High-throughput evaluation of genetic variants with prime editing sensor libraries.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report.

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

Investigative needle core biopsies for multi-omics in Glioblastoma.

Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few effective therapies. The ability to investigate the tumor microenvironment before and during treatment would greatly enhance both understanding of disease response and progression, as well as the delivery and impact of therapeutics. Stereotactic biopsies are a routine surgical procedure performed primarily for diagnostic histopathologic purposes. The role of investigative biopsies - tissue sampling for the purpose of understanding tumor microenvironmental responses to treatment using integrated multi-modal molecular analyses ('Multi-omics") has yet to be defined. Secondly, it is unknown whether comparatively small tissue samples from brain biopsies can yield sufficient information with such methods. Here we adapt stereotactic needle core biopsy tissue in two separate patients. In the first patient with recurrent GBM we performed highly resolved multi-omics analysis methods including single cell RNA sequencing, spatial-transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics from biopsy tissue that was obtained from a single procedure. In a second patient we analyzed multi-regional core biopsies to decipher spatial and genomic variance. We also investigated the utility of stereotactic biopsies as a method for generating patient derived xenograft models in a separate patient cohort. Dataset integration across modalities showed good correspondence between spatial modalities, highlighted immune cell associated metabolic pathways and revealed poor correlation between RNA expression and the tumor MHC Class I immunopeptidome. In conclusion, stereotactic needle biopsy cores are of sufficient quality to generate multi-omics data, provide data rich insight into a patient's disease process and tumor immune microenvironment and can be of value in evaluating treatment responses. One sentence summary: Integrative multi-omics analysis of stereotactic needle core biopsies in glioblastoma.

NExtSEEK: Extending SEEK for Active Management of Interoperable Metadata.

Data management is a critical challenge required to improve the rigor and reproducibility of large projects. Adhering to Findable, Accessible, Interoperable, and Reusable (FAIR) standards provides a baseline for meeting these requirements. Although many existing repositories handle data in a FAIR-compliant manner, there are limited tools in the public domain to handle the metadata burden required to connect data from multi-omic projects that span multiple institutions and are deposited in diverse repositories. One promising approach is the SEEK platform, which allows for diverse metadata and provides an established repository. SEEK is challenged by the assumption of single deposition events where a sample is immutable once entered in the database. This is structured for published data but presents a limitation for ongoing studies where multiple sequential events may occur in a single sample at different sites. To address this issue, we have created a modified wrapper around the SEEK platform that allows for active data management by establishing more discrete sample types that are mutable to permit the expansion of the types of metadata, allowing researchers to track additional information. The use of discrete nodes also converts assays from nodes to edges, creating a network model of the study and more accurately representing the experimental process. With these changes to SEEK, users are able to collect and organize the information that researchers need to improve reusability and reproducibility as well as make data and metadata available to the scientific community through public repositories.

A single-cell liver atlas of Plasmodium vivax infection.

Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years and reactivate to cause recurrent blood-stage infection. Although they are an important target for malaria eradication, little is known about the molecular features of replicative and non-replicative intracellular liver-stage parasites and their host cell dependence. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites for transcriptional profiling. Coupling enrichment strategies with bulk and single-cell analyses, we capture both parasite and host transcripts in individual hepatocytes throughout the course of infection. We define host- and state-dependent transcriptional signatures and identify unappreciated populations of replicative and non-replicative parasites that share features with sexual transmissive forms. We find that infection suppresses the transcription of key hepatocyte function genes and elicits an anti-parasite innate immune response. Our work provides a foundation for understanding host-parasite interactions and reveals insights into the biology of P. vivax dormancy and transmission.

Myeloid cell subsets that express latency-associated peptide promote cancer growth by modulating T cells.

Myeloid suppressor cells promote tumor growth by a variety of mechanisms which are not fully characterized. We identified myeloid cells (MCs) expressing the latency-associated peptide (LAP) of TGF-ß on their surface and LAPHi MCs that stimulate Foxp3+ Tregs while inhibiting effector T cell proliferation and function. Blocking TGF-ß inhibits the tolerogenic ability of LAPHi MCs. Furthermore, adoptive transfer of LAPHi MCs promotes Treg accumulation and tumor growth in vivo. Conversely, anti-LAP antibody, which reduces LAPHi MCs, slows cancer progression. Single-cell RNA-Seq analysis on tumor-derived immune cells revealed LAPHi dominated cell subsets with distinct immunosuppressive signatures, including those with high levels of MHCII and PD-L1 genes. Analogous to mice, LAP is expressed on myeloid suppressor cells in humans, and these cells are increased in glioma patients. Thus, our results identify a previously unknown function by which LAPHi MCs promote tumor growth and offer therapeutic intervention to target these cells in cancer.

Quantitative mapping of the cellular small RNA landscape with AQRNA-seq.

Current next-generation RNA-sequencing (RNA-seq) methods do not provide accurate quantification of small RNAs within a sample, due to sequence-dependent biases in capture, ligation and amplification during library preparation. We present a method, absolute quantification RNA-sequencing (AQRNA-seq), that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member microRNA reference library, oligonucleotide standards of varying length, and RNA blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial transfer RNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced, tRNA-driven, codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells.

High-throughput Minitaturized RNA-Seq Library Preparation.

Advances in next-generation sequencing technologies have allowed RNA sequencing to become an increasingly time efficient, cost-effective, and accessible tool for genomic research. We present here an automated and miniaturized workflow for RNA library preparation that minimizes reagent usage and processing time required per sample to generate Illumina compatible libraries for sequencing. The reduced-volume libraries show similar behavior to full-scale libraries with comparable numbers of genes detected and reproducible clustering of samples.

Cross-Site Evaluation of Commercial Sanger Sequencing Chemistries.

Sanger sequencing remains an essential tool utilized by researchers. Despite competition from commercial sequencing providers, many academic sequencing core facilities continue to offer these services based on a model of competitive pricing, knowledgeable technical support, and rapid turnaround time. In-house Sanger sequencing remains a viable core service and, until recently, Applied Biosystems BigDye Terminator chemistry was the only commercially available solution for Sanger DNA sequencing on Applied Biosystems (ABI) instruments; however, several new products employing novel dye chemistries and reaction configurations have entered the market. As a result, there is a need to benchmark the performance of these new chemistries on various DNA templates, including difficult-to-sequence templates, and their amenability to commonly employed cost-saving measures, such as dye dilution and reaction miniaturization. To evaluate these new reagents, a study was designed to compare the quality of Sanger sequencing data produced by ABI BigDye and commercially available kits from 2 other vendors using both control and difficult-to-sequence DNA templates under various reaction conditions. This study will serve as a valuable resource to core facilities conducting Sanger sequencing that wish to evaluate the use of an alternative chemistry in their sequencing core.

Bioinformatics Core Survey Highlights the Challenges Facing Data Analysis Facilities.

Over the last decade, the cost of -omics data creation has decreased 10-fold, whereas the need for analytical support for those data has increased exponentially. Consequently, bioinformaticians face a second wave of challenges: novel applications of existing approaches (e.g., single-cell RNA sequencing), integration of -omics data sets of differing size and scale (e.g., spatial transcriptomics), as well as novel computational and statistical methods, all of which require more sophisticated pipelines and data management. Nonetheless, bioinformatics cores are often asked to operate under primarily a cost-recovery model, with limited institutional support. Seeing the need to assess bioinformatics core operations, the Association of Biomolecular Resource Facilities Genomics Bioinformatics Research Group conducted a survey to answer questions about staffing, services, financial models, and challenges to better understand the challenges bioinformatics core facilities are currently faced with and will need to address going forward. Of the respondent groups, we chose to focus on the survey data from smaller cores, which made up the majority. Although all cores indicated similar challenges in terms of changing technologies and analysis needs, small cores tended to have the added challenge of funding their operations largely through cost-recovery models with heavy administrative burdens.

Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs in vivo. Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.

SPRI Beads-based Size Selection in the Range of 2-10kb.

Application of solid-phase reversible immobilization (SPRI) beads for size selection in molecular biology should be expanded, in light of the property of the beads to accommodate to high MW intervals of DNA fragment size selection, depending on composition of bead-suspension buffer. Here we show how the conventional size selection interval of 150-800 bp be shifted to 1.5-7 Kbp with by adjusting the concentration of NaCl in the stock suspension buffer. The MW capacity of SPRI beads also change when NaCl replaced with other cations and when the concentration of polyethylene glycol (PEG) 8000 is decreased. Testing the limits of SPRI beads revealed cuts as high as 10 Kbp are possible for some salt/PEG combinations of modified SPRI beads-suspension buffers.

Single-cell transcriptomic profiling of the aging mouse brain.

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.

H3K27me3-mediated silencing of structural genes is required for zebrafish heart regeneration.

Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3K27M-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.

Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.

HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis.

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.

Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription.

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in vivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.

Global transcriptional regulation of innate immunity by ATF-7 in C. elegans.

The nematode Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1- ATF-7 signaling reveals a striking level of control over the innate immune response to infection through a single transcriptional regulator.

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

BACKGROUND: Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. RESULTS: We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. CONCLUSIONS: These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.