR-based WGA and MDA-based WGA is that MDA-based WGA does not rely on amplification cycles of denaturation, annealing and extension, and therefore does not require a thermal cycler. MDA is an example of random and isothermal amplification and uses an enzyme with strand displacement activity, thereby discarding the need for a denaturation step (figure 3). This enzyme, the bacteriophage Phi29 DNA polymerase, extends the DNA with 50-200 bp/s, which is faster then Taq polymerase. 33 During synthesis Phi29 DNA polymerase displaces downstream DNAand exerts 3’ —> 5’ exonuclease proofreading activity, resulting in a low error rate between 1 in 100.000 and 1 in 1.000.000 nt. 4,34 Moreover, MDA does not suffer from inhibitors including hemoglobin and humic acid. 35 As a result, some kits do not require blood samples to be purified
prior to amplification. 36 This is favorable in forensic investigations, as each additional processing
step increases the chance of contamination. Similarly as PCR-based amplification MDA occurs exponentially, and uneven exponential amplification results in amplification bias. However, in general genome coverage of MDA-based WGA is higher compared to PCR-based WGA.5,37
Multiple MDA-based kits for single cell WGA have been developed, including REPLI-g (Qiagen), TruePrime (Sygnis) and GenomiPhi V2 DNA amplification (GE Healthcare). The general principles of these protocols are similar, but some important differences will be discussed below. REPLI-g and TruePrime both use alkaline for denaturation, whereas GenomiPhi V2 DNA amplification uses heat-induced denaturation. 23,24,36 Interestingly, skipping this denaturation step actually
increases the DNA yield. 4,38 An advantage of REPLI-g and GenomiPhi is that the method cannot only be used for single cells, but also for unpurified samples, including whole blood and dried blood samples. 24,36 The highest DNA yield (40 μg) is also achieved by REPLI-g. 36 GenomiPhi V2 DNA
amplification follows with a yield of 4-7 μg and for TruePrime yields between 3 and 10 μg have been reported. 23,24,39 All yields would be sufficient for subsequent STR analysis. For REPLI-g and
GenomiPhi V2 DNA amplification, random primers are added, which could lead to amplification bias. TruePrime uses a different mechanism. Here, a DNA primase called Tth PrimPol synthesizes DNA primers for Phi29 DNA polymerase. The duration of the protocols of TruePrime and GenomiPhi V2 DNA amplification are ~3 h, whereas the duration of the protocol of REPLI-g is ~9 h. 23,24,36
A number of researchers have compared the performance of two or more MDA kits and other WGA methods and some variation is observed between the results obtained in each study.11,13,40 This could be due to the different human celltypes and genetic analyses that were used. For example, in one comparative study the highest genome coverage (91.26 %) was reported when using TruePrime, 39 whereas a very low genome coverage for TruePrime (amplicon count <200) was reported elsewhere. 13 The efficiency of MDA partly depends on the formation of hyperbranched structures and the generation of long amplicons, which requires a long DNA template. Therefore, it was proposed that
MDA is not suitable for WGA of degraded DNA samples. 37 In accordance with this hypothesis,
Maciejewska et al. (2013) showed an increase in DNA amount of 10034x when using an input of 1 ng non-degraded DNA and only an increase in DNA amount of 796x when using an input of 1 ng degraded DNA (100-200 bp). 12 Taken together, MDA could be a valuable tool in forensic
investigations, as DNA yields between 3 and 40 μg can be obtained with single cell WGA. However, the usefulness of MDA for highly degraded DNA samples is limited.
Figure 3. Principle of multiple displacement amplification.
Random hexamer primers and Phi29 polymerase are added to a DNA sample. The random hexamer primers bind to complementary sequences in the DNA and are extended by Phi29 polymerase, which can displace upstream double-stranded DNA. The amplicons serve as new DNA template.
2. 3 Hybrid methods
Two hybrid kits for single cell WGA are multiple annealing and looping-based amplification cycles (MALBAC) (Yikon Genomics) and PicoPlex (Rubicon Genomics). The principle underlying these methods is a combination of displacement pre-amplification followed by PCR amplification. It could be viewed as a combination between MDA and PCR, as the polymerase that is used has strand displacement activity, but the amplification reaction resembles PCR-based methods.
The workflow of MALBAC consists of quasi-linear amplification and exponential amplification. 25 Cell lysis takes ~60 min and is followed by pre-amplification. Here, primers
consisting of 8 random nt at the 3’ end and 27 fixed nt at the 5’ end are added to the DNA sample. During 10 amplification cycles, the 3’ end of the primers will hybridize to complementary DNA sequences and a raise in temperature (65 °C) allows strand extension by Bst polymerase. An advantage of this polymerase is its strand displacement activity and, compared to Taq polymerase, its low error rate of 1 in 10.000 nt. Another raise in temperature (94 °C) will cause dissociation of the amplification products from their templates. The products are then used to generate amplicons with complementary ends, resulting in hairpin formation after dissociation. Hairpin formation protects and thereby prevents the amplification of amplicons. Thus, with MALBAC only amplicons of the original DNA are generated. This quasi-linear amplification is preferred over the exponential
5’ 3’
Random primer binding
5’ 3’
Extension by Phi29 pol
5’ 3’
Strand displacement by Phi29 pol
5’ 3’
amplification of DOP-PCR and MDA, as it does not result in over and/or under amplification of certain sequences. After quasi-linear amplification, the full amplicons undergo multiple rounds of exponential amplificaiton. 5 It is possible to obtain 2-5 μg DNA from 1 cell, which would be sufficient
for subsequent STR analysis.25
Figure 4. Principle of MALBAC and PicoPlex kits for hybrid WGA.
Primers with a fixed 5’ end and a random 3’ end are added to the DNA sample. The random 3’ end binds to complementary DNA sequences and is extended by a DNA polymerase. During the quasi-linear amplification step, multiple rounds of denaturation, primer binding and extension full amplicons with the fixed primer sequence on both ends of the amplicon are produced. These amplicons undergo loop formation, thereby ensuring that only amplicons of the original DNA template are generated. During exponential amplification the full amplicons are amplified.
PicoPLEX is also based on displacement pre-amplification followed by multiple PCR-cycles and the protocol resembles the MALBAC protocol. 41 However, different reagents are used and the
number and temperature of amplification cycles differ. The most recent version of PicoPLEX can be used for the amplification of 1-5 human cells. 41 A DNA yield of 2 μg can be obtained and the
protocol takes ~3 hours. Biezuner et al. (2017) reported an average amplicon count of 750 amplicons, which is slightly higher than the average amplicon count of MALBAC in the same experiment (696.5 amplicons). 13 Taken together, an advantage of hybrid WGA methods is the absence of amplification bias. However, in contrast to MDA-based WGA, a thermal cycler is needed for the amplification cycles. As a result, it could be challenging to perform MALBAC or PicoPLEX on the crime scene. Xu et al., 2019 highlighted the potential of single cell MALBAC prior to STR profiling in forensic investigations, but the influence of DNA degradation remains unknown. 42 Studies with samples resembling real crime traces could provide valuable insights with regard to the success rate of MALBAC and PicoPLEX as a means to increase the amount of input DNA for STR analysis.
Random primer binding
Extension 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ Quasi-linear amplification Looping Exponential amplification
2.4 Performance of single cell WGA analyzed with STR profiling
A number of studies have compared the performance of various WGA methods but few studies used STR analysis to evaluate the performance of single cell WGA. Therefore, some studies in which single cell WGA products were analyzed with other genetic analyses are also included in this report. The performance of each method is most often addressed in literature via the allelic dropout (ADO) rate. Future research should elucidate the effect of single cell WGA not only on ADO rates, but also on allelic drop-in (ADI) rates and the occurrence of stutter peaks in resulting STR profiles.Of all methods described above, the highest ADO (percentage of observed/expected alleles) and ADI (percentage of discordant observed alleles) rate was reported for DOP-PCR. DOP-PCR products from 15 pg DNA input analyzed with multiplex STR showed an ADO+ADI rate of 99%.20 For iDOP-PCR products with the same amount of input DNA analyzed with multiplex STR, the ADO+ADI rate was improved to 50%.20 However, the DNA input of 15 pg DNA corresponds to 2-3 cells. Thus, it
remains unclear whether this method would be suitable for single cell WGA in forensic investigations. For single cell DOPlify analyzed with STR profiling an ADO rate of 60% was reported. 29
This is an improvement relative to conventional DOP-PCR, but does not exceed some MDA-based or hybrid WGA methods.
The performance of LA-PCR methods Ampli1 and GenomePlex WGA4 was evaluated with the aim to develop a non-invasive alternative to prenatal diagnosis. 43 Here, single unfixed and fixed
lymphoblastoid cells amplified with Ampli1 and Genomeplex WGA4 were analyzed with multiplex STR. For Ampli1, relatively low ADO rates of 6% and 19% for unfixed and fixed cells were reported, respectively. However, these results are not in line with the ADO rate of 61% that has been reported elsewhere. 29 The ADO rates reported for GenomePlex WGA4 were much higher, namely 69% and 51% for unfixed and fixed cells, respectively. This could be an effect of random fragmentation, as breakpoints can arise at STR loci. 44 Despite these relatively high ADO rates GenomePlex WGA4 could
still be a useful tool in forensic investigations. Uchigasaki et al. (2018) investigated the use of a GenomePlex kit coupled to STR-PCR to analyze dried human bloodstains that were exposed to ultraviolet (UV) light. 32 UV light induces DNA damage, and thus negatively impacts the success rate
of DNA analysis. As crime traces that have been exposed to UV light could be encountered at crime scenes, insights with regard to the use of WGA for these crime samples are relevant in a forensic setting. In this study, dried blood samples were exposed to multiple cycles of UV light. The results showed that alleles that would be lost with normal STR analysis could be recovered when samples were pre-amplified with WGA prior to STR-PCR. 32 Therefore, LA-PCR methods could be a suitable
WGA method in forensic investigations when DNA degradation has occurred.
The performance of MDA method REPLI-g has been evaluated via STR analysis. Here, an ADO rate of only 8% was reported, which is the lowest ADO rate of all analyzed methods in this study. 29
The performance of other MDA methods has not been evaluated via STR analysis. However, a comparative study including REPLI-g, GenomiPhi and TruePrime reported ADO rates of 60% for REPLI-g and GenomiPhi, and 90% for TruePrime. Here, instead of STR analysis the amplified products were analyzed with whole genome sequencing and SNP genotyping. 11 In order to determine which
MDA method is most suitable in forensic investigations, it will be essential to investigate the performance of each kit using STR analysis as genetic detection method. It should also be taken into account that a disadvantage of MDA is the requirement of a long DNA template. Therefore, it was
proposed that MDA is not suitable for WGA of degraded DNA samples. 37 Consistent with this
hypothesis, no STR profiles could be obtained from biologically degraded DNA that was amplified with REPLI-g prior to STR-PCR. 12 Thus, MDA could become a valuable tool for the amplification of
crime samples containing a small amount of DNA, but its applicability might be limited to non-degraded DNA samples.
Hybrid WGA methods have been developed with the aim to combine some of the advantages of PCR- and MDA-based methods. Xu et al. (2019) evaluated the performance of the hybrid method MALBAC with the Goldeneye™ DNA ID 22NC STR profiling kit, in which 20 autosomal STR loci are analyzed. When performing MALBAC prior to STR analysis on 6.25 pg input DNA, the success of STR analysis increases from 5 to 8.7 correctly detected alleles. 42 For single cell PicoPLEX
coupled with STR-PCR, ADO rates between 36-49% have been reported, which is lower than LA-PCR, but does not exceed the performance of MDA. 29,43 In order to elucidate whether DNA degradation
3 On-chip whole genome amplification
In chapter 2 it was proposed that single cell WGA can improve the succes rate of subsequent STR analysis. For forensic investigations, on-chip single cell WGA could have several advantages, including reducing the chance of contamination and separating DNA from donors in a mixture already at the amplification stage instead of after STR analysis. The following section contains an overview of some available microfluidic devices for the performance of on-chip WGA (table 2).
Chips can be made of inorganic materials, including silicon, glass and ceramics, organic materials, including thermoplastic polymers, polycarbonate (PC) and poly-di-methyl-siloxane (PDMS), or a combination of inorganic and organic materials. 6 Which material is best depends on the application of the chip. For example, chips made from some polymers are inexpensive and often suitable for mass production. However, drawbacks of polymers include ageing of the material, water permeability, which allows evaporation of the sample, and adsorption of hydrophobic molecules. In contrast, glass chips are more expensive to fabricate but are not permeable to water and show less adsorption compared to some polymers. These issues should be taken into account when designing a microfluidic device.3 Various chip designs for isolating and analyzing single cells on-chip have been
developed. For example, a sample can flow through multiple connected chambers and microchannels in a continuous flow, or the flow can be interrupted via valves. Valves are used to regulate the single cell flow through the microfluidic channels and the mixing of reagents. 45 A similar
but less complex design is the use of closed nanowells. The reaction volume can be scaled down even further via the generation of picoliter (pl) droplets on-chip. 46 In this chapter, examples of
microfluidic devices for on-chip MDA and MALBAC with diverse chip designs are discussed.
Table 2. Overview of single cell WGA methods on-chip.
*with DNA input of 4.2 pg. WGA: whole genome amplification; MDA: multiple displacement amplification; MALBAC: multiple annealing and looping based amplification cycles
WGA
technique Approach Cell type Reaction units per chip Duration DNA yield Refs
MDA Chambers separated via valves Bacterial cells 7 ~16 h 40-50 ng 43 Circulating tumor cells 10 ~3 h unknown 20 Primary and cultured cells 96 ~9.5 h 150-200 ng 47
Wells Colorectal cancer cells 8 ~8 h unknown 50
Droplets Human umbilical vein endothelial cells 1 ~10 h 2000 ng 48 Bacterial cells 1 ~18 h ~10 ng* 49 Micropillars HeLa cells 10 ~4 h 60 ng 51 MALBAC Chambers separated via valves Mouse embryonic stem cells 8 ~4 h 50 ng 52
3.1 MDA on-chip
3.1.1 Valve-based MDA on-chip Multiple microfluidic devices for MDA on-chip have been developed in the medical field in order to reduce amplification bias and contamination via reagents or other DNA present in the sample. 17,45,47 When designing a chip for MDA, it should be taken into account that the amplification mixture of MDA can interact with the chip material, resulting in a delay or inhibition of the reaction. This problem could be overcome via coating the surfaces of the chip, for example with bovine serum albumine (BSA). 7 Chips for on-chip MDA often consist of microchannels and chambers connected via valves.17,45 The valves have a regulatory function in sample preparation (e.g. the delivery and mixing of reagents). The actual MDA reaction occurs in a closed chamber. Marcy et al. (2007) developed a microfluidic chip for isolation and subsequent MDA of single bacteria. 45 This microfluidic device is made from PDMS and allows simultaneous single cell MDA of 7 cells plus a negative and positive control (figure 4A). Cells are first pumped into a sorting channel containing an isolating region. When a cell is detected - this could be done via detection of a fluorescent signal from fluorescently labeled cells or manually via microscopy - this cell is isolated via closing the isolation valve. Next, an image is made to confirm that only one cell is isolated. If one cell is detected, this cell will be pumped into the next chamber. From here, single cell MDA will be performed according to a modified REPLI-g protocol in four chambers per reaction, in a reaction volume of 60 nl (figure 4B-I). The first two chambers are used for alkaline cell lysis. When the neutralization buffer is added the third chamber is opened, and the fourth chamber is opened prior to the amplification reaction. For amplification, the chip needs to be heated for 10-16 h at 32 °C.From one single cell ~107 copies of the genome can be generated, corresponding to a DNA yield of
40-50 ng. In order to obtain enough DNA for sequencing, the products from on-chip MDA were re-amplified off-chip. This step makes this method less suitable for forensic purposes, as it reduces the time- and cost-effectiveness and increases the chance of contamination. 45 However, this extra
amplification step might not be necessary for on-chip MDA coupled to off-chip STR analysis, as the minimal DNA input for STR analysis is ~1 ng. 1 Overall, MDA on-chip reduces the amplification bias
and reagent cost per reaction. However, the experiments were performed with bacterial cells, so further research could elucidate whether this method yields similar results with human cells and whether this method could be coupled to off-chip STR-PCR.
Figure 4. Design and workflow for MDA on-chip. Adapted from “Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells”.45
A) Image of the microfluidic device, showing the channels and chambers (blue) and channels to control the valves (red). B-I) Schematic of the chambers in which each step of the reaction is performed. Closed valves are shown in red and opened valves are transparent. Reagents are directed to the reaction chambers via the feed line and the amplified products are collected in a tube. The feed line is flushed between reaction steps. Li et al. (2019) developed a research chip for MDA-based WGA of single circulating tumor cells from whole blood. 17 The chip integrates multiple steps: isolation, enrichment and amplification of circulating tumor cells. The chip consists of a glass layer, a sample processing layer from PDMS and a sample control layer from PDMS (figure 5A/B). A valuable characteristic of this chip is that whole blood samples can be used as input material. This could also be useful for the analysis of blood traces in forensic investigations. The blood sample first enters a filtering chamber, in which the blood is filtered from large impurities (Figure 5C). Next, the filtered blood enters an enrichment chamber which enriches the circulating tumor cells. This is not relevant for forensic purposes, but incorporating an enrichment chamber for specific cell types could be useful for investigations of mixed crime samples. The sample also passes a staining chamber for fluorescent cell labeling. This is of importance for the algorithm controlling the valves and pumps in the WGA chamber. After passing the staining chamber, single cells are isolated/captured in the subchannels of the WGA chamber (figure 5F). The WGA chamber consists of 10 subchannels, enabling WGA in parallel of 10 single cells. MDA is performed in a volume of 600 nl within a time period of ~3 h. Subsequent whole genome sequencing showed that with this method it is possible to identify cancer-related mutations and SNPs between the single circulating tumor cells. The latter suggests that this method might be valuable for forensic SNP analysis. However, first it should be elucidated whether this method could be coupled to off-chip STR-PCR. A B C D E F G H I
Figure 5. Design and workflow for MDA on-chip. Adapted from “Device for whole genome sequencing single circulating tumor cells from whole blood”.17
A) Schematic of the three layers of the microfluidic device: a glass layer, a sample processing layer from PDMS and a sample control layer from PDMS. B) Image of the microfluidic device; the chambers and valves are depicted in red. C) The blood filtering chamber consists of a micropillar array, which traps large impurities. F) Schematic (top) and image (bottom) of the chambers in which each step of the reaction is performed. CTC: circulating tumor cell; MDA: multiple displacement amplification; WGA: whole genome amplification
A commercially available MDA-based chip is the C1 System (Fluidigm). 47 Here, 96 single cells
can be isolated, lysed and amplified on one chip within 9.5 h. An advantage of this chip compared to the chip described by Marcy et al. (2007) is the relatively high DNA yield of 150-200 ng. 45 This yield is
sufficient for subsequent off-chip whole genome sequencing, in which an average genome coverage of more than 90% can be obtained. 47 No data is available with regard to amplification via the C1
system coupled to off-chip STR-PCR. An issue with the C1 system is that cell selection is biased towards a certain cell size. 50 This could be problematic in a forensic setting where traces consisting of multiple cell types are encountered. In addition, the microfluidic chip needs to be placed in the C1 system, thereby limiting the portability of the method. 3.1.2 Well-based MDA on-chip Marie et al. (2018) developed a microfluidic device for the isolation of single cells, cell lysis and MDA with REPLI-g. 51 In this study, single cells from a colorectal cell line were used for analysis. The chip material is a combination of cyclic olefin copolymer and glass and the chip contains 12 wells with a volume of 50 μl (figure 6). The entrapment of single cells in each well can be monitored with a microscope. After single cell entrapment, cell lysis is performed in nl volumes and MDA using REPLI-g is performed in μl volumes. The amplification reaction takes ~8 h. The amplified products were analyzed with off-chip whole genome sequencing and from the obtained results it was concluded that the genome coverage obtained with well-based MDA on-chip is better compared with methods in which cell lysis and amplification are both performed in μl volumes. However, genome coverage is similar compared to methods in which cell lysis and amplification are both performed in nl volumes. The latter are often complex microfluidic devices, which are difficult and costly to produce. Taken
together, this study suggests that it is unnecessary to use complex microfluidic devices for MDA in nl volumes; cell lysis in nl volumes followed by MDA in μl volumes yields similar results. 51 A simple
microfluidic device for MDA on-chip could be suitable for the development of a portable method that could be performed on the crime scene. However, the amplification products have not yet been analyzed with STR-PCR, so the applicability for forensic investigations remains unknown.
Figure 6. Design for MDA on-chip in wells. Adapted from “Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device”.51
A) Image of the single use cyclic olefin copolymer microfluid device B) Schematic of the microfluidic device, which contains a cell inlet, two buffer inlets (B1 and B2), a waste outlet and 8 outlets for single cell entrapment, lysis and MDA.
3.1.3 Droplet-based MDA on-chip
Various researchers have reported the use of a microfluidic device for WGA in droplets. 52 The
method described by Fu et al. (2015) is called emulsion WGA (eWGA). 48 Here, MDA is performed in
pl droplets, resulting in a more uniform amplification of the genome and less contamination via reagents compared to tube-based MDA. In this study, single cells are isolated and lysed off-chip (figure 7). The resulting DNA fragments have a length of ~10 kb and are mixed with the MDA reaction buffer. Droplet generation is achieved via injecting the solution into a microfluidic device made from PDMS containing mineral oil. The emulsification is driven by compressed air. After emulsion, the pl droplets, each containing one or two DNA fragments are transferred to a tube and incubated at 30 °C for 8-10 h for MDA. Fu et al. (2015) observed a higher genome coverage when using eWGA compared to tube-based MDA. 48 An explanation for this observation is that there is no
over amplification of contaminant DNA that is transferred to the sample via the reagents, as it will only be present in a few droplets. A similar approach was described by Rhee et al (2016). 49 This method, called digital droplet MDA (ddMdA), has a similar mechanism: a microfluidic device generates droplets with a volume of 150 pl prior to the off-chip MDA reaction.
Figure 7. Workflow for emulsion whole genome amplification (eWGA). Adapted from “Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification”.48 Single cells are lysed and mixed with MDA reaction buffer. The resulting solution is used for on-chip emulsion generation followed by off-chip MDA, or immediately used for conventional off-chip MDA. In each graph the DNA copy number is plotted against the amplification time. The lower graph (eWGA) shows a more uniform amplification compared to the upper graph (conventional MDA).
It is important to not have too many DNA fragments into one droplet, as the number of fragments per droplet affects the amplification rate. Thus, in order to inject the solution with DNA fragments and MDA reaction buffer into the microfluidic device in a specific dilution, it is important to perform DNA quantification prior to droplet generation. This could be challenging in a forensic setting, where the level of DNA degradation is not known. Furthermore, only droplet generation was performed on-chip; the selection, isolation and lysis of single cells and the actual MDA reaction was performed off-chip. In order to minimize the chance of contamination, which is of great importance in forensic investigations, eWGA could be improved via incorporating these steps into the microfluidic device that was used for droplet generation.
3.1.4 Genomic amplification via micropillar arrays (GAMA)
Another method for MDA on-chip involves flowing fluids through a chamber with micropillars. In this study, a cell suspension with HeLa cells was injected into a microfluidic device from PDMS. 53 From
here, the fluid is flowed through 10 identical micropillar arrays, consisting of micropillars with a height of 1.5 μm, spaced apart in a radius of 2 μm, towards an output port (figure 8A). This way, 10 single cells can be amplified with REPLI-g in parallel. Single cell entrapment is achieved at the apex of the micropillar array, and could be optimized via adjusting the height and spacing of the micropillars (figure 8B). Upon cell lysis, the genomic DNA will become entangled into the micropillar array, whereas smaller molecules will flow towards the output port of the microfluidic device. A previous report showed that entrapped DNA strands extend over 11 mm of the micropillar array. Due to the relatively small length of the amplification products compared to the genomic DNA, the amplicons will not be trapped in the micropillar array, and can be collected in the output ports (figure 8C). The amplification products were analyzed via whole exome sequencing, and the average genome coverage of GAMA was concluded to be higher compared to the average genome coverage of off-chip REPLI-g. A proposed explanation for this difference is that the constant fluid flow results in more uniform amplification. Mixing of reagents does not depend on passive diffusion and the amplification products are carried out of the micropillar array immediately after amplification,
thereby preventing chimera formation. GAMA could be a useful tool in forensic investigations, as the genome coverage is improved compared to off-chip MDA and cell lysis and WGA are performed in one enclosed microchannel. However, the entrapment of genomic DNA via micropillars might not be efficient for degraded DNA and amplification products have not yet been analyzed with off-chip STR-PCR. Furthermore, channels in which multiple cells are entrapped (4 out of 10 channels) are now discarded from further analysis. In forensic investigations, you do not want to lose the information from these cells. Thus, the protocol needs optimization before GAMA could be used for forensic purposes.53 Figur 8. Workflow for genomic amplification via micropillar arrays (GAMA). Reprinted from “Single cell on-chip whole genome amplification via micropillar arrays for reduced amplification bias”.53 Schematics of the device A) the blue arrow represents the fluid flow from left to right B) single cells are trapped in the apex of the micropillar array. Excess cells flow towards the output reservoir via the side walls enclosing the micropillar array C) after single cell entrapment, cell lysis buffer is flowed through the device. The genomic DNA (orange) will become entangled in the micropillars, due to its length. Smaller molecules (RNA, proteins and lipids) are too short to become entangled and will flow through the device. Upon WGA, the amplification products (blue), which are also too short to become entangled, will flow through the device and will be collected in the output reservoir.53
3.2 MALBAC on-chip
Yu et al. (2014) developed a valve-based microfluidic device for single cell MALBAC. 54 Compared to other WGA methods MALBAC provides high genome coverage and does not suffer from substantial under and over amplification of certain sequences. 5,37 The chip is made of PDMS and contains eight fluidic channels (figure 9A). Each channel consists of three separate chambers: a cell lysis chamber, a MALBAC preamplification chamber and a MALBAC PCR chamber (figure 9B). The interfaces between the chambers can be opened and closed. Samples are injected via one injection chamber, from which single cells can enter one of the eight cell lysis chambers. Cells need to be injected in a solution with a concentration of 104-105 cells/mL and a microscope can be used to monitor that only
single cells enter the cell lysis chamber. The total process, including cell lysis with Protease, 10 pre-amplification cycles and 16 PCR single cells enter the cell lysis chamber. The total process, including cell lysis with Protease, 10 pre-amplification cycles, takes ~4 hours and 8 reactions can be performed simultaneously. A microscope can be used to ensure that a single cell is present in each fluidic chamber (figure 9D). Yu et al. (2014) reported a 8000-fold increase in genomic DNA compared to the genomic DNA present in a single human diploid cell. 54
Figure 9. Workflow for MALBAC on-chip. Reprinted from “Microfluidic Whole Genome Amplification Device for Single Cell Sequencing”54 A) Schematic of the microfluidic device containing 8 fluidic channels (purple) and control channels separating the fluidic channels (magenta). B) Schematic of the chambers and volumes in which each step of the reaction is performed. C) Schematic of the set-up for the whole MALBAC reaction. The microfluidic device is placed on a modified thermal cycler and placed under a microscope. D) Image of a single-cell on the microfluidic device. MALBAC: multiple annealing and loopin based amplification cycles; PCR: polymerase chain reaction; PDMS: poly-di-methyl-siloxane.
Compared to MALBAC performed off-chip, the contamination level of on-chip MALBAC was reduced from ~4.8% to ~2.4%. This percentage represents the portion of DNA sequences from other species (including Haemosporida and Primate DNA) that was present in the amplified product.54
Another advantage of on-chip MALBAC is its small reaction volume of 1.1 μl, resulting in a reduction in reagent cost and a reduction in contamination via reagents. The chance of contamination is also reduced via the use of an enclosed microchannel. This is especially of great importance in forensic investigations where the results from the analysis could later be used as evidence in court. Finally, more fluidic channels and/or reaction chambers could be added to the chip, allowing the inclusion of more replicates or more functions, respectively. A drawback of on-chip MALBAC is that, although the chip itself is very small, the whole method is not portable. Non-portable equipment that is needed for on-chip MALBAC includes a modified thermal cycler to heat and cool the whole chip and a microscope to observe the cell flow through the chambers (figure 9C). Taken together, we conclude that on-chip MALBAC could be valuable in forensic investigations due to its small reaction volume, but is currently not suitable to be performed at the crime scene.
4 Conclusions and recommendations
4.1 WGA preceding STR profiling increases success rate
Few studies have investigated the performance of single cell WGA with the aim to increase the success rate of STR analysis for forensic investigations. More often, the aim is to determine whether WGA could be used to obtain enough DNA for subsequent analysis of specific disease-related alleles.17 Due to the different cells and detection methods that are used in each study, it is challenging to compare the results of these studies. However, the findings of these individual studies still provide valuable information to determine which kit would be suitable for forensic purposes. For STR analysis in a forensic context, low ADO and ADI rates and minimal stutter are preferred. In order to achieve this, the single cell WGA method preceding STR-PCR should have a low amplification bias and high genome coverage. Due to the high error-rate of Taq polymerase and the need for a thermal cycler, of all methods described in this review it is proposed that DOP-PCR is least suitable for forensic investigations. Although the ADO+ADI rate of DOP-PCR variants is improved compared to conventional DOP-PCR, they do not surpass the performance of MDA-based or hybrid WGA. Due to the high DNA yields (3-40 μg) and relatively low dropout rates – i.e. 8% for REPLI-g - that have been reported for single cell MDA kits, 29 MDA could become a valuable tool for forensic
analyses. However, these experiments were performed with high quality DNA and it remains unclear whether similar results could be obtained with degraded DNA samples. For example, no DNA profiles could be obtained from biologically degraded DNA amplified with MDA preceding STR-PCR.12
For degraded DNA, LA-PCR might be preferred over MDA, since LA-PCR could increase the success rate of obtaining a DNA profile from these samples. 12 A disadvantage of both PCR- and MDA-based
single cell WGA is the amplification bias that arises due to exponential amplification. The two hybrid methods described in this review, MALBAC and PicoPLEX, make use of quasi-linear amplification, thereby reducing amplification bias. 5 Although, hybrid WGA improves the success rate of STR profiling, ADO rates reported for hybrid methods do not surpass MDA ADO rates.
The lowest ADO rates and the highest genome coverage have been reported for MDA. An advantage of MDA over PCR-based and hybrid WGA is the absence of an amplification step that requires a thermal cycler. This would potentially simplify an on-chip WGA method that could be used at the crime scene. However, the need for long DNA templates could become problematic for degraded DNA samples, such as old DNA samples from cold cases. Here, hybrid or LA-PCR WGA could be more successful.
Taken together, single cell WGA preceding STR-PCR increases the chance of obtaining a useful DNA profile from single cells. Overall, the performance of MDA exceeds the performance of PCR-based or hybrid WGA. However, this is based on experiments with high-quality DNA whereas the quality of DNA encountered in real traces is often impaired. Studies with DNA samples mimicking real crime traces could elucidate whether other methods might be more suitable for forensic casework.
4.2 On-chip WGA reduces contamination and reaction volume
Recently, multiple valve-, droplet- and well-based microfluidic devices for single cell WGA have been developed. Single cell isolation and lysis, which is performed prior to WGA, is often incorporated in the chip. This is advantageous in forensic investigations, as it reduces the chance of contamination. The available microfluidic devices for WGA on-chip all use MDA or a hybrid technique for WGA. The absence of PCR-based WGA chips could be explained by the higher ADO/ADI rates that have been observed for PCR-based methods off-chip and the need for a thermal cycler, which abolishes the portability of a microfluidic device.Time-wise, valve-based MDA on-chip17, GAMA on-chip53, and MALBAC on-chip54, are
preferred to use for forensic purposes as they have reaction times of 3-4 h. This would enable fast analysis on the crime scene. Especially valve-based MDA on-chip17, where whole blood samples can
be used as input material due to the presence of a filtering chamber, could be useful for the analysis of blood crime traces. In MALBAC on-chip the contamination is reduced compared to MALBAC off-chip. However, a thermal cycler is required due to multiple rounds of amplification at various temperatures. 54
As a result, this method is not suitable for analysis on the crime scene. GAMA on-chip could potentially be a useful technique for forensic investigations. However, the single cell entrapment step of the protocol needs optimization in order to prevent the entrapment of multiple cells in one reaction unit and the effect of DNA degradation on entrapment should be investigated. 53
Advantages of droplet-based WGA are the simple chip design and reduced amplification bias. However, the duration of the protocol is long compared to other methods. Moreover, as the MDA reaction volume is similar to conventional MDA, the method does not add value with regard to time, cost and contamination. 48,49
Overall, genome coverage is improved and amplification bias and contamination is reduced in WGA on-chip compared to conventional WGA off-chip. The DNA yield of each method seems to be sufficient for subsequent STR-PCR. 17,45,48,49,53,54 However, on-chip WGA coupled to STR-PCR has not been investigated. Moreover, most methods have been performed with cultured cells, whereas in forensic casework environmental factors could have affected the quality of the DNA. Furthermore, little research has been performed with mixed samples containing multiple cell types, except by Li et al. (2018), who filtered the mixed samples for the cells of interest on-chip prior to WGA. 17 As the size of the microchannel is important for single cell isolation, it could be challenging to design a chip that is compatible with cells of different sizes. Single cell isolation could be problematic, as big cells might not be able to enter the microchannel and small cells can enter the microchannel simultaneously. On the other hand, the ability to select cells within a mixture may also be helpful in some situations, for example in sexual assault cases. This should be taken into account when designing a microfluidic chip for forensic purposes. In conclusion, before on-chip WGA could be applied in forensic casework, it is key to perform on-chip WGA with samples mimicking real crime traces and and optimize the method accordingly, and to evaluate the method with subsequent off-chip STR-PCR.
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