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Single Cell Whole Genome Amplification On-Chip for Forensic Purposes 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. I 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 II 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 1 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.
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