Targeted Sequencing: Single cells and single strand breaks by Navpreet Singh Ranu B.S. Chemical Engineering, University of California, Berkeley, 2011 Submitted to the Department of Biological Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2018 0 Massachusetts Institute of Technology 2018. All rights reserved. Signature redacted A uthor ......................... Department of Biological Engineering 24 May 2018 Signature redacted Certified by........................ Paul .jBlainey Associate rofessor Thesis Supervisor Signature redacted A ccepted by .............. ............. Forest White Chair of Graduate Program, Department of Biological Engineering MASSACHUSES INSTITUTE OF TECHNOWGY C0 AUG 2 8 2018 LIBRARIES Thesis Committee Members Eric J. Alm, Ph.D. (Chair) Professor of Biological Engineering Massachusetts Institute of Technology Deborah Hung MD,Ph.D. Associate Professor in the Department of Microbiology and Immunobiology Harvard Medical School Associate professor in the Department of Molecular Biology Massachusetts General Hospital 2 Targeted Sequencing: Single cells and single strand breaks by Navpreet Singh Ranu Submitted to the Department of Biological Engineering on 24 May 2018, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering Abstract Sequencing the human genome has spurred systematic work on understanding how gene expression and genomic integrity contribute to disease. To date, 3,519 genes have been identified as the underlying cause of specific single gene disorders. However, complex diseases still pose a daunting challenge that require both an understanding of cell function as well as how the genome interacts with its cellular environment. Sequencing technologies are now routinely applied to interrogate gene variants, gene expression patterns, chromosome accessibility, among other measurements to infer gene and cell function. We build upon past work to address the challenge of tar- geting sequencing effort to cells and genomic loci of interest to probe the molecular mechanisms behind disease. In this thesis, we demonstrate two novel targeted se- quencing methods that can enable a greater understanding of cell function. (1) The development of targeted sequencing in pooled single cell RNA-seq libraries and (2) the development of a novel sequencing approach that allows for the quantification and identification of single stranded break (SSB) locations across the genome. First, we introduce a new targeted sequencing approach to identify rare cells of interest in pooled sequence libraries. Improved throughput in single cell sequencing has enabled the transcriptional profiling of thousands of cells at once. However, due to reliance on pooled library construction methods, it is now more difficult to focus on and analyze particular cells of interest, apart from analyzing the library in its entirety. We designed multiplex PCR primers to simultaneously enrich targeted cells from a complex DNA library pool of single cells. We show how molecular enrich- ment can be used to efficiently target rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC). Next, we demonstrate a new targeted sequencing approach, called NickSeq, to locate and quantify DNA SSBs with single nucleotide resolution. SSBs are the most common form of DNA damage at an estimated 10,000 per cell per day, but there is no available method to robustly determine the exact sites of damage. SSB accumulation correlates with disease, but it is unknown how the location and amount of damage relate to health outcomes. We intentionally create a unique mutational signature at the SSB that is a fingerprint for this specific type of DNA damage when the locus is 3 sequenced. Taken as a whole, we introduce two novel strategies to further understand cell func- tion through studying rare cells in single cell populations and analyzing DNA SSB damage in relation to cell health. This work demonstrates that targeted sequenc- ing approaches have promise for understanding the molecular mechanisms behind aberrant cell function, a necessary step in the prevention and treatment of disease. Thesis Supervisor: Paul C. Blainey Title: Associate Professor 4 Acknowledgments I had support from many people throughout my PhD: " from my advisor Paul Blainey for giving me the freedom to pursue my interests and the ability to act on them * from my thesis committee Eric Alm and Deb Hung for helping guide me through the challenges of graduate school " from my collaborators, Chloe Villani, Roi Avraham, and Arnaud Gutierrez for introducing me to new fields and challenges " from my colleagues and friends in the Blainey Lab - it has been wonderful experiencing the thrill of getting all our projects started, including all the ups and downs of science " from the Biological Engineering department, particularly the class of 2012 for being such an open group of people and helping make life outside of graduate school a blast " from Su Vora for the love and support through all our adventures " from my parents and my sister. 5 Contents 1 Introduction 12 1.1 There is a need for targeted sequencing to detect rare cells within a population ................................. 15 1.1.1 Single cell analysis enables insight into population heterogeneity 15 1.1.2 Pooled sequencing approaches have led to major increases in cell throughput ... .......... ........... .. 16 1.1.3 Limitations of current technologies ..... ....... ... 18 1.2 There is a need for targeted sequencing in identifying DNA damage in the genom e .......... ............ .......... 19 1.2.1 Causes and consequences of DNA damage ....... .... 19 1.2.2 SSB measurements enable identification of genotoxicity and patterns of damage ....... ........ ........ 20 1.2.3 SSB damage is difficult to quantify with nucleotide resolution and high sensitivity .. ...... ...... ..... ..... 21 2 Targeting individual cells by barcode in pooled sequence libraries 23 2.1 Abstract ........................ .......... 23 2.2 Introduction ...................... .......... 23 2.3 R esults .... ............. ............ ...... 25 2.3.1 Hemi-specific PCR enables up to 100 fold decrease in sequenc- ing effort to identify cells .... ...... .. ..... 25 2.3.2 Specifically on-target gene expression profiles of cells pre- and post- enrichment are highly correlated .... ......... 25 6 W 1110, 2.3.3 Principal components analysis allows quantification of the noise added to single cell gene expression profiles due to PCR .. .. 26 2.3.4 Marker gene expression profile for AS DCs is faithfully captured 27 2.4 D iscussion ................................. 27 2.5 Methods ................... ............... 31 2.5.1 Cell isolation and sorting ..................... 31 2.5.2 Single-cell library preparation and target cell enrichment .. 31 2.5.3 Sequencing and primary data processing ............ 32 2.5.4 Target cell enrichment calculation ................ 32 2.5.5 Correlation analysis and Bootstrapping ............. 32 2.5.6 Principal Component Analysis (PCA) and clustering ..... 33 2.5.7 UMI-gene pair uniqueness analysis ............... 33 2.5.8 Targeting putative AXL+ SIGLEC6+ DCs (AS DC's) ..... 34 2.5.9 Acknowledgements . ....................... 34 3 Identifying locations of DNA single stranded breaks with single base pair resolution 51 3.1 A bstract .............. .................... 51 3.2 Introduction ................. ............... 52 3.3 R esults .. ........ ......... ........ ........ 53 3.3.1 Overview of technological innovation .. ........... 53 3.3.2 dPTP and dKTP create a unique mutational signature when in templates amplified by PCR ..... ....... ..... 54 3.3.3 Molecules that contain dPTP and dKTP can be enriched through pulldown with biotin dUTP .... ....... ....... 55 3.3.4 Sites of single stranded breaks can be measured through a com- bination of the engineered mutation generation and biotin en- richnient . ..................... ........ 55 3.3.5 In silico analysis shows theoretical sensitivity of one nick in a thousand molecules ............ ............ 56 7 3.4 D iscussion ............ ............. ........ 57 3.5 M ethods ... ............. ............ ...... 58 3.5.1 Template preparation for nicking ..... .... ..... .. 58 3.5.2 Incorporation of nucleotide analogs and biotinylated dNTPs 59 3.5.3 Custom transposome assembly ... ............ ... 59 3.5.4 Targeted pulldown and library construction ....... ... 59 3.5.5 Sequencing ...... ............ .......... 60 3.5.6 Secondary computational analysis ...... .......... 60 3.5.7 ROC and sensitivity analysis .... ...... ..... .... 60 3.5.8 Acknowledgements ........... ............. 61 4 Future directions 68 4.1 Targeting individual cells by barcode in pooled sequence libraries . 68 4.1.1 Limitations, challenges, and next steps of targeting sequencing reads by barcode ....... ......................... 69 4.2 Identifying locations of DNA single stranded breaks with single base pair resolution ...... ....... ...... ....... ..... 72 4.2.1 Limitations and challenges in detecting nicked sites/regions .. 72 4.2.2 Applications to understanding mutation progression and evolu- tion ....... ........ ....... ....... ... 74 8 List of Figures 2-1 Targeted enrichment of single cells within a pooled RNA-seq sequence library ............ ....................... 29 2-2 Single-cell expression profile before and after enrichment ......