Physical Laboratory Automation in Synthetic Biology.

Synthetic Biology has overcome many of the early challenges facing the field and is entering a systems era characterized by adoption of Design-Build-Test-Learn (DBTL) approaches. The need for automation and standardization to enable reproducible, scalable, and translatable research has become increasingly accepted in recent years, and many of the hardware and software tools needed to address these challenges are now in place or under development. However, the lack of connectivity between DBTL modules and barriers to access and adoption remain significant challenges to realizing the full potential of lab automation. In this review, we characterize and classify the state of automation in synthetic biology with a focus on the physical automation of experimental workflows. Though fully autonomous scientific discovery is likely a long way off, impressive progress has been made toward automating critical elements of experimentation by combining intelligent hardware and software tools. It is worth questioning whether total automation that removes humans entirely from the loop should be the ultimate goal, and considerations for appropriate automation versus total automation are discussed in this light while emphasizing areas where further development is needed in both contexts.

A nanopore interface for higher bandwidth DNA computing.

DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies' MinION device). These results increase DSD output bandwidth by an order of magnitude over what is currently feasible with fluorescence spectroscopy.

Combinatorial PCR Method for Efficient, Selective Oligo Retrieval from Complex Oligo Pools.

With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications including gene synthesis, CRISPR-based gene editing, and DNA data storage. The selective retrieval of specific oligos from these complex pools traditionally uses polymerase chain reaction (PCR). Designing a large number of primers to use in PCR presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool is orders of magnitude larger than other applications. Although a nested primer address system was recently developed to increase the number of accessible files for DNA storage, it requires more complicated lab protocols and more expensive reagents to achieve high specificity, as well as more DNA address space. Here, we present a new combinatorial PCR method that has none of those drawbacks and outperforms in retrieval specificity. In experiments, we accessed three files that each comprised 1% of a DNA prototype database that contained 81 different files and enriched them to over 99.9% using our combinatorial primer method. Our method provides a viable path for scaling up DNA data storage systems and has broader utility whenever one must access a specific target oligo and can design their own primer regions.

Integrating DNA Encapsulates and Digital Microfluidics for Automated Data Storage in DNA.

Using DNA as a durable, high-density storage medium with eternal format relevance can address a future data storage deficiency. The proposed storage format incorporates dehydrated particle spots on glass, at a theoretical capacity of more than 20 TB per spot, which can be efficiently retrieved without significant loss of DNA. The authors measure the rapid decay of dried DNA at room temperature and present the synthesis of encapsulated DNA in silica nanoparticles as a possible solution. In this form, the protected DNA can be readily applied to digital microfluidics (DMF) used to handle retrieval operations amenable to full automation. A storage architecture is demonstrated, which can increase the storage capacity of today's archival storage systems by more than three orders of magnitude: A DNA library containing 7373 unique sequences is encapsulated and stored under accelerated aging conditions (4 days at 70 °C, 50% RH) corresponding to 116 years at room temperature and the stored information is successfully recovered.

Synthetic DNA applications in information technology.

Synthetic DNA is a growing alternative to electronic-based technologies in fields such as data storage, product tagging, or signal processing. Its value lies in its characteristic attributes, namely Watson-Crick base pairing, array synthesis, sequencing, toehold displacement and polymerase chain reaction (PCR) capabilities. In this review, we provide an overview of the most prevalent applications of synthetic DNA that could shape the future of information technology. We emphasize the reasons why the biomolecule can be a valuable alternative for conventional electronic-based media, and give insights on where the DNA-analog technology stands with respect to its electronic counterparts.

Passively sensing SARS-CoV-2 RNA in public transit buses.

Affordably tracking the transmission of respiratory infectious diseases in urban transport infrastructures can inform individuals about potential exposure to diseases and guide public policymakers to prepare timely responses based on geographical transmission in different areas in the city. Towards that end, we designed and tested a method to detect SARS-CoV-2 RNA in the air filters of public buses, revealing that air filters could be used as passive fabric sensors for the detection of viral presence. We placed and retrieved filters in the existing HVAC systems of public buses to test for the presence of trapped SARS-CoV-2 RNA using phenol-chloroform extraction and RT-qPCR. SARS-CoV-2 RNA was detected in 14% (5/37) of public bus filters tested in Seattle, Washington, from August 2020 to March 2021. These results indicate that this sensing system is feasible and that, if scaled, this method could provide a unique lens into the geographically relevant transmission of SARS-CoV-2 through public transit rider vectors, pooling samples of riders over time in a passive manner without installing any additional systems on transit vehicles.

Multiplexed direct detection of barcoded protein reporters on a nanopore array.

Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.

An Empirical Comparison of Preservation Methods for Synthetic DNA Data Storage.

Synthetic DNA has recently risen as a viable alternative for long-term digital data storage. To ensure that information is safely recovered after storage, it is essential to appropriately preserve the physical DNA molecules encoding the data. While preservation of biological DNA has been studied previously, synthetic DNA differs in that it is typically much shorter in length, it has different sequence profiles with fewer, if any, repeats (or homopolymers), and it has different contaminants. In this paper, nine different methods used to preserve data files encoded in synthetic DNA are evaluated by accelerated aging of nearly 29 000 DNA sequences. In addition to a molecular count comparison, the DNA is also sequenced and analyzed after aging. These findings show that errors and erasures are stochastic and show no practical distribution difference between preservation methods. Finally, the physical density of these methods is compared and a stability versus density trade-offs discussion provided.

Scaling DNA data storage with nanoscale electrode wells.

Synthetic DNA is an attractive medium for long-term data storage because of its density, ease of copying, sustainability, and longevity. Recent advances have focused on the development of new encoding algorithms, automation, preservation, and sequencing technologies. Despite progress in these areas, the most challenging hurdle in deployment of DNA data storage remains the write throughput, which limits data storage capacity. We have developed the first nanoscale DNA storage writer, which we expect to scale DNA write density to 25 × 106 sequences per square centimeter, three orders of magnitude improvement over existing DNA synthesis arrays. We show confinement of DNA synthesis to an area under 1 square micrometer, parallelized over millions of nanoelectrode wells and then successfully write and decode a message in DNA. DNA synthesis on this scale will enable write throughputs to reach megabytes per second and is a key enabler to a practical DNA data storage system.

Molecular-level similarity search brings computing to DNA data storage.

As global demand for digital storage capacity grows, storage technologies based on synthetic DNA have emerged as a dense and durable alternative to traditional media. Existing approaches leverage robust error correcting codes and precise molecular mechanisms to reliably retrieve specific files from large databases. Typically, files are retrieved using a pre-specified key, analogous to a filename. However, these approaches lack the ability to perform more complex computations over the stored data, such as similarity search: e.g., finding images that look similar to an image of interest without prior knowledge of their file names. Here we demonstrate a technique for executing similarity search over a DNA-based database of 1.6 million images. Queries are implemented as hybridization probes, and a key step in our approach was to learn an image-to-sequence encoding ensuring that queries preferentially bind to targets representing visually similar images. Experimental results show that our molecular implementation performs comparably to state-of-the-art in silico algorithms for similarity search.

Rapid and robust assembly and decoding of molecular tags with DNA-based nanopore signatures.

Molecular tagging is an approach to labeling physical objects using DNA or other molecules that can be used when methods such as RFID tags and QR codes are unsuitable. No molecular tagging method exists that is inexpensive, fast and reliable to decode, and usable in minimal resource environments to create or read tags. To address this, we present Porcupine, an end-user molecular tagging system featuring DNA-based tags readable within seconds using a portable nanopore device. Porcupine's digital bits are represented by the presence or absence of distinct DNA strands, called molecular bits (molbits). We classify molbits directly from raw nanopore signal, avoiding basecalling. To extend shelf life, decrease readout time, and make tags robust to environmental contamination, molbits are prepared for readout during tag assembly and can be stabilized by dehydration. The result is an extensible, real-time, high accuracy tagging system that includes an approach to developing highly separable barcodes.

Quantifying molecular bias in DNA data storage.

DNA has recently emerged as an attractive medium for archival data storage. Recent work has demonstrated proof-of-principle prototype systems; however, very uneven (biased) sequencing coverage has been reported, which indicates inefficiencies in the storage process. Deviations from the average coverage in the sequence copy distribution can either cause wasteful provisioning in sequencing or excessive number of missing sequences. Here, we use millions of unique sequences from a DNA-based digital data archival system to study the oligonucleotide copy unevenness problem and show that the two paramount sources of bias are the synthesis and amplification (PCR) processes. Based on these findings, we develop a statistical model for each molecular process as well as the overall process. We further use our model to explore the trade-offs between synthesis bias, storage physical density, logical redundancy, and sequencing redundancy, providing insights for engineering efficient, robust DNA data storage systems.

Using Strand Displacing Polymerase To Program Chemical Reaction Networks.

Chemical reaction networks (CRNs) provide a powerful abstraction to formally represent complex biochemical processes. DNA provides a promising substrate to implement the abstract representation (or programming language) of CRNs due to its programmable nature. Prior works that used DNA to implement CRNs either used DNA-only systems or multienzyme DNA circuits. Architectures with DNA-only components had the rationale of being biologically simple systems. Multienzyme systems, on the other hand, aimed at using natural enzymes to improve circuit performance, although, at the cost of increased complexity. In this work, we explore an alternative architecture that lies along the spectrum in between DNA-only systems and multienzyme DNA systems. Our architecture relies on only a strand displacing polymerase enzyme and DNA hybridization reactions for implementing CRNs. First, we briefly introduce the theory and DNA design of simple CRNs and then explore the fundamental properties of polymerase-based strand displacement systems. Finally, we engineer a catalytic amplifier in vitro as a use-case of our framework since such amplifiers require the intricate design of DNA sequences and reaction conditions.

Probing the physical limits of reliable DNA data retrieval.

Synthetic DNA is gaining momentum as a potential storage medium for archival data storage. In this process, digital information is translated into sequences of nucleotides and the resulting synthetic DNA strands are then stored for later retrieval. Here, we demonstrate reliable file recovery with PCR-based random access when as few as ten copies per sequence are stored, on average. This results in density of about 17 exabytes/gram, nearly two orders of magnitude greater than prior work has shown. We successfully retrieve the same data in a complex pool of over 1010 unique sequences per microliter with no evidence that we have begun to approach complexity limits. Finally, we also investigate the effects of file size and sequencing coverage on successful file retrieval and look for systematic DNA strand drop out. These findings substantiate the robustness and high data density of the process examined here.

Author Correction: Probing the physical limits of reliable DNA data retrieval.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stabilizing synthetic DNA for long-term data storage with earth alkaline salts.

Rapid aging tests (70 °C, 50% RH) of solid state DNA dried in the presence of various salt formulations, showed the strong stabilizing effect of calcium phosphate, calcium chloride and magnesium chloride, even at high DNA loadings (>20 wt%). A DNA-based digital information storage system utilizing the stabilizing effect of MgCl2 was tested by storing a DNA file, encoding 115 kB of digital data, and the successful readout of the file by sequencing after accelerated aging.

DNA assembly for nanopore data storage readout.

Synthetic DNA is becoming an attractive substrate for digital data storage due to its density, durability, and relevance in biological research. A major challenge in making DNA data storage a reality is that reading DNA back into data using sequencing by synthesis remains a laborious, slow and expensive process. Here, we demonstrate successful decoding of 1.67 megabytes of information stored in short fragments of synthetic DNA using a portable nanopore sequencing platform. We design and validate an assembly strategy for DNA storage that drastically increases the throughput of nanopore sequencing. Importantly, this assembly strategy is generalizable to any application that requires nanopore sequencing of small DNA amplicons.

Molecular digital data storage using DNA.

Molecular data storage is an attractive alternative for dense and durable information storage, which is sorely needed to deal with the growing gap between information production and the ability to store data. DNA is a clear example of effective archival data storage in molecular form. In this Review, we provide an overview of the process, the state of the art in this area and challenges for mainstream adoption. We also survey the field of in vivo molecular memory systems that record and store information within the DNA of living cells, which, together with in vitro DNA data storage, lie at the growing intersection of computer systems and biotechnology.

High density DNA data storage library via dehydration with digital microfluidic retrieval.

DNA promises to be a high density data storage medium, but physical storage poses a challenge. To store large amounts of data, pools must be physically isolated so they can share the same addressing scheme. We propose the storage of dehydrated DNA spots on glass as an approach for scalable DNA data storage. The dried spots can then be retrieved by a water droplet using a digital microfluidic device. Here we show that this storage schema works with varying spot organization, spotted masses of DNA, and droplet retrieval dwell times. In all cases, the majority of the DNA was retrieved and successfully sequenced. We demonstrate that the spots can be densely arranged on a microfluidic device without significant contamination of the retrieval. We also demonstrate that 1 TB of data could be stored in a single spot of DNA and successfully retrieved using this method.

Demonstration of End-to-End Automation of DNA Data Storage.

Synthetic DNA has emerged as a novel substrate to encode computer data with the potential to be orders of magnitude denser than contemporary cutting edge techniques. However, even with the help of automated synthesis and sequencing devices, many intermediate steps still require expert laboratory technicians to execute. We have developed an automated end-to-end DNA data storage device to explore the challenges of automation within the constraints of this unique application. Our device encodes data into a DNA sequence, which is then written to a DNA oligonucleotide using a custom DNA synthesizer, pooled for liquid storage, and read using a nanopore sequencer and a novel, minimal preparation protocol. We demonstrate an automated 5-byte write, store, and read cycle with a modular design enabling expansion as new technology becomes available.