The nonsense-mediated decay (NMD) surveillance pathway can recognize erroneous transcripts and physiological mRNAs, such as precursor mRNA alternative splicing (AS) variants. Currently, information on the global extent of coupled AS and NMD remains scarce and even absent for any plant species. To address this, we conducted transcriptome-wide splicing studies using Arabidopsis thaliana mutants in the NMD factor homologs UP FRAMESHIFT1 (UPF1) and UPF3 as well as wild-type samples treated with the translation inhibitor cycloheximide. Our analyses revealed that at least 17.4% of all multi-exon, protein-coding genes produce splicing variants that are targeted by NMD. Moreover, we provide evidence that UPF1 and UPF3 act in a translation-independent mRNA decay pathway. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. Genes generating NMD-sensitive AS variants function in diverse biological processes, including signaling and protein modification, for which NaCl stress-modulated AS-NMD was found. Besides mRNAs, numerous noncoding RNAs and transcripts derived from intergenic regions were shown to be NMD responsive. In summary, we provide evidence for a major function of AS-coupled NMD in shaping the Arabidopsis transcriptome, having fundamental implications in gene regulation and quality control of transcript processing.
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 compounds2,3. 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.
Motivation: Understanding the occurrence and regulation of alternative splicing (AS) is a key task towards explaining the regulatory processes that shape the complex transcriptomes of higher eukaryotes. With the advent of high-throughput sequencing of RNA (RNA-Seq), the diversity of AS transcripts could be measured at an unprecedented depth. Although the catalog of known AS events has grown ever since, novel transcripts are commonly observed when working with less well annotated organisms, in the context of disease, or within large populations. Whereas an identification of complete transcripts is technically challenging and computationally expensive, focusing on single splicing events as a proxy for transcriptome characteristics is fruitful and sufficient for a wide range of analyses. Results: We present SplAdder, an alternative splicing toolbox, that takes RNA-Seq alignments and an annotation file as input to (i) augment the annotation based on RNA-Seq evidence, (ii) identify alternative splicing events present in the augmented annotation graph, (iii) quantify and confirm these events based on the RNA-Seq data and (iv) test for significant quantitative differences between samples. Thereby, our main focus lies on performance, accuracy and usability. Availability: Source code and documentation are available for download at http://github.com/rats chlab/spladder. Example data, introductory information and a small tutorial are accessible via
Sequencing data are 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 building compressed representations of annotated (or colored) de Bruijn graphs for efficiently indexing k-mer sets. However, approaches for representing quantitative attributes such as gene expression or genome positions in a general manner have remained underexplored. In this work, we propose counting de Bruijn graphs, a notion generalizing annotated de Bruijn graphs by supplementing each node–label relation with one or many attributes (e.g., a k-mer count or its positions). Counting de Bruijn graphs index k-mer abundances from 2652 human RNA-seq samples in over eightfold smaller representations compared with state-of-the-art bioinformatics tools and is faster to construct and query. Furthermore, counting de Bruijn graphs with positional annotations losslessly represent entire reads in indexes on average 27% smaller than the input compressed with gzip for human Illumina RNA-seq and 57% smaller for Pacific Biosciences (PacBio) HiFi sequencing of viral samples. A complete searchable index of all viral PacBio SMRT reads from NCBI's Sequence Read Archive (SRA) (152,884 samples, 875 Gbp) comprises only 178 GB. Finally, on the full RefSeq collection, we generate a lossless and fully queryable index that is 4.6-fold smaller than the MegaBLAST index. The techniques proposed in this work naturally complement existing methods and tools using 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 graph-based sequence indexes.
We present an algorithm for the optimal alignment of sequences to genome graphs. It works by phrasing the edit distance minimization task as finding a shortest path on an implicit alignment graph. To find a shortest path, we instantiate the A paradigm with a novel domain-specific heuristic function that accounts for the upcoming subsequence in the query to be aligned, resulting in a provably optimal alignment algorithm called AStarix. Experimental evaluation of AStarix shows that it is 1-2 orders of magnitude faster than state-of-the-art optimal algorithms on the task of aligning Illumina reads to reference genome graphs. Implementations and evaluations are available at https://github.com/eth-sri/astarix.
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.
Structural Rearrangements can have unexpected effects on quantitative phenotypes. Surprisingly, these rearrangements can also be considered as...
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.
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