Methods for Multiplexed Biology

Our ability to read and write DNA is fundamental for understanding Biology. While the past decade has brought about exponential improvements in our DNA sequencing and synthesis capabilities, major challenges remain. First, many DNA sequencers are hindered by their short read lengths, which has hindered genome assemblies, molecular haplotyping, and more recently, multiplexed functional assays. Synthetic Long Reads (SLRs) are a recently developed method that address this issue. SLRs leverage molecular barcodes to guide the computational reassembly of multiple short reads into a longer contiguous molecule. Here we present a novel SLR technology, BAGEL-seq, that can theoretically sequence molecules up to 40 kb, and achieve read lengths of ~1 kb in a proof-of-principle experiment. Second, large-scale synthesis of gene-length, synthetic DNA is cost-prohibitive for many research applications. We present two complementary methods to address this limitation – one to quantify errors in synthetic gene constructs using next-generation sequencing (NGS), and another, DropSynth, to synthesize > 10,000 ~1 kb genes using emulsions and DNA microarrays. Despite these limitations, researcher have recently leveraged DNA sequencing and synthesis to test the functional effects of thousands of variants in multiplex. Known as multiplexed functional assays (MFAs), these experiments have revolutionized the investigation of biological processes across the Central Dogma. In this dissertation we present three different MFAs. In the first, we used DropSynth to build homologogs of an essential E. coli protein, and tested their function in a complimentation assay. In the second, we measured the response of 39 murine olfactory receptors against hundreds of different odorants. Lastly, we assessed the effects of ~7,800 single amino acid changes to the Beta2-adrenergic receptor in the presence of increasing agonist concentration. Taken together, this dissertation represents a fundamental improvement in our ability to read and write DNA, and pushes the state of the art in combining these technologies for large-scale, multiplexed experiments