Method of the Year 2022: long-read sequencing
Long-read sequencing powers a more complete reading of genomic information.
Long-read sequencing powers a more complete reading of genomic information.
Many scientists are active on social media, especially Twitter. The social media world is changing, but these researchers want to stay socially connected.
Planaria are a group of worms within the phylum Platyhelminthes (flatworms). Many species, including Schmidtea mediterranea, have the ability to regenerate their body from small pieces of tissue and are easy to keep in the laboratory, which makes them a prime model system for studying whole-body regeneration.
To large-scale projects and individual labs, long-read sequencing has delivered new vistas and long wish lists for this technology’s future.
The year 2022 will be remembered as the turning point for accurate long-read sequencing, which now establishes the gold standard for speed and accuracy at competitive costs. We discuss the key bioinformatics techniques needed to power long reads across application areas and close with our vision for long-read sequencing over the coming years.
Advances in long-read sequencing technologies have broadened our understanding of genetic variation in the human population, uncovered new complex structural variants and offered an opportunity to elucidate new variant associations with disease.
Long-read sequencing has become a widely employed technology that enables a comprehensive view of RNA transcripts. Here, we discuss the importance of long-read sequencing in interpreting the variables along RNA molecules, such as polyadenylation sites, transcription start sites, splice sites and other RNA modifications. In addition, we highlight the history of short-read and long-read technologies and their advantages and disadvantages, as well as future directions in the field.
As long-read sequencing technologies continue to advance, the possibility of obtaining maps of DNA and RNA modifications at single-molecule resolution has become a reality. Here we highlight the opportunities and challenges posed by the use of long-read sequencing technologies to study epigenetic and epitranscriptomic marks and how this will affect the way in which we approach the study of health and disease states.
Long-read sequencing has made closed microbial genomes a routine task, and the dramatic increase in quality and quantity now paves the way to a complete microbial tree of life through genome-centric metagenomics.
In vitro models and single-cell atlases help uncover the mysteries of human embryogenesis.
The combination of microscopy, targeted illumination and single-cell sequencing is driving applications from direct evolution to spatial omics.
CRISPR systems can be leveraged to direct recombinases to integrate gene-sized DNA sequences.
Computational approaches are pushing the limits of unknown metabolite annotation.
Data integration methods weave genomics data into holistic pictures of biological systems.
Strategies to overcome poor reproducibility in MRI studies are needed.
Advances in fluorescence microscopy and spectroscopy show their promise for applications that complement in situ structural biology methods like cryoelectron tomography.
Mechanoimmunology methods help dissect the mechanical forces within the immune system.
Spatially resolved and single-cell transcriptomics provide insights into tumor heterogeneity and tumor–microbiome interactions.
During the first two years of postnatal development, the human brain undergoes rapid, pronounced changes in size, shape and content. Using high-resolution MRI, we constructed month-to-month atlases of infants 2 weeks to 2 years old, capturing key spatiotemporal traits of early brain development in terms of cortical geometries and tissue properties.
We trained DEDAL, an algorithm based on deep-learning language models, to generate pairwise alignments of protein sequences taking into account the sequence-specific context of amino acid substitutions or gaps. DEDAL improved the alignment correctness on remote homologs by up to threefold and the discrimination of remote homologs from evolutionarily unrelated sequences.
To accelerate data acquisition for in situ cryo-electron tomography, we created a method that takes into consideration sample geometry for the robust prediction of sample movement while the microscope stage is tilted. This approach enabled the parallel collection of tens to hundreds of tilt series.
Localization Model Fit (LocMoFit) is a tool that enables fitting of super-resolution microscopy data to an arbitrary geometric model. The fit extracts quantitative parameters of individual cellular structures, which can be used to investigate dynamic and heterogenous protein assemblies and to create average protein distribution maps.
We engineered a 3D outer-blood-retina-barrier (3D-oBRB) with a fully polarized retinal pigment epithelium (RPE) monolayer on top of a Bruch’s membrane and a fenestrated choriocapillaris network. This 3D-oBRB tissue faithfully recapitulates RPE– choriocapillaris interactions, dry age-related macular degeneration (AMD) phenotypes (including sub-RPE drusen deposits and choriocapillaris degeneration) and the wet AMD phenotype of choriocapillaris neovascularization.
This Resource presents surface and volume atlases of human brain development during early infancy, at monthly intervals.
Antibody-barcode eCLIP (ABC) uses proximity ligation to couple DNA-barcoded antibodies to RNA-binding protein (RBP)-protected RNA fragments for multiplexed eCLIP. ABC can be used to interrogate several RBPs in a single tube with results on par with eCLIP.
miRFP718nano is a rationally designed small near-infrared fluorescent protein with an emission tail that extends into the short-wave infrared range for improved multiplexed and deep-tissue imaging applications.
Nano3P-seq presents a nanopore-based sequencing tool to profile polyA-tailed and non-polyA-tailed transcripts, as well as capture polyA tail length and composition.
This work introduces CaRPool-seq that utilizes the RNA-targeting CRISPR–Cas13d system to perform combinatorial perturbations in single-cell screens.
Virus-assisted directed evolution of tRNAs (VADER) enables selection of improved suppressor tRNAs in mammalian cells. Improved pyrrolysyl- and tyrosyl-tRNA variants enhance unnatural amino acid incorporation in mammalian systems.
DEDAL is a deep learning-based protein sequence alignment method that improves the quality of predicted alignment for remote homologs and better discriminates remote homologs from evolutionarily unrelated sequences.
Chemically activated protein domains are chemogenetic tools that inhibit the activity of short peptides in the absence of a small molecule. Their versatility was demonstrated on a range of peptides and in both cells and mice.
This paper shows that the uniformity of vitreous ice thickness relies on the surface flatness of the supporting film, and presents a method to use ultraflat graphene as the support for cryo-EM specimen preparation.
The parallel cryo electron tomography (PACE-tomo) method increases the throughput on in situ samples by parallelizing acquisition. It maximizes the usable sample area on individual lamellae without compromising data quality.
Localization Model Fit (LocMoFit) is an open-source tool for extracting meaningful parameters from individual structures in localization microscopy data. The framework was used for quantitative analysis of diverse biological structures.
A physiologically relevant 3D model of the outer-blood-retina-barrier recapitulates healthy and disease phenotypes.