Rapid Nanopore DNA Sequencing: potential for field applications and forensic sample analysis

Abstract

Nanopore MinION sequencing shows prospects for application in forensic science because of its portability and low initial cost. The research in nanopore sequencing of forensic biological markers is limited and it is still in the exploratory phase (Cornelis, Gansemans, Deleye, Deforce, & Van Nieuwerburgh, 2017; Cornelis et al., 2019). This thesis builds and optimises a workflow that analyses long-range mitochondrial amplicons for forensic reference type buccal swab samples. Then the workflow was tested by applying it to mock case type samples. The workflow was optimised around ease of use, time of analysis and DNA retention. DNA retention was optimised to overcome the nanopore library preparation DNA input amounts which require a large amount of DNA or a high concentration of DNA. DNA IQ, Genomic tip, Chelex and FTA paper extraction methodologies were compared to optimise for ease of use, time of analysis and the ability for the extracts to be amplified to produce long-range mitochondrial amplicons. The extraction methodology that was selected for the workflow tested on case type samples was the FTA paper extraction because of the minimal time of analysis and improved ease of use when compared to the other extraction methodologies. When applying the FTA paper extraction to the case type samples a reduction of the DNA amount after amplification was observed. This is most likely due to inconsistent DNA because of the distribution of the DNA on the FTA paper. The AMPure clean-up steps of the nanopore library preparation methodology were altered to increase the retention of the DNA to allow for a DNA amount that could be reanalysed if necessary. Increasing he elution temperature, washing with 80% ethanol, and using elution buffer instead of nuclease free water increased the amount of DNA that was retained after the AMPure clean-up steps during library preparation. The different library preparation kits and methodologies of the Ligation Library Preparation Kit with the Barcoding PCR Addon, Rapid PCR Barcoding Kit and the VolTRAX Sequencing Kit were compared to determine applicability, ease of use and time of analysis. The VolTRAX Sequencing Kit had the lowest time of analysis and was the easiest to perform but at the time of analysis it could only prepare one sample into a library and it was not compatible with barcoding. The Ligation Library Preparation Kit with the Barcoding PCR Addon took longer than the Rapid PCR Barcoding Kit. All the library kits compared successfully sequenced. The Rapid PCR Barcoding Kit was selected for the case type samples because of its ability to include barcoding and the lower time of analysis when compared to the ligation library preparation. The samples that were used to be mock case type samples were saliva samples on the rims of bottles and cans and small blood stains on paper. There was variability in the amount of DNA after long-range mitochondrial amplification but all samples that were prepared into a sequencing library could be aligned to the reference mitochondrial genome. Using MinION sequencing to facilitate sequencing in a decentralised location at the scene or a portable laboratory would speed up the rate of analysis so that results are obtained faster. One of the reasons biological sample analysis if conducted in a centralised location because the methodologies used require large machinery that is not portable. Obtaining quicker results would help influence and guide decision making for other testing of evidence or the investigation that the police is conducting. Sequencing of biological samples short tandem repeat (STR) markers provides more discriminatory power when compared to length based (STR) analysis because both differences in length and differences in sequence can be compared.