Development and implementation of novel applications of massively parallel sequencing in precision medicine

Next-generation sequencing (NGS) based on “sequencing by synthesis”, such as Illumina’s MiSeq, NextSeq500 or HiSeq instruments has reached a state where consistency, throughput and quality make it a mature technology to use for cancer genome research, such as single-cell sequencing or in clinical diagnostics. Within the scope of this thesis, we designed and tested three protocols to mobilise high-throughput sequencing for precision medicine to provide optimal solutions for doctors to help with understanding, diagnosing and determining states of human cancer cells. Targeted re-sequencing of clinically relevant cancer genes using NGS ensures an economical use of tissue, by only doing one experiment for testing many drug-resistance tests. In collaboration with oncologists, three protocols are introduced and discussed depending on the respective use case. First, a comprehensive cancer panel based on targeted bait capture technology was developed, for a quick and in-depth genetic variant detection of 69 commonly examined cancer genes. After designing, over 300 clinical formalin-fixed and paraffin-embedded (FFPE) samples from patients, with known mutations in KRAS, NRAS, BRAF and EGFR at varying rates were sequenced. Sequencing data was collected and analysed in-depth to define assay properties and to determine sensitivity, specificity and overall reliability. Input material was evaluated by measuring DNA integrity (DIN) scores for each sample and correlated with overall performance. In doing so, we were able to set precise input criteria to ensure experimental success. The second step was the development of a diagnostic cancer panel of nine genes, based on enzymatic digestion and target amplification for a rapid and cost-effective testing of multiple genetic markers, who have an immediate impact on doctor’s decision on cancer drug therapies. The method introduces a novel approach by adding a molecular barcode to each molecular fragment sequenced, which is supposed to increase sensitivity as it allows removal of PCR artefacts and sequencing errors. The new panel was tested on 48 clinical FFPE samples that were previously genotyped on common cancer mutation hotspots with validated pyrosequencing. Strengths and imitations of this library-preparation method could be identified and considered. The third protocol describes a method to quickly extract and prepare DNA from thousands of individual cells simultaneously by capturing each cell in water droplets in an emulsion. Each droplet contains a collection of barcoded primer pairs that allows not only-barcoding individual fragments, but entire cells. The system has been tested by mixing mouse NIH3T3 cells with human KRAS insert, which carries a heterozygous mutation and human K562 cells with no known mutation in KRAS in an 80:20 ratio. Cells were emulsified with barcoded primer, a polymerase chain reaction amplified exons two and three, added cell-specific barcodes and prepared a library for sequencing in one step. After emulsion-breaking and clean-up and a second PCR, library was ready for sequencing. Around ten percent of a standard MiSeq run is enough to genotype over 10,000 cells. A developed script extracts barcodes from the sequencing data, which is then aligned with a standard alignment software, such as Burroughs-Wheeler-Aligner (BWA). Subsequently the introduced single-cell barcodes clustered according to cellular sub-populations to give deep insight into heterogeneity of the prepared cell population. This will prove a valuable tool in the assessment of diversity amongst cells in a variety of disease backgrounds and contribute to the development of precision medicine through informing patient-specific, personalised diagnostic approaches.