טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKovac Omer
SubjectNanopore Sequencing of the Cancer Biomarker MACC1
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Amit Meller


Abstract

Cancer is a widespread disease, affecting millions of people every year worldwide. Cancer develops when abnormal cells proliferate in an uncontrolled manner, ultimately forming a tumor. It is a progressive disease, defined by multiple stages, which relates to the severity of the patient’s condition. When tumor cells detach from the primary tumor site, travel through the bloodstream and produce a tumor at a secondary site, the cancer has metastasized. Metastasis is the most frequent cause of treatment failure and death in many cancer patients. Hence, detecting cancer at an early stage or identifying the risk for metastasis formation is crucial and could lead to improved treatment and survival of patients.

About a decade ago, our collaborator, Prof. Ulrike Stein, has discovered an important biomarker for the onset of metastasis, called MACC1. MACC1 overexpression promotes cell proliferation, invasion and metastasis formation by activation of several cancer-related signaling pathways. Prof. Stein had developed a non-invasive blood test, based on quantitative Real Time Reverse Transcription PCR (qRT-PCR) assay, for circulating metastasis biomarker MACC1 transcript level, demonstrating its value for diagnosis and prognosis of disease outcome for colorectal cancer patients.

Albeit its importance, the most up-to-date entry of the MACC1 transcript in the NCBI database was attained by fusing four different sequences found in distinctly different tissues. The reason for that was, most probably, due to difficulties to obtain the full-length transcript with the prevalent short-read sequencing techniques. Single-molecule nanopore sensing has gained tremendous traction over the past two decades. Research in this field had recently evolved into an affordable, real-time sequencer, the MinION (ONT), capable of producing ultra-long reads. We hypothesized that by employing this advantageous 3rd-generation sequencing technique, the full-length transcript of MACC1 may be resolved for the first time, potentially revealing important features in the sequence that might be relevant to clinical applications.

            In this study we've introduced and established this nanopore sequencing technique in our lab. Using the MinION sequencer device we are able to sequence near to full-length MACC1 transcripts. Moreover, single nucleotide variations in MACC1 transcripts from metastatic and non-metastatic cell lines were observed. We are currently investigating the clinical relevance of these SNPs.