Abstract Motivation Exponential growth in sequencing databases has motivated scalable De Bruijn graph-based (DBG) indexing for searching these data, using annotations to label nodes with sample IDs. Low-depth sequencing samples correspond to fragmented subgraphs, complicating finding the long contiguous walks required for alignment queries. Aligners that target single-labelled subgraphs reduce alignment lengths due to fragmentation, leading to low recall for long reads. While some (e.g. label-free) aligners partially overcome fragmentation by combining information from multiple samples, biologically irrelevant combinations in such approaches can inflate the search space or reduce accuracy. Results We introduce a new scoring model, ‘multi-label alignment’ (MLA), for annotated DBGs. MLA leverages two new operations: To promote biologically relevant sample combinations, ‘Label Change’ incorporates more informative global sample similarity into local scores. To improve connectivity, ‘Node Length Change’ dynamically adjusts the DBG node length during traversal. Our fast, approximate, yet accurate MLA implementation has two key steps: a single-label seed-chain-extend aligner (SCA) and a multi-label chainer (MLC). SCA uses a traditional scoring model adapting recent chaining improvements to assembly graphs and provides a curated pool of alignments. MLC extracts seed anchors from SCAs alignments, produces multi-label chains using MLA scoring, then finally forms multi-label alignments. We show via substantial improvements in taxonomic classification accuracy that MLA produces biologically relevant alignments, decreasing average weighted UniFrac errors by 63.1%–66.8% and covering 45.5%–47.4% (median) more long-read query characters than state-of-the-art aligners. MLAs runtimes are competitive with label-combining alignment and substantially faster than single-label alignment.

Authors Harun Mustafa, Mikhail Karasikov, Nika Mansouri Ghiasi, Gunnar Rätsch, André Kahles

Submitted Bioinformatics, ISMB 2024

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Abstract The amount of biological sequencing data available in public repositories is growing exponentially, forming an invaluable biomedical research resource. Yet, making it full-text searchable and easily accessible to researchers in life and data science is an unsolved problem. In this work, we take advantage of recently developed, very efficient data structures and algorithms for representing sequence sets. We make Petabases of DNA sequences across all clades of life, including viruses, bacteria, fungi, plants, animals, and humans, fully searchable. Our indexes are freely available to the research community. This highly compressed representation of the input sequences (up to 5800×) fits on a single consumer hard drive (≈100 USD), making this valuable resource cost-effective to use and easily transportable. We present the underlying methodological framework, called MetaGraph, that allows us to scalably index very large sets of DNA or protein sequences using annotated De Bruijn graphs. We demonstrate the feasibility of indexing the full extent of existing sequencing data and present new approaches for efficient and cost-effective full-text search at an on-demand cost of $0.10 per queried Mbp. We explore several practical use cases to mine existing archives for interesting associations and demonstrate the utility of our indexes for integrative analyses.

Authors Mikhail Karasikov, Harun Mustafa, Daniel Danciu, Marc Zimmermann, Christopher Barber, Gunnar Rätsch, André Kahles

Submitted bioRxiv

Link DOI

Abstract The amount of data stored in genomic sequence databases is growing exponentially, far exceeding traditional indexing strategies' processing capabilities. Many recent indexing methods organize sequence data into a sequence graph to succinctly represent large genomic data sets from reference genome and sequencing read set databases. These methods typically use De Bruijn graphs as the graph model or the underlying index model, with auxiliary graph annotation data structures to associate graph nodes with various metadata. Examples of metadata can include a node's source samples (called labels), genomic coordinates, expression levels, etc. An important property of these graphs is that the set of sequences spelled by graph walks is a superset of the set of input sequences. Thus, when aligning to graphs indexing samples derived from low-coverage sequencing sets, sequence information present in many target samples can compensate for missing information resulting from a lack of sequence coverage. Aligning a query against an entire sequence graph (as in traditional sequence-to-graph alignment) using state-of-the-art algorithms can be computationally intractable for graphs constructed from thousands of samples, potentially searching through many non-biological combinations of samples before converging on the best alignment. To address this problem, we propose a novel alignment strategy called multi-label alignment (MLA) and an algorithm implementing this strategy using annotated De Bruijn graphs within the MetaGraph framework, called MetaGraph-MLA. MLA extends current sequence alignment scoring models with additional label change operations for incorporating mixtures of samples into an alignment, penalizing mixtures that are dissimilar in their sequence content. To overcome disconnects in the graph that result from a lack of sequencing coverage, we further extend our graph index to utilize a variable-order De Bruijn graph and introduce node length change as an operation. In this model, traversal between nodes that share a suffix of length < k-1 acts as a proxy for inserting nodes into the graph. MetaGraph-MLA constructs an MLA of a query by chaining single-label alignments using sparse dynamic programming. We evaluate MetaGraph-MLA on simulated data against state-of-the-art sequence-to-graph aligners. We demonstrate increases in alignment lengths for simulated viral Illumina-type (by 6.5%), PacBio CLR-type (by 6.2%), and PacBio CCS-type (by 6.7%) sequencing reads, respectively, and show that the graph walks incorporated into our MLAs originate predominantly from samples of the same strain as the reads' ground-truth samples. We envision MetaGraph-MLA as a step towards establishing sequence graph tools for sequence search against a wide variety of target sequence types.

Authors Harun Mustafa, Mikhail Karasikov, Gunnar Rätsch, André Kahles

Submitted bioRxiv

Link DOI

Abstract Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters (‘Candidatus Eudoremicrobiaceae’) that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments.

Authors Lucas Paoli, Hans-Joachim Ruscheweyh, Clarissa C. Forneris, Florian Hubrich, Satria Kautsar, Agneya Bhushan, Alessandro Lotti, Quentin Clayssen, Guillem Salazar, Alessio Milanese, Charlotte I. Carlström, Chrysa Papadopoulou, Daniel Gehrig, Mikhail Karasikov, Harun Mustafa, Martin Larralde, Laura M. Carroll, Pablo Sánchez, Ahmed A. Zayed, Dylan R. Cronin, Silvia G. Acinas, Peer Bork, Chris Bowler, Tom O. Delmont, Josep M. Gasol, Alvar D. Gossert, Andre Kahles, Matthew B. Sullivan, Patrick Wincker, Georg Zeller, Serina L. Robinson, Jörn Piel, and Shinichi Sunagawa

Submitted Nature

Link DOI

Abstract High-throughput sequencing data is rapidly accumulating in public repositories. Making this resource accessible for interactive analysis at scale requires efficient approaches for its storage and indexing. There have recently been remarkable advances in solving the experiment discovery problem and building compressed representations of annotated de Bruijn graphs where k-mer sets can be efficiently indexed and interactively queried. However, approaches for representing and retrieving other quantitative attributes such as gene expression or genome positions in a general manner have yet to be developed. In this work, we propose the concept of Counting de Bruijn graphs generalizing the notion of annotated (or colored) de Bruijn graphs. Counting de Bruijn graphs supplement each node-label relation with one or many attributes (e.g., a k-mer count or its positions in genome). To represent them, we first observe that many schemes for the representation of compressed binary matrices already support the rank operation on the columns or rows, which can be used to define an inherent indexing of any additional quantitative attributes. Based on this property, we generalize these schemes and introduce a new approach for representing non-binary sparse matrices in compressed data structures. Finally, we notice that relation attributes are often easily predictable from a node’s local neighborhood in the graph. Notable examples are genome positions shifting by 1 for neighboring nodes in the graph, or expression levels that are often shared across neighbors. We exploit this regularity of graph annotations and apply an invertible delta-like coding to achieve better compression. We show that Counting de Bruijn graphs index k-mer counts from 2,652 human RNA-Seq read sets in representations over 8-fold smaller and yet faster to query compared to state-of-the-art bioinformatics tools. Furthermore, Counting de Bruijn graphs with positional annotations losslessly represent entire reads in indexes on average 27% smaller than the input compressed with gzip -9 for human Illumina RNA-Seq and 57% smaller for PacBio HiFi sequencing of viral samples. A complete joint searchable index of all viral PacBio SMRT reads from NCBI’s SRA (152,884 read sets, 875 Gbp) comprises only 178 GB. Finally, on the full RefSeq collection, they generate a lossless and fully queryable index that is 4.4-fold smaller compared to the MegaBLAST index. The techniques proposed in this work naturally complement existing methods and tools employing de Bruijn graphs and significantly broaden their applicability: from indexing k-mer counts and genome positions to implementing novel sequence alignment algorithms on top of highly compressed and fully searchable graph-based sequence indexes.

Authors Mikhail Karasikov, Harun Mustafa, Gunnar Rätsch, André Kahles

Submitted RECOMB 2022

Link DOI

Abstract We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.

Authors David Danko, Daniela Bezdan, Evan E. Afshin, Sofia Ahsanuddin, Chandrima Bhattacharya, Daniel J. Butler, Kern Rei Chng, Daisy Donnellan, Jochen Hecht, Katelyn Jackson, Katerina Kuchin, Mikhail Karasikov, Abigail Lyons, Lauren Mak, Dmitry Meleshko, Harun Mustafa, Beth Mutai, Russell Y. Neches, Amanda Ng, Olga Nikolayeva, Tatyana Nikolayeva, Eileen Png, Krista A. Ryon, Jorge L. Sanchez, Heba Shaaban, Maria A. Sierra, Dominique Thomas, Ben Young, Omar O. Abudayyeh, Josue Alicea, Malay Bhattacharyya, Ran Blekhman, Eduardo Castro-Nallar, Ana M. Cañas, Aspassia D. Chatziefthimiou, Robert W. Crawford, Francesca De Filippis, Youping Deng, Christelle Desnues, Emmanuel Dias-Neto, Marius Dybwad, Eran Elhaik, Danilo Ercolini, Alina Frolova, Dennis Gankin, Jonathan S. Gootenberg, Alexandra B. Graf, David C. Green, Iman Hajirasouliha, Jaden J.A. Hastings, Mark Hernandez, Gregorio Iraola, Soojin Jang, Andre Kahles, Frank J. Kelly, Kaymisha Knights, Nikos C. Kyrpides, Paweł P. Łabaj, Patrick K.H. Lee, Marcus H.Y. Leung, Per O. Ljungdahl, Gabriella Mason-Buck, Ken McGrath, Cem Meydan, Emmanuel F. Mongodin, Milton Ozorio Moraes, Niranjan Nagarajan, Marina Nieto-Caballero, Houtan Noushmehr, Manuela Oliveira, Stephan Ossowski, Olayinka O. Osuolale, Orhan Özcan, David Paez-Espino, Nicolás Rascovan, Hugues Richard, Gunnar Rätsch, Lynn M. Schriml, Torsten Semmler, Osman U. Sezerman, Leming Shi, Tieliu Shi, Rania Siam, Le Huu Song, Haruo Suzuki, Denise Syndercombe Court, Scott W. Tighe, Xinzhao Tong, Klas I. Udekwu, Juan A. Ugalde, Brandon Valentine, Dimitar I. Vassilev, Elena M. Vayndorf, Thirumalaisamy P. Velavan, Jun Wu, María M. Zambrano, Jifeng Zhu, Sibo Zhu, Christopher E. Mason, The International MetaSUB Consortium

Submitted Cell

Link DOI

Abstract Since the amount of published biological sequencing data is growing exponentially, efficient methods for storing and indexing this data are more needed than ever to truly benefit from this invaluable resource for biomedical research. Labeled de Bruijn graphs are a frequently-used approach for representing large sets of sequencing data. While significant progress has been made to succinctly represent the graph itself, efficient methods for storing labels on such graphs are still rapidly evolving. In this paper, we present RowDiff, a new technique for compacting graph labels by leveraging expected similarities in annotations of vertices adjacent in the graph. RowDiff can be constructed in linear time relative to the number of vertices and labels in the graph, and in space proportional to the graph size. In addition, construction can be efficiently parallelized and distributed, making the technique applicable to graphs with trillions of nodes. RowDiff can be viewed as an intermediary sparsification step of the original annotation matrix and can thus naturally be combined with existing generic schemes for compressed binary matrices. Experiments on 10,000 RNA-seq datasets show that RowDiff combined with Multi-BRWT results in a 30% reduction in annotation footprint over Mantis-MST, the previously known most compact annotation representation. Experiments on the sparser Fungi subset of the RefSeq collection show that applying RowDiff sparsification reduces the size of individual annotation columns stored as compressed bit vectors by an average factor of 42. When combining RowDiff with a Multi-BRWT representation, the resulting annotation is 26 times smaller than Mantis-MST.

Authors Daniel Danciu, Mikhail Karasikov, Harun Mustafa, André Kahles, Gunnar Rätsch

Submitted ISMB/ECCB 2021

Link DOI

Abstract The amount of biological sequencing data available in public repositories is growing exponentially, forming an invaluable biomedical research resource. Yet, making all this sequencing data searchable and easily accessible to life science and data science researchers is an unsolved problem. We present MetaGraph, a versatile framework for the scalable analysis of extensive sequence repositories. MetaGraph efficiently indexes vast collections of sequences to enable fast search and comprehensive analysis. A wide range of underlying data structures offer different practically relevant trade-offs between the space taken by the index and its query performance. MetaGraph provides a flexible methodological framework allowing for index construction to be scaled from consumer laptops to distribution onto a cloud compute cluster for processing terabases to petabases of input data. Achieving compression ratios of up to 1,000-fold over the already compressed raw input data, MetaGraph can represent the content of large sequencing archives in the working memory of a single compute server. We demonstrate our framework’s scalability by indexing over 1.4 million whole genome sequencing (WGS) records from NCBI’s Sequence Read Archive, representing a total input of more than three petabases. Besides demonstrating the utility of MetaGraph indexes on key applications, such as experiment discovery, sequence alignment, error correction, and differential assembly, we make a wide range of indexes available as a community resource, including those over 450,000 microbial WGS records, more than 110,000 fungi WGS records, and more than 20,000 whole metagenome sequencing records. A subset of these indexes is made available online for interactive queries. All indexes created from public data comprising in total more than 1 million records are available for download or usage in the cloud. As an example of our indexes’ integrative analysis capabilities, we introduce the concept of differential assembly, which allows for the extraction of sequences present in a foreground set of samples but absent in a given background set. We apply this technique to differentially assemble contigs to identify pathogenic agents transfected via human kidney transplants. In a second example, we indexed more than 20,000 human RNA-Seq records from the TCGA and GTEx cohorts and use them to extract transcriptome features that are hard to characterize using a classical linear reference. We discovered over 200 trans-splicing events in GTEx and found broad evidence for tissue-specific non-A-to-I RNA-editing in GTEx and TCGA.

Authors Mikhail Karasikov, Harun Mustafa, Daniel Danciu, Marc Zimmermann, Christopher Barber, Gunnar Rätsch, André Kahles

Submitted bioRxiv

Link

Abstract Jaccard Similarity index is an important measure of the overlap of two sets, widely used in machine learning, computational genomics, information retrieval, and many other areas. However, little efforts have been made to develop a scalable and high-performance scheme for computing the Jaccard Similarity for today's large data sets. To address this issue, we design and implement SimilarityAtScale, the first communicationefficient distributed algorithm for computing the Jaccard Similarity. The key idea is to express the problem algebraically, as a sequence of matrix operations, and implement these operations with communication-avoiding distributed routines to minimize the amount of transferred data and ensure both high scalability and low latency. We then apply our algorithm to the problem of obtaining distances between whole-genome sequencing samples, a key part of modern metagenomics analysis and an evergrowing need due to the increasing availability of high-throughput DNA sequencing data. The resulting scheme is the first to enable accurate Jaccard distance derivations for massive datasets, using large-scale distributed-memory systems. We package our routines in a tool, called GenomeAtScale, that combines the proposed algorithm with tools for processing input sequences. Our evaluation on real data illustrates that one can use GenomeAtScale to effectively employ tens of thousands of processors to reach new frontiers in large-scale genomic and metagenomic analysis. While GenomeAtScale can be used to foster DNA research, the more general underlying SimilarityAtScale algorithm may be used for high-performance distributed similarity computations in other data analytics application domains.

Authors Maciej Besta, Raghavendra Kanakagiri, Harun Mustafa, Mikhail Karasikov, Gunnar Rätsch, Torsten Hoefler, Edgar Solomonik

Submitted IPDPS 2020

Link DOI

Abstract High-throughput DNA sequencing data are accumulating in public repositories, and efficient approaches for storing and indexing such data are in high demand. In recent research, several graph data structures have been proposed to represent large sets of sequencing data and to allow for efficient querying of sequences. In particular, the concept of labeled de Bruijn graphs has been explored by several groups. Although there has been good progress toward representing the sequence graph in small space, methods for storing a set of labels on top of such graphs are still not sufficiently explored. It is also currently not clear how characteristics of the input data, such as the sparsity and correlations of labels, can help to inform the choice of method to compress the graph labeling. In this study, we present a new compression approach, Multi-binary relation wavelet tree (BRWT), which is adaptive to different kinds of input data. We show an up to 29% improvement in compression performance over the basic BRWT method, and up to a 68% improvement over the current state-of-the-art for de Bruijn graph label compression. To put our results into perspective, we present a systematic analysis of five different state-of-the-art annotation compression schemes, evaluate key metrics on both artificial and real-world data, and discuss how different data characteristics influence the compression performance. We show that the improvements of our new method can be robustly reproduced for different representative real-world data sets.

Authors Mikhail Karasikov, Harun Mustafa, Amir Joudaki, Sara Javadzadeh-No, Gunnar Rätsch, André Kahles

Submitted Journal of Computational Biology

Link DOI

Abstract Motivation Protein quality assessment (QA) is a crucial element of protein structure prediction, a fundamental and yet open problem in structural bioinformatics. QA aims at ranking predicted protein models to select the best candidates. The assessment can be performed based either on a single model or on a consensus derived from an ensemble of models. The latter strategy can yield very high performance but substantially depends on the pool of available candidate models, which limits its applicability. Hence, single-model QA methods remain an important research target, also because they can assist the sampling of candidate models. Results We present a novel single-model QA method called SBROD. The SBROD (Smooth Backbone-Reliant Orientation-Dependent) method uses only the backbone protein conformation, and hence it can be applied to scoring coarse-grained protein models. The proposed method deduces its scoring function from a training set of protein models. The SBROD scoring function is composed of four terms related to different structural features: residue-residue orientations, contacts between backbone atoms, hydrogen bonding, and solvent-solute interactions. It is smooth with respect to atomic coordinates and thus is potentially applicable to continuous gradient-based optimization of protein conformations. Furthermore, it can also be used for coarse-grained protein modeling and computational protein design. SBROD proved to achieve similar performance to state-of-the-art single-model QA methods on diverse datasets (CASP11, CASP12, and MOULDER). Availability The standalone application implemented in C++ and Python is freely available at https://gitlab.inria.fr/grudinin/sbrod and supported on Linux, MacOS, and Windows.

Authors Mikhail Karasikov, Guillaume Pagès, Sergei Grudinin

Submitted Bioinformatics

Link DOI

Abstract High-throughput DNA sequencing data is accumulating in public repositories, and efficient approaches for storing and indexing such data are in high demand. In recent research, several graph data structures have been proposed to represent large sets of sequencing data and to allow for efficient querying of sequences. In particular, the concept of labeled de Bruijn graphs has been explored by several groups. While there has been good progress towards representing the sequence graph in small space, methods for storing a set of labels on top of such graphs are still not sufficiently explored. It is also currently not clear how characteristics of the input data, such as the sparsity and correlations of labels, can help to inform the choice of method to compress the graph labeling. In this work, we present a new compression approach, Multi-BRWT, which is adaptive to different kinds of input data. We show an up to 29% improvement in compression performance over the basic BRWT method, and up to a 68% improvement over the current state-of-the-art for de Bruijn graph label compression. To put our results into perspective, we present a systematic analysis of five different state-of-the-art annotation compression schemes, evaluate key metrics on both artificial and real-world data and discuss how different data characteristics influence the compression performance. We show that the improvements of our new method can be robustly reproduced for different representative real-world datasets.

Authors Mikhail Karasikov, Harun Mustafa, Amir Joudaki, Sara Javadzadeh-No, Gunnar Rätsch, Andre Kahles

Submitted RECOMB 2019

Link DOI

Abstract Motivation: Technological advancements in high-throughput DNA sequencing have led to an exponential growth of sequencing data being produced and stored as a byproduct of biomedical research. Despite its public availability, a majority of this data remains hard to query for the research community due to a lack of efficient data representation and indexing solutions. One of the available techniques to represent read data is a condensed form as an assembly graph. Such a representation contains all sequence information but does not store contextual information and metadata. Results: We present two new approaches for a compressed representation of a graph coloring: a lossless compression scheme based on a novel application of wavelet tries as well as a highly accurate lossy compression based on a set of Bloom filters. Both strategies retain a coloring even when adding to the underlying graph topology. We present construction and merge procedures for both methods and evaluate their performance on a wide range of different datasets. By dropping the requirement of a fully lossless compression and using the topological information of the underlying graph, we can reduce memory requirements by up to three orders of magnitude. Representing individual colors as independently stored modules, our approaches can be efficiently parallelized and provide strategies for dynamic use. These properties allow for an easy upscaling to the problem sizes common to the biomedical domain. Availability: We provide prototype implementations in C++, summaries of our experiments as well as links to all datasets publicly at https://github.com/ratschlab/graph_annotation.

Authors Harun Mustafa, Ingo Schilken, Mikhail Karasikov, Carsten Eickhoff, Gunnar Rätsch, Andre Kahles

Submitted Bioinformatics

Link DOI

Abstract Much of the DNA and RNA sequencing data available is in the form of high-throughput sequencing (HTS) reads and is currently unindexed by established sequence search databases. Recent succinct data structures for indexing both reference sequences and HTS data, along with associated metadata, have been based on either hashing or graph models, but many of these structures are static in nature, and thus, not well-suited as backends for dynamic databases. We propose a parallel construction method for and novel application of the wavelet trie as a dynamic data structure for compressing and indexing graph metadata. By developing an algorithm for merging wavelet tries, we are able to construct large tries in parallel by merging smaller tries constructed concurrently from batches of data. When compared against general compression algorithms and those developed specifically for graph colors (VARI and Rainbowfish), our method achieves compression ratios superior to gzip and VARI, converging to compression ratios of 6.5% to 2% on data sets constructed from over 600 virus genomes. While marginally worse than compression by bzip2 or Rainbowfish, this structure allows for both fast extension and query. We also found that additionally encoding graph topology metadata improved compression ratios, particularly on data sets consisting of several mutually-exclusive reference genomes. It was also observed that the compression ratio of wavelet tries grew sublinearly with the density of the annotation matrices. This work is a significant step towards implementing a dynamic data structure for indexing large annotated sequence data sets that supports fast query and update operations. At the time of writing, no established standard tool has filled this niche.

Authors Harun Mustafa, Andre Kahles, Mikhail Karasikov, Gunnar Raetsch

Submitted bioRxiv

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