Single cell genomics in alginate microspheres [electronic resource]
Keywords: Chemical Engineering

Current interest in single cell analysis stems from the desire to individually moni-tor cell response rather than averaging over the entire system. Individual cells inside a heterogeneous system, for example a tumor, may differ dramatically, and characterizing the single cells provides useful information about the function and the behavior of the whole. Microbial communities are another example where individual isolates contribute to the function of a complex ecosystem. The three main steps in single cell analysis include: 1) isolation, 2) amplification, and 3) nucleic acid analysis. Whereas isolation and analysis strategies are relatively well established, amplification from a single cell is limi-ted by a low amount of starting material, the potential risk of genome fragment loss, and an exacerbation of noise due to background DNA contamination. These limitations, result in failed reactions, poor sequencing coverage, and unacceptably high rates of contamina-tion in the prepared genomic libraries. The goal of this dissertation is to develop an efficient means of spatially isolating and amplifying the whole genome from a single cell, in order to produce sequencing-ready DNA with minimal contamination. To make this possible, we first isolated single cells in barium-alginate hydrogel microspheres. Barium-alginate serves as an ideal PCR reactor due to its temperature stability, and the ability to undergo reversible catalytic crosslinking. It is also permeable to PCR reagents and preferentially retains amplified DNA products while excluding contaminating DNA by size exclusion. We simplified the microsphere preparation step using disposable fingertip aerosol sprayers, producing Ba-alginate microspheres that ranged from 50-300µm in diameter. By optimizing the cell concentration and selecting a given size of microspheres by sieving, we were able to easily produce a population of microspheres that contained one cell or less. Next, we optimized the reagents and reaction conditions for PCR and whole genome amplification (WGA) in order to amplify the isolated genomes inside the microspheres. We demonstra-ted conventional PCR amplification of specific E.coli genes as well as WGA of isolated human and E.coli genomes. We developed a simple approach to sort and recover single amplified microspheres by serial dilution in a 384 well plate and analysis by PicoGreen nucleic acid analysis using a fluorescent plate reader. In order to recover the amplified DNA from the microspheres, we developed a rapid and simple method of microsphere lysis that relied on chelation of the barium crosslinking ion. We observed that by-pro-ducts of chelation inhibited PCR using commercial master mixes, and thus we were requ-ired to optimize a new set of PCR reagents. We then analyzed the amplified PCR and WGA products extracted from the microspheres by using quantitative PCR (q-PCR) and next generation sequencing (NGS). Our final improvement was to incorporate the library preparation steps for NGS inside an alginate microsphere, providing sequencing-ready material. In summary, we established a simple strategy for single cell encapsulation, lysis, and amplification inside Ba-alginate microspheres, and demonstrated that DNA products could be easily extracted and analyzed by PCR, q-PCR or NG sequencing.