Title: Multiplex polony sequencing for analysis of genomes, transcriptomes, and exosomes
Description: This dissertation is focused on the development of the accurate, inexpensive cyclic array DNA sequencing technology commonly referred to as multiplex polony sequencing and its application to bacterial genome resequencing, digital expression profiling, and selection of genomic subsets by targeted multiplex amplification. Chapter 1 briefly reviews the field of DNA sequencing technology development from 1954 to the present, with emphasis on the salient features of "next-generation", or cyclic array, sequencing platforms. Chapter 2 describes a novel DNA sequencing technology in which a commonly-available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. Multiplex amplification is used to generate millions of sequencing features in parallel, which are arrayed and subjected to rounds of multiplex ligation-based sequencing biochemistry followed by epiflourescence microscopy digital imaging to determine the sequence of each feature in parallel. We applied this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. Cost per base was roughly one-ninth as much as that of conventional sequencing. Chapter 3 describes a digital expression profiling technology, "polony multiplex analysis of gene expression (PMAGE)", which detects mRNAs as rare as one transcript per three cells by incorporating an improved ligation-based sequencing method to sequence 14-nucleotide tags derived from individual mRNA molecules. Using PMAGE, we identified early transcriptional changes that preceded pathological manifestations of hypertrophic cardiomyopathy in mice carrying a disease-causing mutation.

Chapter 4 describes two methods by which select subsets of a genome can be amplified in a multiplex reaction compatible with cyclic array sequencing platforms to enable applications including resequencing of mammalian exonomes. By converting genomic targets to covalently-closed circles in a ligation reaction driven by synthetic 'targeting' oligonucleotides, we selected subsets of the human genome and quantified the extent to which the process was specific and uniform. Polony tag sequencing to sample over one million circles per selection reaction allowed precise quantitation of the dynamic range of the reaction products. Appendix A presents detailed protocols for all molecular biology tasks associated with polony sequencing. Appendix B presents an overview of the hardware and software components of the sequencing instrument. Appendix C presents drawings of the specific flowcell components I designed. Appendices D, E, and F present source code I wrote for the majority of the software that operates the sequencing instrument.