Purpose This Funding Opportunity Announcement (FOA) solicits R43/R44 grant applications to innovate and develop the early commercial stages of novel technologies that will enable greater than a one order of magnitude improvement in DNA sequencing, or methods for direct sequencing of the diversity of entire RNA molecules. Advances in genomics and more broadly in biomedical research have been greatly facilitated by cycles of commercial technology innovation and disruption that have driven significant and sustained nucleic acid sequencing throughput and assembly quality increases combined with cost decreases and quality improvements. The goal now is to dramatically advance commercial DNA sequencing and direct RNA sequencing technologies at reasonable costs with the anticipation that significant innovation in any of these and related areas would make significant contributions to the mission of NHGRI and the field of genomics, including to many of NHGRI’s other technology development goals. Background The ability to rapidly sequence large numbers of increasingly complete genomes and transcriptomes utilizing commercially available equipment coupled with the free dissemination of sequence data have dramatically changed the nature of biological and biomedical research. DNA and direct RNA sequence in combination with other genomic data have the potential to lead to remarkable improvements in many facets of human life and society, including the understanding, diagnosis, treatment and prevention of disease; advances in agriculture, environmental science and remediation; and our understanding of evolution and ecological systems. The ability to sequence many genomes and transcriptomes has been made possible by the enormous reduction of the costs and increased capabilities of sequencing in the past four decades, from tens of dollars per base in the 1980s to very small fractions of a cent per base today. Advances through technology innovations, and the subsequent development of new generations of commercial sequencing systems, have enabled many projects that have provided stunning insights into biology and disease. Nevertheless, the cost to completely sequence very large numbers of completely phased mammalian-size genomes or complete end to end comprehensive transcriptome analysis remains very high and technically challenging. There is room for transformative commercial innovation efforts to enable new technologies to achieve the low costs and high quality needed to fully enable the comprehensive use of extensive genomic and transcriptomic sequence information in research and individual health care. One of the major contributions by NHGRI has been in the genome technology domain. Short read sequencing has driven the field to and beyond a $1,000 genome to hundreds of dollars today enabling tremendous research usage and medical applications. Juxtaposed single molecule-based sequencing has provided insights into still emerging extensive amounts and complexity of transcript diversity, provided a means of achieving telomere to telomere sequencing of entire human chromosomes (X and 8 today) for the first time with the entire genome within sight, allowed direct determinations of modified bases within their sequence context, achieved tremendous read lengths of 4.1 Mb, and accuracies of 99.9% for tens of kb long DNA molecules. Novel and emerging commercial technologies are still needed to complement and advance on these successes. These achievements and the work to still be accomplished suggest bright prospects for future successful commercial technology innovation and early-development in novel nucleic acid sequencing technologies. Objectives NHGRI seeks to fund small-business research efforts in novel chemistries, physical approaches and instrumentation for DNA and direct RNA sequencing. Transformative methodologies and early-development of innovations are sought that would, if successful, significantly propel forward the field of genomics. Applicants may propose work on DNA or direct RNA sequencing, or both, in a single application. A model system or question can be tested as proof of principle for the transformative technology proposed, but should not be the primary focus on this technology development application. Applications that are not 1) proposing new sequencing methodology innovations or 2) working on early-development of existing innovations should consider applying under other mechanisms (e.g., NOT-HG-21-018) and elsewhere at the NIH. The FOA deliberately does not specify goals for cost, quality, throughput or read lengths since achievable endpoints are likely to improve over the life of the opportunity, and applicants are encouraged to optimize and balance these key attributes for the technology innovation or early development proposed. It is expected that applicants will develop scientific and practical definitions of optimal cost, quality, capabilities and read lengths relative to enabling significant genomics opportunity advancements. Priority will be given to applications that propose innovation or early development of technologies enabling the greatest improvements based on state of the art. Such improvements may be achieved by focusing on one critical factor, or a combination of important ones. For DNA sequencing, transformative commercial innovation and early development of technologies to generate large numbers of long reads of high quality with a low cost are sought. New physical or chemical detection methods for sequencing are especially encouraged. Those new commercial technologies that can yield novel sequence-based insights or that solve existing limitations in the field (e.g., routine de novo assembly of telomere to telomere phased human genomes, direct simultaneous determination of multiple and specific base modifications within a sequenced context, complete and quantitative sequencing of all the DNA in a sample, essentially complete genomes of single cells, utilize very small quantities of starting material down to a single cell or less, long reads of 99.9% accuracy or better for base calling and phasing of at least hundreds of kilobases to several megabases or more, etc.) are of especially high interest. For RNA sequencing, the need is for commercial quantitative, accurate and high-throughput direct sequencing of entire transcripts from the complete transcriptome. Applicants are expected to develop novel methods for quantitatively assessing the sequence of a diversity of full-length RNA molecules without an entirely cDNA intermediate. Enabling new approaches to commercial RNA analysis is a key goal of direct RNA sequencing (e.g., exhaustive sequencing of every RNA molecule in a sample or precise quantification across the entire very large dynamic range of the diversity of RNA transcripts, direct simultaneous determination of specific and multiple base modifications within a sequenced context, determining RNA secondary structural elements while sequencing, cost-effective and statistically-robust single cell transcriptomics, etc.). High-risk and high reward proposed research may plan to develop complete commercial systems or novel key components for nucleic acid sequencing. Very novel physical or chemical approaches to sequencing are solicited along with novel enzymatic methodologies. The technology innovation can either develop an entirely new commercial way of sequencing or advance the early development of an existing commercial technology for a proposed new application. If an existing technology is being modified for a new sequencing application, the work proposed should involve significant technical modification and early development efforts well beyond optimization. Applicants are also expected to work with a coordinating center NHGRI is seeking to establish to accelerate technology development and progress in the field of genomics (RFA-HG-20-019). Grantee efforts will include actively and fully participating in a yearly meeting; as well as collaborating to synthesize findings and disseminate advances; developing and promoting standards for genomic technologies; facilitating technology transfer to research and clinical utilization, as appropriate; and working with the coordinating center as needed to assist in achieving the goals of this FOA. Non-responsive projects Proposed projects with the following properties will be considered non-responsive and not reviewed: Applications that fail to clearly and specifically identify the transformative commercial innovation or early development activities proposed. Applications that fail to explicitly address how over an order of magnitude improvement in commercial sequencing technology will be achieved. Applications focused exclusively on methods for sample and library preparation. Projects that focus on addressing a specific biological questions or hypotheses. For related research topics that for example seek to improve the quality or efficiency of existing sequencing methods should consider applying via other avenues of support from NHGRI (e.g., NOT-HG-21-018) or elsewhere at the NIH.