Genotyping tools for forensic DNA phenotyping : from low- to high-throughput

Abstract

Forensic analysis of deoxyribonucleic acid (DNA) evidence is a powerful tool for law enforcement that can provide a link between a suspect and a crime or eliminate a suspect from suspicion. Forensic analysis of DNA has two broad purposes: forensic identification and forensic DNA phenotyping (FDP). The purpose of identification is to associate a suspect with a crime based on DNA evidence, a feat that is achieved using genetic markers, such as short tandem repeats and single nucleotide polymorphisms (SNPs). Effective DNA identification relies on comparison between a reference DNA profile and an evidentiary DNA profile, which allows for the inclusion or exclusion of a suspect. If no reference profiles are available or there is a large pool of suspects, DNA profiling has limited capacity to resolve a crime. In these cases, police investigators often depend on eyewitness statements, which are notoriously unreliable. By contrast, FDP is the process of inferring phenotypic traits from DNA and can be used as a biological witness. FDP utilises SNPs along with other markers such as insertions-deletions and microhaplotypes. SNPs are the most common markers for FDP because of their low mutation rates and account for more than 85 percent of variance in the human genome.
Genome-wide association studies have identified a variety of SNPs associated with phenotypic traits, such as eye colour, hair colour, skin colour, baldness and freckles. These SNPs must be typed appropriately to generate FDP profiles. A range of SNP genotyping technologies exist, including: real-time polymerase chain reaction (PCR) based assays with probe hybridisation such as TaqMan (Thermo Fisher Scientific—TFS); microfluidic technology such as Fluidigm Biomark or Open array (TFS); single base primer extension assays such as SNaPshot™ (TFS); and post-PCR assays such as high resolution melt (HRM) analysis. Most recently, massively parallel sequencing (MPS) assays incorporating sequencing by synthesis are represented in the forensic field by Ion Torrent (TFS) and Illumina technologies. These SNP-typing technologies differ in cost, throughput, detection methods and run times, which can make it difficult to choose between them for FDP purposes. Some forensically important criteria include simplicity of operation, reliability, reproducibility, flexibility and modularity. An ideal method should be cost effective, able to process degraded samples and have the ability to sequence a large battery of FDP SNPs. This thesis compares forensic SNP genotyping techniques for three categories of throughput: low, medium and high HRM analysis is a low-throughput genotyping method and was applied to the IrisPlex eye colour FDP panel of six SNPs. It is a simple and fast post-PCR real-time method. HRM produced reproducible profiles at 0.5 ng DNA input amounts. Its cost-effectiveness can be further increased by using half-volume reactions. IrisPlex includes a symmetrical SNP (rs16891982) and a SNP with high guanine-cytosine content regions (rs1800407) critical to eye colour inference. HRM underperformed in genotyping these SNPs, which might present a challenge in terms of their application for these types of panels. HRM also possesses limited multiplexing capability.
SNaPshot™ (TFS) is the most common forensic SNP-typing tool and was assessed as a medium-throughput genotyping method. This evaluation was also performed using the IrisPlex eye colour panel. The workflow involved a PCR step (amplification of templates) and minisequencing step (single base extension) that introduced a contamination risk due to multiple tube-to-tube transfers. SNaPshot generated reproducible profiles at 0.1 ng DNA and other studies confirmed their reproducibility at 0.062 ng. The assay is able to multiplex up to 40 SNPs and can be applied to both forensic identification—using identity informative SNPs—and FDP. This thesis includes a published review of SNaPshot forensic SNP genotyping assays.
The Illumina MiSeq MPS platform was evaluated as a high-throughput tool. It was used to simultaneously genotype 136 SNPs from five SNaPshot assays: the SNPforID 52-plex, SNPforID 34-plex, Eurasiaplex, Pacifiplex and IrisPlex. MPS libraries were generated from 0.05 ng input amounts for each multiplex. A total of 24 samples were pooled in a single run using unique oligonucleotide barcodes as sample identifiers. MPS was demonstrated to be applicable to degraded samples, UV-exposed samples and humic acid inhibited samples. Sequencing on the MiSeq produced genotypes that were 98 percent concordant with genotypes derived from SNaPshot and Ion Torrent sequencing. It generated 100 percent reproducible profiles. This unique approach demonstrated the capacity to multiplex SNP panels from existing SNaPshot assays (identity and phenotyping) and apply them to multiple samples with no requirement for investing in new panel designs. Further, this thesis describes an automated workflow in a forensic laboratory for routine application of MPS. Two major library normalisation procedures—magnetic bead-based and real-time PCR-based—were compared with real-time PCR to demonstrate the best performance.
In summary, this thesis compares and contrasts three FDP SNP genotyping methods available for forensic applications with different throughput requirements. It is anticipated that the findings may serve as a starting point and guide for forensic laboratories in implementing FDP SNP-typing for routine cases.

Date of Award	2019
Original language	English
Awarding Institution	University of Canberra
Supervisor	Dennis Mcnevin (Supervisor), James Robertson (Supervisor), Tamsin Kelly (Supervisor), Chris Lennard (Supervisor) & Runa Daniel (Supervisor)