Targeted sequencing : single cells and single strand breaks
Name
1080641893-MIT.pdf
Description
Full printable version
Size
8.16 MB
Format
Adobe PDF
Checksum (MD5)
a5f1b6fe1e70c773893ecf98facf252b
Author(s)
Ranu, Navpreet Singh
Advisor(s)
Paul C. Blainey.
Date Issued
2018
Publisher
Massachusetts Institute of Technology
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 targeting sequencing effort to cells and genomic loci of interest to probe the molecular mechanisms behind disease. In this thesis, we demonstrate two novel targeted sequencing 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 enrichment 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 sequenced. Taken as a whole, we introduce two novel strategies to further understand cell function through studying rare cells in single cell populations and analyzing DNA SSB damage in relation to cell health. This work demonstrates that targeted sequencing approaches have promise for understanding the molecular mechanisms behind aberrant cell function, a necessary step in the prevention and treatment of disease.
Description
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 77-92).
Subjects
Biological Engineering.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
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