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Chapter 2: a Technique for Generating Unbiased Whole Genome UC San Diego UC San Diego Electronic Theses and Dissertations Title Massively Parallel Polymerase Cloning and Genome Sequencing of Single Cells Using the Microwell Displacement Amplification System (MIDAS) / Permalink https://escholarship.org/uc/item/8kn4n1wd Author Gole, Jeffrey Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Massively Parallel Polymerase Cloning and Genome Sequencing of Single Cells Using the Microwell Displacement Amplification System (MIDAS) A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Jeffrey Gole Committee in charge: Professor Kun Zhang, Chair Professor Vineet Bafna Professor Michael Heller Professor Xiaohua Huang Professor Yu-Hwa Lo 2013 Copyright Jeffrey Gole, 2013 All rights reserved The Dissertation of Jeffrey Gole is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2013 iii DEDICATION For my parents iv TABLE OF CONTENTS SIGNATURE PAGE………………………………………………………………....iii DEDICATION ................................................................................................... iv TABLE OF CONTENTS ....................................................................................v LIST OF FIGURES.......................................................................................... vii LIST OF TABLES............................................................................................viii ACKNOWLEDGEMENTS ................................................................................ ix VITA ................................................................................................................ xii ABSTRACT OF THE DISSERTATION ...........................................................xiii Chapter 1: Introduction......................................................................................1 1.1: Next Generation Sequencing..............................................................1 1.2: Single Cell Isolation.............................................................................2 1.3: Single Cell Amplification......................................................................4 1.4: Library Construction............................................................................7 1.5: De Novo Assembly of Microbial Genomes from Single Cells ..............9 1.6: Copy Number Variation......................................................................11 1.7: Scope of the Dissertation..................................................................12 Chapter 2: A Technique for Generating Unbiased Whole Genome Amplification Sequencing Libraries from Single Cells.....................................14 2.1: Abstract.............................................................................................14 2.2: Introduction .......................................................................................14 2.3 Methods ..............................................................................................16 2.3.1 Microwell Array Fabrication.............................................................16 2.3.2 Cell Seeding, Lysis, and Multiple Displacement Amplification .....17 2.3.4 Image Analysis .............................................................................18 2.3.5 Amplicon Extraction......................................................................19 2.3.5 Amplicon Quantification................................................................20 v 2.3.6 Low Input Library Construction.....................................................20 2.3.7 Bulk Library Construction .............................................................22 2.3.8 Data Analysis................................................................................23 2.4: Results ..............................................................................................24 2.5: Conclusions.......................................................................................31 2.6: Acknowlegements.............................................................................32 Chapter 3: De Novo Assembly of Single E. Coli Cell Genomes.....................45 3.1: Abstract.............................................................................................45 3.2: Introduction .......................................................................................45 3.3: Methods ............................................................................................48 3.3.1 Bacterial Preparation....................................................................48 3.3.2 Amplification and Library preparation...........................................49 3.3.3 Mapping and De Novo Assembly of Bacterial Genomes..............49 3.3.4 Data Analysis................................................................................50 3.4: Results ..............................................................................................51 3.5: Conclusions.......................................................................................56 3.6: Acknowledgements...........................................................................57 Chapter 4: Identification of Copy Number Variants in Single Neurons...........64 4.1: Abstract.............................................................................................64 4.2: Introduction .......................................................................................64 4.3: Methods ............................................................................................67 4.3.1 Neuron Preparation ......................................................................67 4.3.2 Amplification and Library preparation...........................................68 4.3.3 Identification of CNVs in MIDAS and MDA data...........................68 4.3.4 Identification of Artificial CNVs in MDA and MIDAS data .............69 4.3.5 Identification of true CNVs in MIDAS data .....................................70 4.4: Results ..............................................................................................70 4.5: Conclusions.......................................................................................76 4.6: Acknowledgements...........................................................................77 Chapter 5: Discussion and Future Directions.................................................96 5.1: Discussion.........................................................................................96 5.2: Future Directions...............................................................................99 References....................................................................................................103 vi LIST OF FIGURES Figure 2.1: Overall Process of MIDAS.………………………………………... 34 Figure 2.2: Cell seeding in microwells ………………………………………… 35 Figure 2.3: SEM images of single cells……………………………………….. 36 Figure 2.4: Real time MDA …………………………………………………….. 37 Figure 2.5: Amplicon extraction ………………………………………………. 38 Figure 2.6: Genomic coverage of single bacterial and mammalian cells post MDA and MIDAS ………………………………………………………………... 39 Figure 2.7: Distribution of coverage of amplified single mammalian cells with larger bin size.……………………………………………………………………. 40 Figure 2.8: Comparison of MIDAS to in-tube MDA, microfluidic MDA, and MALBAC………………………………………………………………………… 41 Figure 2.9: Comparison of distributions of coverage……………………….. 42 Figure 3.1: Coverage vs depth plots …………………………………………. 58 Figure 3.2: Depth of coverage of assembled contigs aligned to the reference E. coli genome ………………………………………………………………… 59 Figure 3.3: Comparison of assembly to mapped reads across genome….. 60 Figure 3.4: Assembly Comparison to an In-tube MDA Derived Library…… 61 Figure 4.1: Detection of copy number variants using MIDAS and in-tube MDA……………………………………………………………………..………… 79 Figure 4.2: MIDAS identifies 2 Mb spike-in CNVs even with 20% additional technical noise………………………………………………………………….. 80 vii LIST OF TABLES Table 2.1: Cost of library preparation per sample…………………………..... 43 Table 2.2: Cross-well contamination is not present in mixed-sample MIDAS …………………………………………………………………………………….... 44 Table 3.1: Chimera statistics…………………………………………………..... 62 Table 3.2: Single E. coli assembly statistics…………………………………… 63 Table 4.1: Single neuron and unamplified neuron cellular pool sequencing statistics…………………………………………………………………………….. 81 Table 4.2: Artificial CNV transplantation statistics…………………………….. 82 Table 4.3: Copy number events called in each single neuron or pooled sample …………………………………………………………………………...… 86 Table 4.4: List of genes identified to possess somatic copy number changes in single neurons ……………………………………………………………..……... 90 viii ACKNOWLEDGEMENTS I would like to thank everyone who helped and supported me throughout my graduate school experience. Without the support of the following people, this dissertation would not be possible. First, my advisor, Kun Zhang, decided to take a chance on me five years ago. Though I still believe he just picked the first naïve graduate student who asked him about rotating in his lab, his trust and faith in me over the past five years has allowed me to succeed. He taught me how to be a successful researcher and scientist. More importantly, he taught me to keep a level mind as research does not always go as
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