Long-read single-molecule RNA structure sequencing using nanopore.

RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

Accurate analysis of genuine CRISPR editing events with ampliCan.

We present ampliCan, an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, and off-target read detection and quantifies insertions, deletions, HDR repair, as well as targeted base editing. It is scalable to thousands of amplicon sequencing-based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis. We benchmarked ampliCan on both real and simulated data sets against other leading tools, demonstrating that it outperformed all in the face of common confounding factors.

ORFik: a comprehensive R toolkit for the analysis of translation.

BACKGROUND: With the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. RESULTS: Here, we introduce ORFik, a user-friendly R/Bioconductor API and toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5'UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames (uORFs). As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5' UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. CONCLUSION: In summary, ORFik introduces hundreds of tested, documented and optimized methods. ORFik is designed to be easily customizable, enabling users to create complete workflows from raw data to publication-ready figures for several types of sequencing data. Finally, by improving speed and scope of many core Bioconductor functions, ORFik offers enhancement benefiting the entire Bioconductor environment. AVAILABILITY: http://bioconductor.org/packages/ORFik .

tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing.

Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3'-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr's performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.

CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing.

The CRISPR-Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR-Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR-Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.no.

CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering.

In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing. Its overarching principle is to provide an intuitive and powerful tool that can serve both novice and experienced users. In this major update we introduce tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases. We support a number of new features that improve the targeting power, usability and efficiency of CHOPCHOP. To increase targeting range and specificity we provide support for custom length sgRNAs, and we evaluate the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies. These and other new features, coupled with an updated interface for increased usability and support for a continually growing list of organisms, maintain CHOPCHOP as one of the leading tools for CRISPR genome editing. CHOPCHOP v2 can be found at http://chopchop.cbu.uib.no.

RareVariantVis: new tool for visualization of causative variants in rare monogenic disorders using whole genome sequencing data.

MOTIVATION: The search for causative genetic variants in rare diseases of presumed monogenic inheritance has been boosted by the implementation of whole exome (WES) and whole genome (WGS) sequencing. In many cases, WGS seems to be superior to WES, but the analysis and visualization of the vast amounts of data is demanding. RESULTS: To aid this challenge, we have developed a new tool-RareVariantVis-for analysis of genome sequence data (including non-coding regions) for both germ line and somatic variants. It visualizes variants along their respective chromosomes, providing information about exact chromosomal position, zygosity and frequency, with point-and-click information regarding dbSNP IDs, gene association and variant inheritance. Rare variants as well as de novo variants can be flagged in different colors. We show the performance of the RareVariantVis tool in the Genome in a Bottle WGS data set. AVAILABILITY AND IMPLEMENTATION: https://www.bioconductor.org/packages/3.3/bioc/html/RareVariantVis.html CONTACT: tomasz.stokowy@k2.uib.no SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Deep conservation of ribosome stall sites across RNA processing genes.

The rate of translation can vary depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pauses and stalls, but due to library-specific biases, these predictions are often unreliable. Here, we take advantage of the deep conservation of protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important locations of ribosome slowdown, here collectively called stall sites. We analyze multiple ribosome profiling datasets from phylogenetically diverse eukaryotes: yeast, fruit fly, zebrafish, mouse and human to identify conserved stall sites. We find thousands of stall sites across multiple species, with the enrichment of proline, glycine and negatively charged amino acids around conserved stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved role in RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.

CRISPR Genome Editing Made Easy Through the CHOPCHOP Website.

The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on-target editing) with minimal editing at unintended locations (off-target editing). Although laboratory validation should ideally be used to detect off-target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA-DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user-friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock-out, sequence knock-in, and RNA knock-down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of gRNAs for gene knock-out Alternate Protocol 1: Design of gRNAs for dCas9 fusion/effector targeting Support Protocol: Design of gRNAs for targeting transgenic or plasmid sequences Basic Protocol 2: Design of gRNAs for RNA targeting Basic Protocol 3: Design of gRNAs for sequence knock-in Alternate Protocol 2: Design of gRNAs for knock-in using non-homologous end joining Basic Protocol 4: Design of gRNAs for knock-in using Cas9 nickases.

Rapid genome editing by CRISPR-Cas9-POLD3 fusion.

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.