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2024 • bioRxiv

Artifacts in single-cell mitochondrial DNA mutation analyses misinform phylogenetic inference

Caleb A Lareau, Michael S Chapman, Livius Penter, Tal Nawy, Dana Pe'er, Leif S Ludwig

Sequencing mitochondrial DNA (mtDNA) variants from single cells has resolved clonality and lineage in native human samples and clinical specimens. Prior work established that heteroplasmic mtDNA variants can be used to delineate clonality in hematopoiesis, but they have limited ability to reconstruct cellular phylogenies. However, a recent report by Weng et al. challenges the current paradigm by describing an unprecedented number of shared mtDNA variants between cells that reportedly resolve high-resolution phylogenetic trees. We re-examined the claims of Weng et al., and identified two major points of concern regarding this unprecedented connectedness. First, shared variants between cells are disproportionately detected in a single molecule per cell, and second, these variants are enriched 10-20-fold at the edges of mtDNA molecules, reminiscent of artifacts reported in other sequencing approaches. Further, our analyses show that pruning low support and likely artificial mtDNA variants removes nearly all of the reported phylogenetic structure. Thus, we strongly caution against using mtDNA variant calling workflows that rely on minimal evidence, including the computational pipeline introduced in Weng et al., as variants with high connectedness and low evidence are likely artifacts that lead to the construction of false phylogenies.

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Dec 2023 • Cancer Research

Mitochondrial DNA mutations as natural barcodes for lineage tracing of murine tumor models

Livius Penter, Elisa Ten Hacken, Jackson Southard, Caleb A Lareau, Leif S Ludwig, Shuqiang Li, Donna S Neuberg, Kenneth J Livak, Catherine J Wu

Murine models are indispensable tools for functional genomic studies and preclinical testing of novel therapeutic approaches. Mitochondrial single-cell assay for transposase-accessible chromatin using sequencing (mtscATAC-seq) enables the dissection of cellular heterogeneity and clonal dynamics by capturing chromatin accessibility, copy-number variations (CNV), and mitochondrial DNA (mtDNA) mutations, yet its applicability to murine studies remains unexplored. By leveraging mtscATAC-seq in novel chronic lymphocytic leukemia and Richter syndrome mouse models, we report the detection of mtDNA mutations, particularly in highly proliferative murine cells, alongside CNV and chromatin state changes indicative of clonal evolution upon secondary transplant. This study thus demonstrates the feasibility and utility of multi-modal single-cell and natural barcoding approaches to characterize …

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Dec 2023 • Blood Cancer Discovery

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities

Elisa ten Hacken, Tomasz Sewastianik, Shanye Yin, Gabriela Brunsting Hoffmann, Michaela Gruber, Kendell Clement, Livius Penter, Robert A Redd, Neil Ruthen, Sebastien Hergalant, Alanna Sholokhova, Geoffrey Fell, Erin M Parry, Julien Broseus, Romain Guieze, Fabienne Lucas, Maria Hernandez-Sanchez, Kaitlyn Baranowski, Jackson Southard, Heather Joyal, Leah Billington, Fara Faye D Regis, Elizabeth Witten, Mohamed Uduman, Binyamin A Knisbacher, Shuqiang Li, Haoxiang Lyu, Tiziana Vaisitti, Silvia Deaglio, Giorgio Inghirami, Pierre Feugier, Stephan Stilgenbauer, Eugen Tausch, Matthew S Davids, Gad Getz, Kenneth J Livak, Ivana Bozic, Donna S Neuberg, Ruben D Carrasco, Catherine J Wu

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice, and recapitulated the process of transformation from indolent CLL into large cell lymphoma (i.e. Richter’s syndrome [RS]). Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed co-selection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, down-modulation of the receptor …

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Nov 2023 • Blood

Systematic Prediction of Minor Histocompatibility Antigens to Inform GvHD Outcomes after Allogeneic Stem Cell Transplantation

Nicoletta Cieri, Nidhi Hookeri, Kari Stromhaug, Liang Li, Julia Keating, Jonathan Stevens, Kameron Kooshesh, Helen Ji, Gabrielle M Hernandez, Jennifer Abelin, Susan Klaeger, Cleo Forman, Karl Clauser, Siranush Sarkizova, Giacomo Oliveira, Livius Penter, Haesook T Kim, William Lane, Shuqiang Li, Kenneth J Livak, Steven A Carr, Derin B Keskin, Vincent T Ho, Jerome Ritz, Robert J Soiffer, Donna S Neuberg, Chip Stewart, Gad Getz, Catherine J Wu

T cell alloreactivity against minor histocompatibility antigens (mHAgs), polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms (SNPs, indels, frameshifts), is at the core of the therapeutic effect of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite the crucial role of mHAgs in graft-versus-leukemia and graft-versus-host (GvHD) reactions, it has not been possible thus far to consistently link patient-specific mHAg repertoires to clinical outcomes. As a result, only D-R HLA matching and the activity of GvHD prophylaxis strategies are currently available to help clinicians in this challenge.We hypothesized that post-transplant outcomes could be impacted by genome-wide mHAg load, delineated in an organ- and malignancy-specific fashion. We have therefore devised an analytic framework to systematically identify autosomal and Y-encoded mHAgs, based …

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Nov 2023 • Blood

Ipilimumab plus decitabine for patients with MDS or AML in posttransplant or transplant-naïve settings

Jacqueline S Garcia, Yael Flamand, Livius Penter, Michael Keng, Benjamin K Tomlinson, Lourdes M Mendez, Paul Koller, Nicole Cullen, Yohei Arihara, Kathleen Pfaff, Jacquelyn O Wolff, Andrew M Brunner, Ilene Galinsky, Asad Bashey, Joseph H Antin, Corey Cutler, Vincent Ho, Brian A Jonas, Marlise R Luskin, Martha Wadleigh, Eric S Winer, Alexandra Savell, Rebecca Leonard, Taylor Robertson, Matthew S Davids, Howard Streicher, Scott J Rodig, Jerome Ritz, Catherine J Wu, Daniel J DeAngelo, Donna Neuberg, Richard M Stone, Robert J Soiffer


Nov 2023 • Blood

Tracking of Leukemia and Immune Single Cell Phenotypes during Ipilimumab-Based Treatment By Long-Read Sequencing

Livius Penter, Mehdi Borji, Adi Nagler, Haoxiang Lyu, Wesley S Lu, Nicoletta Cieri, Alexandria Maurer, Giacomo Oliveira, Aziz M Al'Khafaji, Kiran Garimella, Shuqiang Li, Donna S Neuberg, Jerome Ritz, Robert J Soiffer, Jacqueline S Garcia, Kenneth J Livak, Catherine J Wu

Activity of ipilimumab in bone marrow-involved AML has been modest compared to extramedullary AML relapse or solid tumors like melanoma. Single cell transcriptomics (scRNA-seq) could provide deeper understanding of these differences, yet leukemia and immune cell phenotypes defined by somatic mutations or differential isoform expression often cannot be resolved with native short-read scRNA-seq data due to insufficient read coverage. To overcome this limitation, we devised a workflow ( nanoranger) that leverages improved accuracy of long-read sequencing (Oxford Nanopore, ONT) and enables targeted read-out of molecular features including single-nucleotide variants, gene fusions, isoforms, or T cell receptor (TCR) sequences from intermediate scRNA-seq cDNA libraries.To determine the performance of nanoranger, we undertook a series of assessments. First, we established its genotyping accuracy …

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Nov 2023 • Blood

An Analytic Framework for the Prediction of Gvl Minor Histocompatibility Antigens to Prevent or Treat Post-Transplant Relapse

Nicoletta Cieri, Liang Li, Nidhi Hookeri, Kari Stromhaug, Julia Keating, Jonathan Stevens, Kameron Kooshesh, Helen Ji, Gabrielle M Hernandez, Jennifer Abelin, Susan Klaeger, Cleo Forman, Karl Clauser, Siranush Sarkizova, Giacomo Oliveira, Livius Penter, Haesook T Kim, William Lane, Shuqiang Li, Kenneth J Livak, Steven A Carr, Derin B Keskin, Vincent T Ho, Jerome Ritz, Robert J Soiffer, Donna S Neuberg, Chip Stewart, Gad Getz, Catherine J Wu

The therapeutic effect of allo-HSCT relies on T cell alloreactivity driven by genetic nonidentity between donor and recipient (D-R) pairs. In the setting of HLA-matched transplants, endogenous proteins in recipient cells differing from those of the donor due to genetic polymorphisms, can provide distinct HLA-binding peptides, serving as minor histocompatibility antigens (mHAgs). Hematopoietically-restricted mHAgs represent ideal targets to separate graft-versus-leukemia (GvL) from graft-versus-host-disease (GvHD) effects. To date,however, the quest for optimal GvL mHAgs has resulted in the identification of only a handful of targets, raising concerns on the clinical feasibility of their targeting.We hypothesized that systematic mHAg prediction from whole-exome sequencing (WES) of D-R pairs could help identifying novel candidate mHAgs with an acceptable safety profile for therapeutical targeting. We focused on …

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Nov 2023 • BLOOD 142, 2023

Cellular Immunotherapies: Early Phase and Investigational TherapiesLeukemia Blasts Induce NK-like Dysfunction in AML-Specific T Cells

Francesco Mazziotta, Lauren E Martin, Miranda Lahman, Daniel N Egan, Sinead Kinsella, Kelly Paulson, Yapeng Su, Valentin Voillet, Livius Penter, Catherine J Wu, Philip Greenberg, Aude G Chapuis


Nov 2023 • Blood

Coordinated immune cell networks in the bone marrow microenvironment define the graft versus leukemia response with adoptive cellular therapy

Alexandria Maurer, Cameron Y Park, Shouvik Mani, Mehdi Borji, Crystal Shin, Livius Penter, James R Brenner, Jackson Southard, Wesley S Lu, Haoxiang Lyu, Shuqiang Li, Kenneth J Livak, Jerome Ritz, Robert J Soiffer, Elham Azizi, Catherine J Wu

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow.To infer cell-cell interactions in a …

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Nov 2023 • Blood

Leukemia Blasts Induce NK-like Dysfunction in AML-Specific T Cells

Francesco Mazziotta, Lauren E Martin, Miranda Lahman, Daniel N Egan, Sinéad Kinsella, Kelly Paulson, Yapeng Su, Valentin Voillet, Livius Penter, Catherine J Wu, Philip Greenberg, Aude G Chapuis

IntroductionEfforts to enhance T cell-mediated acute myeloid leukemia (AML) eradication face challenges possibly due to unique mechanisms of leukemia-induced T-cell dysfunction compared to exhaustion observed in solid tumors(Penter et al., Blood 2023). We previously showed the efficacy of Wilms' Tumor (WT) Antigen 1-specific T-cell receptor (TCR C4) inserted into CD8 + T cells (T TCR-C4) in preventing AML recurrence after hematopoietic cell transplantation (HCT) (Chapuis et al., Nat Med 2019). This led us to investigate the safety and efficacy of T TCR-C4 in post-HCT relapsed patients.MethodsIn this Phase I/II study conducted at Fred Hutchinson Cancer Center, 15 patients were enrolled and prospectively monitored for disease response. We manufactured T TCR-C4 using a lentiviral transduction system (Chapuis et al, Nat Med 2019) and performed comprehensive analyses of bone marrow (BM) and …

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Nov 2023 • Blood

Integrative Single-Cell Tracking of Clonal Evolution, Leukemia Phenotypes and Donor Engraftment Following Allogeneic Stem Cell Transplant Using Mitochondrial DNA Mutations

Livius Penter, Nicoletta Cieri, Katie Maurer, Haoxiang Lyu, Wesley S Lu, Giacomo Oliveira, Satyen H Gohil, Caleb Lareau, Leif S Ludwig, Shuqiang Li, Jerome Ritz, Jacqueline S Garcia, Robert J Soiffer, Kenneth J Livak, Catherine J Wu

Mitochondrial DNA (mtDNA) mutations can distinguish cells from unrelated individuals and their spectrum per patient sample can evolve following therapeutic bottlenecks. As such, these natural barcodes can potentially enable integrated single cell tracking of chimeric cellular populations and clonal evolution following unrelated allogeneic hematopoietic stem cell transplantation (HSCT). However, the feasibility of mtDNA-based donor-recipient deconvolution or identification of leukemia-specific phenotypes and the extent of co-evolution with somatic mutations remains unknown. If confirmed, post-transplant monitoring using mtDNA mutations could enable detection of early post-HSCT relapse and provide qualitative information on leukemia phenotypes, clonal evolution, or residual donor engraftment via a combined single cell assay.To investigate the extent of co-evolution of somatic nuclear mutations with mtDNA …

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Nov 2023 • Blood

Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft versus Leukemia Response with Adoptive Cellular Therapy

Alexandria Maurer, Cameron Y Park, Shouvik Mani, Mehdi Borji, Crystal Shin, Livius Penter, James R Brenner, Jackson Southard, Wesley S Lu, Haoxiang Lyu, Shuqiang Li, Kenneth J Livak, Jerome Ritz, Robert J Soiffer, Elham Azizi, Catherine J Wu

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow.To infer cell-cell interactions in a …

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Nov 2023 • Blood

Integrative Single-Cell Tracking of Clonal Evolution, Leukemia Phenotypes and Donor Engraftment Following Allogeneic Stem Cell Transplant Using Mitochondrial DNA Mutations

Livius Penter, Nicoletta Cieri, Katie Maurer, Haoxiang Lyu, Wesley S Lu, Giacomo Oliveira, Satyen H Gohil, Caleb Lareau, Leif S Ludwig, Shuqiang Li, Jerome Ritz, Jacqueline S Garcia, Robert J Soiffer, Kenneth J Livak, Catherine J Wu

Mitochondrial DNA (mtDNA) mutations can distinguish cells from unrelated individuals and their spectrum per patient sample can evolve following therapeutic bottlenecks. As such, these natural barcodes can potentially enable integrated single cell tracking of chimeric cellular populations and clonal evolution following unrelated allogeneic hematopoietic stem cell transplantation (HSCT). However, the feasibility of mtDNA-based donor-recipient deconvolution or identification of leukemia-specific phenotypes and the extent of co-evolution with somatic mutations remains unknown. If confirmed, post-transplant monitoring using mtDNA mutations could enable detection of early post-HSCT relapse and provide qualitative information on leukemia phenotypes, clonal evolution, or residual donor engraftment via a combined single cell assay.To investigate the extent of co-evolution of somatic nuclear mutations with mtDNA …

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Oct 2023 • Medrxiv: the Preprint Server for Health Sciences

A multidimensional analysis reveals distinct immune phenotypes and tertiary lymphoid structure-like aggregates in the bone marrow of pediatric acute myeloid leukemia.

Joost B Koedijk, Inge van der Werf, Livius Penter, Marijn A Vermeulen, Farnaz Barneh, Alicia Perzolli, Joyce I Meesters-Ensing, Marta Fiocco, Hester A de Groot-Kruseman, Rubina Moeniralam, B Porter, K Pfaff, JS Garcia, SJ Rodig, CJ Wu, H Hasle, S Nierkens, ME Belderbos, CM Zwaan, O Heidenreich

Because of the low mutational burden, children with acute myeloid leukemia (AML) are thought to have a'cold'tumor microenvironment and consequently, a low likelihood of response to T cell-directed immunotherapies. Here, we provide a multidimensional overview of the tumor immune microenvironment in newly diagnosed pediatric AML. On a cohort level, we demonstrate wide variation in T cell infiltration with nearly one-third of cases harboring an immune-infiltrated bone marrow. These immune-infiltrated cases are characterized by a decreased abundance of M2-like macrophages, which we find to be associated with response to T cell-directed immunotherapy in adult AML. On an organizational level, we reveal the composition of spatially organized immune aggregates in pediatric AML, and show that in the adult setting such aggregates in post-treatment bone marrow and extramedullary sites associate with response to ipilimumab-based therapy. Altogether, our study provides immune correlates of response to T cell-directed immunotherapies and indicates starting points for further investigations into immunomodulatory mechanisms in AML.

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Oct 2023 • medRxiv

A multidimensional analysis reveals distinct immune phenotypes and tertiary lymphoid structure-like aggregates in the bone marrow of pediatric acute myeloid leukemia

Joost B Koedijk, Inge van der Werf, Livius Penter, Marijn A Vermeulen, Farnaz Barneh, Alicia Perzolli, Joyce I Meesters-Ensing, Marta Fiocco, Hester A de Groot-Kruseman, Rubina Moeniralam, Kristina Bang Christensen, Billie Porter, Kathleen Pfaff, Jacqueline S Garcia, Scott J Rodig, Catherine J Wu, Henrik Hasle, Stefan Nierkens, Mirjam E Belderbos, C Michel Zwaan, Olaf Heidenreich

Because of the low mutational burden, children with acute myeloid leukemia (AML) are thought to have a ‘cold’tumor microenvironment and consequently, a low likelihood of response to T cell-directed immunotherapies. Here, we provide a multidimensional overview of the tumor immune microenvironment in newly diagnosed pediatric AML. On a cohort level, we demonstrate wide variation in T cell infiltration with nearly one-third of cases harboring an immune-infiltrated bone marrow. These immune-infiltrated cases are characterized by a decreased abundance of M2-like macrophages, which we find to be associated with response to T cell-directed immunotherapy in adult AML. On an organizational level, we reveal the composition of spatially organized immune aggregates in pediatric AML, and show that in the adult setting such aggregates in post-treatment bone marrow and extramedullary sites associate with …

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Sep 2023 • Science Immunology

Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti–PD-1 in head and neck cancer

Giacomo Oliveira, Ann Marie Egloff, Alexander B Afeyan, Jacquelyn O Wolff, Zexiang Zeng, Rebecca D Chernock, Liye Zhou, Cameron Messier, Patrick Lizotte, Kathleen L Pfaff, Kari Stromhaug, Livius Penter, Robert I Haddad, Glenn J Hanna, Jonathan D Schoenfeld, Laura A Goguen, Donald J Annino, Vickie Jo, Peter Oppelt, Patrik Pipkorn, Ryan Jackson, Sidharth V Puram, Randal C Paniello, Jason T Rich, Jason Webb, Jose P Zevallos, Mena Mansour, Jingxin Fu, Gavin P Dunn, Scott J Rodig, Jessica Ley, Luc GT Morris, Lara Dunn, Cloud P Paweletz, Dorina Kallogjeri, Jay F Piccirillo, Douglas R Adkins, Catherine J Wu, Ravindra Uppaluri

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti–programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating …

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Jul 2023 • arXiv preprint arXiv:2307.13708

The Impact of Genomic Variation on Function (IGVF) Consortium

IGVF Consortium

Our genomes influence nearly every aspect of human biology from molecular and cellular functions to phenotypes in health and disease. Human genetics studies have now associated hundreds of thousands of differences in our DNA sequence ("genomic variation") with disease risk and other phenotypes, many of which could reveal novel mechanisms of human biology and uncover the basis of genetic predispositions to diseases, thereby guiding the development of new diagnostics and therapeutics. Yet, understanding how genomic variation alters genome function to influence phenotype has proven challenging. To unlock these insights, we need a systematic and comprehensive catalog of genome function and the molecular and cellular effects of genomic variants. Toward this goal, the Impact of Genomic Variation on Function (IGVF) Consortium will combine approaches in single-cell mapping, genomic perturbations, and predictive modeling to investigate the relationships among genomic variation, genome function, and phenotypes. Through systematic comparisons and benchmarking of experimental and computational methods, we aim to create maps across hundreds of cell types and states describing how coding variants alter protein activity, how noncoding variants change the regulation of gene expression, and how both coding and noncoding variants may connect through gene regulatory and protein interaction networks. These experimental data, computational predictions, and accompanying standards and pipelines will be integrated into an open resource that will catalyze community efforts to explore genome function and the impact …

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Jul 2023 • arXiv preprint arXiv:2307.13708

The Impact of Genomic Variation on Function (IGVF) Consortium

IGVF Consortium

Our genomes influence nearly every aspect of human biology from molecular and cellular functions to phenotypes in health and disease. Human genetics studies have now associated hundreds of thousands of differences in our DNA sequence ("genomic variation") with disease risk and other phenotypes, many of which could reveal novel mechanisms of human biology and uncover the basis of genetic predispositions to diseases, thereby guiding the development of new diagnostics and therapeutics. Yet, understanding how genomic variation alters genome function to influence phenotype has proven challenging. To unlock these insights, we need a systematic and comprehensive catalog of genome function and the molecular and cellular effects of genomic variants. Toward this goal, the Impact of Genomic Variation on Function (IGVF) Consortium will combine approaches in single-cell mapping, genomic perturbations, and predictive modeling to investigate the relationships among genomic variation, genome function, and phenotypes. Through systematic comparisons and benchmarking of experimental and computational methods, we aim to create maps across hundreds of cell types and states describing how coding variants alter protein activity, how noncoding variants change the regulation of gene expression, and how both coding and noncoding variants may connect through gene regulatory and protein interaction networks. These experimental data, computational predictions, and accompanying standards and pipelines will be integrated into an open resource that will catalyze community efforts to explore genome function and the impact …

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Jun 2023 • Nature

Transfer learning enables predictions in network biology

Christina V Theodoris, Ling Xiao, Anant Chopra, Mark D Chaffin, Zeina R Al Sayed, Matthew C Hill, Helene Mantineo, Elizabeth M Brydon, Zexian Zeng, X Shirley Liu, Patrick T Ellinor

Mapping gene networks requires large amounts of transcriptomic data to learn the connections between genes, which impedes discoveries in settings with limited data, including rare diseases and diseases affecting clinically inaccessible tissues. Recently, transfer learning has revolutionized fields such as natural language understanding, and computer vision by leveraging deep learning models pretrained on large-scale general datasets that can then be fine-tuned towards a vast array of downstream tasks with limited task-specific data. Here, we developed a context-aware, attention-based deep learning model, Geneformer, pretrained on a large-scale corpus of about 30 million single-cell transcriptomes to enable context-specific predictions in settings with limited data in network biology. During pretraining, Geneformer gained a fundamental understanding of network dynamics, encoding network hierarchy in the …

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Jun 2023 • Nature Protocols, 1-11, 2023

Tutorial: integrative computational analysis of bulk RNA-sequencing data to characterize tumor immunity using RIMA

Lin Yang, Jin Wang, Jennifer Altreuter, Aashna Jhaveri, Cheryl J Wong, Li Song, Jingxin Fu, Len Taing, Sudheshna Bodapati, Avinash Sahu, Collin Tokheim, Yi Zhang, Zexian Zeng, Gali Bai, Ming Tang, Xintao Qiu, Henry W Long, Franziska Michor, Yang Liu, X Shirley Liu

RNA-sequencing (RNA-seq) has become an increasingly cost-effective technique for molecular profiling and immune characterization of tumors. In the past decade, many computational tools have been developed to characterize tumor immunity from gene expression data. However, the analysis of large-scale RNA-seq data requires bioinformatics proficiency, large computational resources and cancer genomics and immunology knowledge. In this tutorial, we provide an overview of computational analysis of bulk RNA-seq data for immune characterization of tumors and introduce commonly used computational tools with relevance to cancer immunology and immunotherapy. These tools have diverse functions such as evaluation of expression signatures, estimation of immune infiltration, inference of the immune repertoire, prediction of immunotherapy response, neoantigen detection and microbiome quantification …

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Jun 2023 • bioRxiv

scATAnno: automated cell type annotation for single-cell ATAC sequencing data

Yijia Jiang, Zhirui Hu, Allen W Lynch, Junchen Jiang, Alexander Zhu, Ziqi Zeng, Yi Zhang, Gongwei Wu, Yingtian Xie, Rong Li, Ningxuan Zhou, Cliff Meyer, Paloma Cejas, Myles Brown, Henry W Long, Xintao Qiu

Recent advances in single-cell epigenomic techniques have created a growing demand for scATAC-seq analysis. One key analysis task is to determine cell type identity based on the epigenetic data. We introduce scATAnno, a python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates the reference atlases from publicly available datasets enabling accurate cell type annotation by integrating query data with reference atlases, without the use of scRNA-seq data. To enhance annotation accuracy, we have incorporated KNN-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compare and benchmark scATAnno against 7 other published approaches for cell annotation and show superior performance in multiple data sets and metrics. We showcase the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cell (PBMC), Triple Negative Breast Cancer (TNBC), and basal cell carcinoma (BCC), and demonstrate that scATAnno accurately annotates cell types across conditions. Overall, scATAnno is a useful tool for scATAC-seq reference building and cell type annotation in scATAC-seq data and can aid in the interpretation of new scATAC-seq datasets in complex biological systems.

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