Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation.

It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity.

A high-resolution enhancer atlas of the developing telencephalon.

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

Predicting chromosomal compartments directly from the nucleotide sequence with DNA-DDA.

Three-dimensional (3D) genome architecture is characterized by multi-scale patterns and plays an essential role in gene regulation. Chromatin conformation capturing experiments have revealed many properties underlying 3D genome architecture, such as the compartmentalization of chromatin based on transcriptional states. However, they are complex, costly and time consuming, and therefore only a limited number of cell types have been examined using these techniques. Increasing effort is being directed towards deriving computational methods that can predict chromatin conformation and associated structures. Here we present DNA-delay differential analysis (DDA), a purely sequence-based method based on chaos theory to predict genome-wide A and B compartments. We show that DNA-DDA models derived from a 20 Mb sequence are sufficient to predict genome wide compartmentalization at the scale of 100 kb in four different cell types. Although this is a proof-of-concept study, our method shows promise in elucidating the mechanisms responsible for genome folding as well as modeling the impact of genetic variation on 3D genome architecture and the processes regulated thereby.

Disregarding multimappers leads to biases in the functional assessment of NGS data.

BACKGROUND: Standard ChIP-seq and RNA-seq processing pipelines typically disregard sequencing reads whose origin is ambiguous ("multimappers"). This usual practice has potentially important consequences for the functional interpretation of the data: genomic elements belonging to clusters composed of highly similar members are left unexplored. RESULTS: In particular, disregarding multimappers leads to the underrepresentation in epigenetic studies of recently active transposable elements, such as AluYa5, L1HS and SVAs. Furthermore, this common strategy also has implications for transcriptomic analysis: members of repetitive gene families, such the ones including major histocompatibility complex (MHC) class I and II genes, are under-quantified. CONCLUSION: Revealing inherent biases that permeate routine tasks such as functional enrichment analysis, our results underscore the urgency of broadly adopting multimapper-aware bioinformatic pipelines -currently restricted to specific contexts or communities- to ensure the reliability of genomic and transcriptomic studies.

Butyrophilin 2a2 (Btn2a2) expression on thymic epithelial cells promotes central T cell tolerance and prevents autoimmune disease.

Butyrophilins are surface receptors belonging to the immunoglobulin superfamily. While several members of the butyrophilin family have been implicated in the development of unconventional T cells, butyrophilin 2a2 (Btn2a2) has been shown to inhibit conventional T cell activation. Here, we demonstrate that in steady state, the primary source of Btn2a2 are thymic epithelial cells (TEC). Absence of Btn2a2 alters thymic T cell maturation and bypasses central tolerance mechanisms. Furthermore, Btn2a2-/- mice develop spontaneous autoimmunity resembling human primary Sjögren's Syndrome (pSS), including formation of tertiary lymphoid structures (TLS) in target organs. Ligation of Btn2a2 on developing thymocytes is associated with reduced TCR signaling and CD5 levels, while absence of Btn2a2 results in increased TCR signaling and CD5 levels. These results define a novel role for Btn2a2 in promoting central tolerance by modulating TCR signaling strength and indicate a potential mechanism of pSS development.

RNA-seq of nine canine prostate cancer cell lines reveals diverse therapeutic target signatures.

BACKGROUND: Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount. METHODS: This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin. RESULTS: Independent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing. CONCLUSION: Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.

An RNA-Seq-Based Framework for Characterizing Canine Prostate Cancer and Prioritizing Clinically Relevant Biomarker Candidate Genes.

Prostate cancer (PCa) in dogs is a highly malignant disease akin to its human counterpart. In contrast to the situation in humans, multi-gene approaches facilitating risk stratification of canine PCa are barely established. The aims of this study were the characterization of the transcriptional landscape of canine PCa and the identification of diagnostic, prognostic and/or therapeutic biomarkers through a multi-step screening approach. RNA-Sequencing of ten malignant tissues and fine-needle aspirations (FNA), and 14 nonmalignant tissues and FNAs was performed to find differentially expressed genes (DEGs) and deregulated pathways. The 4098 observed DEGs were involved in 49 pathways. These 49 pathways could be grouped into five superpathways summarizing the hallmarks of canine PCa: (i) inflammatory response and cytokines; (ii) regulation of the immune system and cell death; (iii) cell surface and PI3K signaling; (iv) cell cycle; and (v) phagosome and autophagy. Among the highly deregulated, moderately to strongly expressed DEGs that were members of one or more superpathways, 169 DEGs were listed in relevant databases and/or the literature and included members of the PCa pathway, oncogenes, prostate-specific genes, and druggable genes. These genes are novel and promising candidate diagnostic, prognostic and/or therapeutic canine PCa biomarkers.

BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells.

Bruton's tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3ß, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.

CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines.

In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.

Differences in embryo quality are associated with differences in oocyte composition: a proteomic study in inbred mice.

Current models of early mouse development assign roles to stochastic processes and epigenetic regulation, which are considered to be as influential as the genetic differences that exist between strains of the species Mus musculus. The aim of this study was to test whether mouse oocytes vary from each other in the abundance of gene products that could influence, prime, or even predetermine developmental trajectories and features of derivative embryos. Using the paradigm of inbred mouse strains, we quantified 2010 protein groups (SILAC LC-MS/MS) and 15205 transcripts (RNA deep sequencing) present simultaneously in oocytes of four strains tested (129/Sv, C57Bl/6J, C3H/HeN, DBA/2J). Oocytes differed according to donor strain in the abundance of catalytic and regulatory proteins, as confirmed for a subset (bromodomain adjacent to zinc finger domain, 1B [BAZ1B], heme oxygenase 1 [HMOX1], estrogen related receptor, beta [ESRRB]) via immunofluorescence in situ. Given a Pearson's r correlation coefficient of 0.18-0.20, the abundance of oocytic proteins could not be predicted from that of cognate mRNAs. Our results document that a prerequisite to generate embryo diversity, namely the different abundances of maternal proteins in oocytes, can be studied in the model of inbred mouse strains. Thus, we highlight the importance of proteomic quantifications in modern embryology. All MS data have been deposited in the ProteomeXchange with identifier PXD001059 (http://proteomecentral.proteomexchange.org/dataset/PXD001059).

Systematic elucidation and in vivo validation of sequences enriched in hindbrain transcriptional control.

Illuminating the primary sequence encryption of enhancers is central to understanding the regulatory architecture of genomes. We have developed a machine learning approach to decipher motif patterns of hindbrain enhancers and identify 40,000 sequences in the human genome that we predict display regulatory control that includes the hindbrain. Consistent with their roles in hindbrain patterning, MEIS1, NKX6-1, as well as HOX and POU family binding motifs contributed strongly to this enhancer model. Predicted hindbrain enhancers are overrepresented at genes expressed in hindbrain and associated with nervous system development, and primarily reside in the areas of open chromatin. In addition, 77 (0.2%) of these predictions are identified as hindbrain enhancers on the VISTA Enhancer Browser, and 26,000 (60%) overlap enhancer marks (H3K4me1 or H3K27ac). To validate these putative hindbrain enhancers, we selected 55 elements distributed throughout our predictions and six low scoring controls for evaluation in a zebrafish transgenic assay. When assayed in mosaic transgenic embryos, 51/55 elements directed expression in the central nervous system. Furthermore, 30/34 (88%) predicted enhancers analyzed in stable zebrafish transgenic lines directed expression in the larval zebrafish hindbrain. Subsequent analysis of sequence fragments selected based upon motif clustering further confirmed the critical role of the motifs contributing to the classifier. Our results demonstrate the existence of a primary sequence code characteristic to hindbrain enhancers. This code can be accurately extracted using machine-learning approaches and applied successfully for de novo identification of hindbrain enhancers. This study represents a critical step toward the dissection of regulatory control in specific neuronal subtypes.

Bioinformatics approaches to single-blastomere transcriptomics.

The totipotent zygote gives rise to cells with differing identities during mouse preimplantation development. Many studies have focused on analyzing the spatio-temporal dependencies during these lineage decision processes and much has been learnt by tracing transgenic marker gene expression up to the blastocyst stage and by analyzing the effects of genetic manipulations (knockout/ overexpression) on embryo development. However, until recently, it has not been possible to get broader overviews on the gene expression networks that distinguish one cell from the other within the same embryo. With the advent of whole genome amplification methodology and microfluidics-based quantitative RT-PCR it became possible to generate transcriptomes of single cells. Here we review the current state of the art of single-cell transcriptomics applied to mouse preimplantation embryo blastomeres and summarize findings made by pioneering studies in recent years. Furthermore we use the PluriNetWork and ExprEssence to investigate cell transitions based on published data.

A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis.

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA-based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type-specific developmental gene expression patterns.

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner.

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.

Genome-wide identification of conserved regulatory function in diverged sequences.

Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term "covert."

ENT3C: an entropy-based similarity measure for Hi-C and micro-C derived contact matrices.

Hi-C and micro-C sequencing have shed light on the profound importance of 3D genome organization in cellular function by probing 3D contact frequencies across the linear genome. The resulting contact matrices are extremely sparse and susceptible to technical- and sequence-based biases, making their comparison challenging. The development of reliable, robust and efficient methods for quantifying similarity between contact matrices is crucial for investigating variations in the 3D genome organization in different cell types or under different conditions, as well as evaluating experimental reproducibility. We present a novel method, ENT3C, which measures the change in pattern complexity in the vicinity of contact matrix diagonals to quantify their similarity. ENT3C provides a robust, user-friendly Hi-C or micro-C contact matrix similarity metric and a characteristic entropy signal that can be used to gain detailed biological insights into 3D genome organization.

CLARE: Cracking the LAnguage of Regulatory Elements.

UNLABELLED: CLARE is a computational method designed to reveal sequence encryption of tissue-specific regulatory elements. Starting with a set of regulatory elements known to be active in a particular tissue/process, it learns the sequence code of the input set and builds a predictive model from features specific to those elements. The resulting model can then be applied to user-supplied genomic regions to identify novel candidate regulatory elements. CLARE's model also provides a detailed analysis of transcription factors that most likely bind to the elements, making it an invaluable tool for understanding mechanisms of tissue-specific gene regulation. AVAILABILITY: CLARE is freely accessible at http://clare.dcode.org/.

Effects of HMGN variants on the cellular transcription profile.

High mobility group N (HMGN) is a family of intrinsically disordered nuclear proteins that bind to nucleosomes, alters the structure of chromatin and affects transcription. A major unresolved question is the extent of functional specificity, or redundancy, between the various members of the HMGN protein family. Here, we analyze the transcriptional profile of cells in which the expression of various HMGN proteins has been either deleted or doubled. We find that both up- and downregulation of HMGN expression altered the cellular transcription profile. Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation. Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain. Doubling the amount of HMGN had a significantly larger effect on the transcription profile than total deletion, suggesting that the intrinsically disordered structure of HMGN proteins plays an important role in their function. The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant.

How enhancers regulate wavelike gene expression patterns.

A key problem in development is to understand how genes turn on or off at the right place and right time during embryogenesis. Such decisions are made by non-coding sequences called 'enhancers.' Much of our models of how enhancers work rely on the assumption that genes are activated de novo as stable domains across embryonic tissues. Such a view has been strengthened by the intensive landmark studies of the early patterning of the anterior-posterior (AP) axis of the Drosophila embryo, where indeed gene expression domains seem to arise more or less stably. However, careful analysis of gene expression patterns in other model systems (including the AP patterning in vertebrates and short-germ insects like the beetle Tribolium castaneum) painted a different, very dynamic view of gene regulation, where genes are oftentimes expressed in a wavelike fashion. How such gene expression waves are mediated at the enhancer level is so far unclear. Here, we establish the AP patterning of the short-germ beetle Tribolium as a model system to study dynamic and temporal pattern formation at the enhancer level. To that end, we established an enhancer prediction system in Tribolium based on time- and tissue-specific ATAC-seq and an enhancer live reporter system based on MS2 tagging. Using this experimental framework, we discovered several Tribolium enhancers, and assessed the spatiotemporal activities of some of them in live embryos. We found our data consistent with a model in which the timing of gene expression during embryonic pattern formation is mediated by a balancing act between enhancers that induce rapid changes in gene expression patterns (that we call 'dynamic enhancers') and enhancers that stabilize gene expression patterns (that we call 'static enhancers'). However, more data is needed for a strong support for this or any other alternative models.

The TiHoCL panel for canine lymphoma: a feasibility study integrating functional genomics and network biology approaches for comparative oncology targeted NGS panel design.

Targeted next-generation sequencing (NGS) enables the identification of genomic variants in cancer patients with high sensitivity at relatively low costs, and has thus opened the era to personalized human oncology. Veterinary medicine tends to adopt new technologies at a slower pace compared to human medicine due to lower funding, nonetheless it embraces technological advancements over time. Hence, it is reasonable to assume that targeted NGS will be incorporated into routine veterinary practice in the foreseeable future. Many animal diseases have well-researched human counterparts and hence, insights gained from the latter might, in principle, be harnessed to elucidate the former. Here, we present the TiHoCL targeted NGS panel as a proof of concept, exemplifying how functional genomics and network approaches can be effectively used to leverage the wealth of information available for human diseases in the development of targeted sequencing panels for veterinary medicine. Specifically, the TiHoCL targeted NGS panel is a molecular tool for characterizing and stratifying canine lymphoma (CL) patients designed based on human non-Hodgkin lymphoma (NHL) research outputs. While various single nucleotide polymorphisms (SNPs) have been associated with high risk of developing NHL, poor prognosis and resistance to treatment in NHL patients, little is known about the genetics of CL. Thus, the ~100 SNPs featured in the TiHoCL targeted NGS panel were selected using functional genomics and network approaches following a literature and database search that shielded ~500 SNPs associated with, in nearly all cases, human hematologic malignancies. The TiHoCL targeted NGS panel underwent technical validation and preliminary functional assessment by sequencing DNA samples isolated from blood of 29 lymphoma dogs using an Ion Torrent™ PGM System achieving good sequencing run metrics. Our design framework holds new possibilities for the design of similar molecular tools applied to other diseases for which limited knowledge is available and will improve drug target discovery and patient care.