학위논문 (박사)-- 서울대학교 대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2019. 2. 권성훈.지금까지는 기술적인 한계로 인해 NGS 를 이용하여 생산된 세포의 유전체 정보가 세포의 조직학적 정보와 연결될 수 없었다. 본 박사학위 논문에서는 단일 세포 혹은 소량의 세포를 조직으로부터 고속으로 분리하여 유전체 분석을 수행하는 PHLI- seq 기술을 소개한다. 그리고 PHLI-seq 기술을 이용하여 암 조직을 대상으로 유전체 정보와 조직학적 정보를 연결하는 연구를 소개한다. 유방암 조직에 대하여 PHLI-seq 을 적용하여 유방암 세포의 유전체 분석을 수행하고 분석된 세포의 유전체 정보와 조직학적 이미지를 연동하여 유방암 조직의 유전적 다양성을 시각화 한다. 또한, 난소암에 대하여 PHLI-seq 을 적용하여 암의 발생 및 진행의 과정을 연구한 내용을 소개한다. 이 연구들을 통하여 암 세포들의 서브클론 형성 및 공간적인 배열이 암 발생 초기에 이루어짐을 알 수 있다.Despite advances in next-generation sequencing technologies, it has been challenging to map molecular information of cells to their tissue context due to technical limitations. In this dissertation, I describe PHLI-seq, a novel approach that enables high-throughput isolation and genome-wide sequence analysis of single-cells or small numbers of cells from a tissue to construct genomic maps within biological samples in relation to the images or phenotypes of the cells. By applying PHLI-seq to breast cancer tissues, genetic heterogeneity of cancer is revealed at a high spatial resolution. Also, in a case of ovarian cancer, a history of tumor development is explored using PHLI-seq. Through these studies, it is concluded that genetic subclonality and spatial separation of the subclones are formed in early stage of cancer development.Table of Contents
Abstract____________________________________________________ i
List of Figures ______________________________________________ iv
List of Tables_______________________________________________ xi
Chapter 1 Introduction _______________________________________ 1
1.1 Identifying Molecular Information of Cells ____________________ 3
1.2 Spatially Resolved Sequencing of Cells_______________________ 6
1.3 Comparison of Technologies _______________________________ 7
Chapter 2 Techical Overview of PHLI-seq ______________________ 11
2.1 Phenotype-based High-throughput Laser-aided Isolation and Sequencing (PHLI-seq) _____________________________________ 12
2.2 Instrumentation_________________________________________ 14
2.3 Whole Genome Amplification _____________________________ 20
2.4 Performance Validation of PHLI-seq________________________ 28
2.5 Performance Comparison with Commercial Laser Microdissection 36
Chapter 3 Spatially Resolved Sequencing of Breast Cancer ________ 42
3.1 PHLI-seq Analysis of a HER2 Positive Breast Tumor___________ 43
3.1.1 Experimental Procedure ______________________________ 43
3.1.2 Somatic Copy Number Alteration (CNA) Analysis _________ 45
3.1.3 Somatic Single Nucleotide Variant (SNV) Analysis_________ 50
3.1.4 Inferring Tumor Evolution ____________________________ 57
3.2 PHLI-seq Analysis of a Triple-negative Breast Tumor __________ 60
3.3 A Summary____________________________________________ 65
Chapter 4 Inferring Tumor Evolution of Ovarian Cancer__________ 67
4.1 Sample Preparation______________________________________ 70
4.2 Somatic CNA and SNV Analysis___________________________ 73
4.3 Inferring Evolutionary Trajectory of the Tumor Cells ___________ 80
4.4 A Summary____________________________________________ 85
Chapter 5 Conclusion________________________________________ 87
Bibliography _______________________________________________ 90
Abstract in Korean _________________________________________ 100Docto
