Leveraging of single molecule sequencing methods for less invasive cancer detection

In the field of paediatric neuro-oncology, the positions of tumours within the central nervous system of the patients makes the acquisition of solid tumour biopsies risky. For many tumour types, monitoring of treatment response is restricted to Magnetic Resonance Imaging (MRI), or cerebrospinal fluid (CSF) cytology in the cases with leptomeningeal dissemination. Both of these lack sensitivity, leaving room for improvement. Recent advances in molecular barcoding sensitivity and error suppression have made the sequencing of DNA derived from liquid biopsies possible. Liquid biopsies offer an alternative to solid biopsies, since the collection of bodily fluids is much less invasive by comparison, and liquid biopsies contain cell-free DNA (cfDNA). In cancer patients, it has been shown that a fraction of the cfDNA in multiple liquid biopsies, such as plasma and CSF, harbour the genetic alterations present within the tumour. This circulating tumour DNA (ctDNA) can be used as a biomarker for diagnosis, stratification, and surveillance of the tumour. The monitoring of treatment response, and the detection of minimal residual disease, is of particular importance in paediatric brain tumours, given the low sensitivity of existing methods. This project created a versatile system, utilising molecular barcoding, which was able to detect Single Nucleotide Variants (SNVs), Insertions/Deletions and Copy-Number Variants in a single assay. A wet-lab workflow was created and iteratively improved, such that it could handle a diverse range of liquid biopsy types, including plasma, cystic fluid and CSF. This workflow was coupled with a bioinformatic pipeline, designed to process the data for all three variant calling processes simultaneously. For SNV calling, a custom variant caller was created to aid in the suppression of errors in barcoded sequencing, and the system was used in the first documented tracking of Adamantinomatous Craniopharyngioma treatment response using cystic fluid liquid biopsies