Characterization of genetic functional elements physically coupled to NGS sequencing

Abstract

The construction of complex systems in living organisms depends on standardized parts. This main paradigm rests on long established standards in other, very successful, engineering disciplines such as electrical and mechanical engineering, which allow the integration of well-characterized components into subsystems and systems that perform as expected by the designer. Being a relatively young field, the shortage – or even complete lack - of such fundamental parts has fueled some controversy regarding the feasibility of this paradigm. Registries of standard parts have been established as open projects or by companies with the goal of commercialization. Nonetheless, characterization and standardization of genetic elements is currently a time consuming effort with little impact for academic research and does not offer a clear model for commercialization. These points make it clear that new tools are required to provide better ways to address these problems, arguably with the help of automation technology and high throughput techniques. In biology, parts are encoded as DNA, and reading DNA sequence allows inferring part properties. However, this inference is incomplete and the information in the DNA sequence must be red out and physically tested. Recently, methods coupling next generation sequencers to functional assays have become available. The underlying idea is to use the massively parallel capabilities of sequencers to read large numbers of different DNA molecules and couple these molecules to information obtained from a posterior characterization of the sequence on the same instrument and in a fully automated setup. The differences in behavior of a large number of similar instances should then allow inferring critical information on part behavior and subsequently either selection of a correctly behaving part or its (future) design. Examples of these methods have been shown using the Illumina sequencing-by-synthesis platform and included characterization of binding properties of DNA or RNA sequences and proteins. Due to the complexity of the setups and the required infrastructure, these approaches are rather difficult to implement, time consuming (each experiment taking up to one month) and costly, and they need major experimental adaptations to include freely diffusible elements, as the physical structure of the DNA sequence determination unit provides no mechanical means to limit diffusion. Here, we present a method to characterize genetic elements, linking sequence to genetic function in a high throughput setup in picoliter-sized reaction wells, and illustrate its potential by characterizing a bacterial promoter library. We couple components of NGS sequencing technology (Sequencing by Ligation, SBL) in a miniaturized well plate with the in situ and in vitro characterization of the functions that are encoded by the DNA-molecule, either at the level of the informational polymer itself or at the protein level. Our approach delivers a slightly lower throughput than the previously mentioned examples, but requires only a microscope with a motorized stage and 4 fluorescent filters, with a turnaround of only one day. Samples are immobilized on a glass plate with picoliter wells, allowing sampling between around 10^5 and 10^6 library elements in parallel. This scope of the platform allows for the comprehensive characterization of libraries with up to 7 mutation-saturated bases per run, including suitable oversampling. Critically, the geometry of the wells has beneficial effects regarding diffusion and crosstalk between samples and allows for a variety of applications including characterization of transcriptional elements, RNA-ribozymes, protein libraries and even alternative DNA chemistries, which would not be possible with other technologies. In the long run, the principles underlying this platform, in particular the combination of NGS sequencing and in vitro approaches for characterization, could become a widely used approach of biosystems design, in which the rapid characterization in a specific context complements standardization efforts in order to have readily available reliable parts for design.