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Sequencing technologies, in particular RNASeq, have become critical tools in the design, build, test and learn cycle of synthetic biology. They provide a better understanding of synthetic designs, and they help identify ways to improve and select designs. While these data are beneficial to design, their collection and analysis is a complex, multistep process that has implications on both discovery and reproducibility of experiments. Additionally, tool parameters, experimental metadata, normalization of data and standardization of file formats present challenges that are computationally intensive. This calls for high-throughput pipelines expressly designed to handle the combinatorial and longitudinal nature of synthetic biology. In this paper, we present a pipeline to maximize the analytical reproducibility of RNASeq for synthetic biologists. We also explore the impact of reproducibility on the validation of machine learning models. We present the design of a pipeline that combines traditional RNASeq data processing tools with structured metadata tracking to allow for the exploration of the combinatorial design in a high-throughput and reproducible manner. We then demonstrate utility via two different experiments: a control comparison experiment and a machine learning model experiment. The first experiment compares datasets collected from identical biological controls across multiple days for two different organisms. It shows that a reproducible experimental protocol for one organism does not guarantee reproducibility in another. The second experiment quantifies the differences in experimental runs from multiple perspectives. It shows that the lack of reproducibility from these different perspectives can place an upper bound on the validation of machine learning models trained on RNASeq data. Graphical Abstract

Background Approximately 30–40% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options.1 2 Rare patients that naturally develop an HA-1-specific graft-versus-leukemia T cell response, however, show substantially lower relapse rates.3 4 HA-1 (VLHDDLLEA, genotype RS_1801284 A/G or A/A) is an HLA-A*02:01-and hematopoietically restricted minor histocompatibility antigen, making it an ideal candidate for TCR immunotherapy for liquid tumors.5 Methods We developed a high-throughput TCR discovery platform that enables rapid cloning of antigen-specific TCRs from healthy donors. We then used this platform to screen 178.3 million naive CD8+ T cells from six unique HA-1- (VLRDDLLEA, genotype RS_1801284 G/G) donors, identifying 329 HA-1-specific TCRs. We tested each TCR for expression and the ability to kill HA-1+ target cells, using a previously published, clinical-stage HA-1-specific TCR as a benchmark for these studies.6 In parallel, we tested TCR constant region modifications to promote expression and proper pairing of exogenous TCR alpha and beta chains and designed a lentiviral vector to co-deliver CD8 coreceptors as well as a CD34 enrichment tag to enable purification of engineered T cells. The top 11 candidates were cloned into our optimized backbone and evaluated for cytotoxicity, cytokine production, and T cell proliferation using a panel of HLA-A*02:01+ HA-1+ cell lines. Finally, the top two TCRs were evaluated for allo-reactivity and off-target cross-reactivity using our proprietary genome-wide T-Scan platform. Results The TCR discovery and evaluation platform described here identified 329 HA-1-specific TCRs from a total of 178.3 million naive T cells, and TSC-100 as the most active TCR. Defined mutations in the constant region of TSC-100 enhanced its surface expression while decreasing expression of endogenous TCRs, and co-introduction of CD8 enabled efficient engagement and function of engineered CD4 cells. Overall, TSC-100 exhibited comparable activity to a clinical-stage benchmark TCR when challenged with cell lines expressing moderate to high levels of HA-1, and superior activity when incubated with cell lines expressing low levels of both HA-1 and MHC-I.6 In addition, TSC-100 exhibited no detectable allo-reactivity to 108 different HLA types tested, and minimal off-target effects when challenged with a genome-wide library expressing peptides derived from human proteins. Conclusions TSC-100 exhibits comparable or superior activity to a clinical-stage therapeutic TCR, with minimal allo-reactivity or off-target effects. Based on these results, TSC-100 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2021. Ethics Approval All clinical samples used in the study were collected by STEMCELL Technologies, StemExpress and HemaCare using their IRB approved protocols. References Pavletic SZ, Kumar S, Mohty M, et al. NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from The Committee on the Epidemiology and Natural History of Relapse following Allogeneic Cell Transplantation. Biol Blood Marrow Transplant. 2010;16(7):871–890. Miller JS, Warren EH, van den Brink MR, et al. NCI First International Workshop on The Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on the Biology Underlying Recurrence of Malignant Disease following Allogeneic HSCT: Graft-versus-Tumor/Leukemia Reaction. Biol Blood Marrow Transplant 2010;16(5):565–586. Marijt WAE, Heemskerk MHM, Kloosterboer FM, et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. PNAS 2003;100:2742–2747. Spierings E, Kim Y, Hendriks M, et al. Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-Matched related and unrelated hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 1244–1253. Bleakley M, Riddell SR. Exploiting T cells specific for human minor histocompatibility antigens for therapy of leukemia. Immunol Cell Biol 2011;89(3):396–407. Dossa RG, Cunningham T, Sommermeyer D, et al. Development of T-cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse. Blood 2018;131(1):108–120.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.