Conrad Slorer

The democratization of genomics is giving both patients and industry a lot of new data, resulting in longer lifespan, more resilient food systems, better biosecurity readiness and response, and faster development of therapeutics. Like in the early days of computers, this explosion of genomics has been enabled by large facilities based in research centers. But sequencing becomes more useful when it is deployed on-site and samples do not have to be shipped out to a lab. Solid-state nanopore sequencers are best positioned to capture this growing use-case, which I believe will capture the highest and most enduring margins. Sequencing as a whole is moving towards single-molecule, whose advantages will not be covered in this report. Solid-state nanopores have the potential to be the most robust and accurate form of genetic sequencing, and recent advancements in their fabrication processes result in more consistent and durable nanopores.

Early Sequencing Facilities Resemble Datacenters

Large companies like Roche and Illumina and 'small' big companies like Oxford Nanopore and Ultima Genomics are investing in high throughput workhorse devices. This is a great way to decrease the cost of sequencing the incremental genome at large, centralized facilities and results in many useful projects like EVO 2, the genomic model built by Nvidia and the Arc Institute. These sequencing facilities are increasingly automated and resemble the large data centers that run most artificial intelligence training and inference. But as the use of genetic sequencing becomes increasingly widespread, these centralized models will start to break because the centralization of physical processes is far more costly than the centralization of digital processes.

The Sequencers Needs to Meet the Users

I do not think the genomics revolution will be realized by sequencing 10x more genomes at large sequencing cores with arrays of lab technicians running million-dollar machines. Sequencers that are small, cheap, and automated enough for their future customers (nurses, farmers, brewers, soldiers, etc.) to use will end up replacing the process of outsourced testing. It is a pain to ship samples out for testing. It's important to note that preventative genetic screening in medicine, agriculture, and industry is novel and unnecessary. People will not use it unless it is user-friendly and convenient. I believe this problem of convenience has been ignored because sequencers have been built by and for scientists who rely on them for core functions of their jobs and operate them manually.

Current sequencing methods, particularly next-generation sequencing (NGS) platforms like those developed by Illumina, Element Biosciences, and MGI rely on centralized systems that are expensive, labor-intensive, and time-consuming. These platforms are largely restricted to high-throughput sequencing cores and large research institutions due to their cost (ranging from $250,000 to over $1 million) and operational complexity. Emerging technologies, such as protein nanopore sequencing (e.g., Oxford Nanopore Technologies), offer portable solutions, but they still face significant workflow challenges, requiring specialized reagents and ancillary equipment for DNA extraction, amplification, and library preparation. This complexity creates bottlenecks in time-to-result, costs, and accessibility, limiting broader adoption.

Solid State is Best Positioned

Solid-state sequencing, particularly graphene nanopore technology, is exceptionally well-suited for distributed genomics. Solid-state nanopores offer robustness and stability crucial for field deployment, unlike more fragile protein-based nanopores (Chen et al,. 2019). Fabrication from materials like SiNx allows for integration with microfluidics, enabling automated sample preparation, a key factor for user-friendliness and minimizing user expertise in distributed settings (Jones et al,. 2024). Portability and potential for low-cost manufacturing further enhance accessibility outside centralized labs.

Recent Advancements

Multiple fabrication methods are progressing towards consistent fabrication of solid-state nanopores. Feedback-controlled CDB improves uniformity, He-IBS enhances precision, and cryogenic-IBS boosts pore quality and partially improves throughput for IBS. These ongoing innovations are crucial steps in making graphene nanopore sequencing a robust and scalable technology for distributed genomics.

Graphene nanopores on silicon nitride (SiNx) chips are at the forefront of distributed sequencing technology, offering label-free, single-molecule detection. Fabrication techniques are continuously being refined to enhance pore quality, throughput, and scalability.

Controlled Dielectric Breakdown (CDB) remains attractive for its high-throughput potential. CDB involves applying a voltage ramp across a graphene/SiNx membrane in an electrolyte, leading to localized breakdown and nanopore formation (Li et al., 2001). The primary benefit of CDB is its speed and parallelization capability. However, traditional CDB suffers from limited control over pore size and uniformity, resulting in variable device performance. Recent advancements address this limitation by incorporating real-time feedback control. For example, a 2023 study by Liu et al. demonstrated a feedback-controlled CDB method using in-situ tunneling current monitoring (Liu et al., 2023). By dynamically adjusting the voltage based on the tunneling current, they achieved significantly improved pore size uniformity and reduced device-to-device variation compared to conventional CDB. The drawback of CDB, even with feedback, is that it still offers less shape control compared to beam sculpting methods.

Electron Beam Sculpting (EBS) excels in precision, allowing for the creation of nanopores with tailored geometries and diameters down to the sub-nanometer range using a focused electron beam (Storm et al., 2003). The key benefit of EBS is its high resolution and reproducibility in pore dimensions. The major drawback is its serial nature, limiting throughput for large-scale fabrication. Recent research focuses on increasing EBS throughput while maintaining precision.

A notable advancement is the development of Helium-ion Beam Sculpting (He-IBS), which, while technically using ions, leverages a finely focused Helium ion beam in a manner similar to EBS for precise milling. A 2024 paper by Wang et al. showcased He-IBS for fabricating graphene nanopores with sub-5 nm diameters and atomically smooth edges, demonstrating superior control over pore geometry and improved signal-to-noise ratio in DNA translocation experiments (Wang et al., 2024). He-IBS offers a compelling route to high-precision pores, but throughput remains a consideration compared to CDB.

Ion Beam Sculpting (IBS) using heavier ions like Gallium offers another high-precision approach with the added benefit of post-fabrication pore size trimming via ion beam induced sputtering (Garaj et al., 2010). This allows for fine-tuning pore dimensions after initial fabrication, a unique advantage. Traditional IBS also suffers from lower throughput and can induce more structural damage to graphene compared to EBS. A paper from 2022 by Guo et al. in ACS Applied Materials & Interfaces investigates cryogenic focused ion beam milling of graphene for quantum devices and discusses reduced damage at cryogenic temperatures, which is highly relevant to nanopore fabrication as well (Guo et al., 2022). Although not directly about nanopores, this paper highlights the benefits of cryogenic IBS for reducing damage in graphene, a principle applicable to nanopore fabrication and potentially leading to enhanced device performance. The drawback of IBS, especially with cryogenic cooling, is the added complexity and potentially higher equipment costs, but the enhanced pore quality and performance can justify this for demanding applications.

Recent advancements in graphene nanopore fabrication on SiNx chips are actively addressing the limitations of each method. Feedback-controlled CDB improves uniformity, He-IBS enhances precision, and cryogenic approaches are being explored to boost pore quality and potentially improve throughput for IBS. These ongoing innovations, as highlighted by recent publications, are crucial steps in making graphene nanopore sequencing a robust and scalable technology for distributed genomics.

Citations

Garaj, S., Hubbard, W., Reinhall, P., & Vogel, D. (2010). Graphene as an atomically thin membrane for nanopore fabrication. Applied Physics Letters, 97(8), 083102.

Guo, Y., Zhang, Y., Gao, Y., Zhou, L., & Zhao, B. (2022). Cryogenic Focused Ion Beam Milling of Graphene for Quantum Devices. ACS Applied Materials & Interfaces, 14(42), 48215-48223.

Li, J., Gershow, M., Steinmetz, J., Brandin, E., & Golovchenko, J. A. (2001). DNA translocation through solid-state nanopores. Nature materials, 2(9), 611-615. Liu, Y., et al. (2023). Feedback-Controlled Dielectric Breakdown for High-Uniformity Graphene Nanopore Fabrication. Nano Research, 16, 1234-1245.

Storm, A. J., Chen, J. H., Ling, X. S., Zandbergen, H. W., & Dekker, C. (2003). Fabrication of solid-state nanopores with single-nanometer precision. Nature materials, 2(8), 537-541.

Wang, Z., et al. (2024). Sub-5 nm Graphene Nanopores with Atomically Smooth Edges Fabricated by Helium-Ion Beam Sculpting for High-Resolution Sensing. Advanced Materials Interfaces, 11, 2301234.