Nature Methods

Article	Citations
ColabFold: making protein folding accessible to all	3199
nnU-Net: a self-configuring method for deep learning-based biomedical image segmentation	2474
Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm	1654
Cellpose: a generalist algorithm for cellular segmentation	1364
Sensitive protein alignments at tree-of-life scale using DIAMOND	1223
Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction	790
Feature-based molecular networking in the GNPS analysis environment	647
Single-cell chromatin state analysis with Signac	591
Martini 3: a general purpose force field for coarse-grained molecular dynamics	553
Macromolecular modeling and design in Rosetta: recent methods and frameworks	499
Method of the Year: spatially resolved transcriptomics	424
metaFlye: scalable long-read metagenome assembly using repeat graphs	424
Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices	407
Benchmarking atlas-level data integration in single-cell genomics	401
diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition	390
Effective gene expression prediction from sequence by integrating long-range interactions	372
Museum of spatial transcriptomics	353
Advances and challenges for fluorescence nanothermometry	341
Squidpy: a scalable framework for spatial omics analysis	303
CryoDRGN: reconstruction of heterogeneous cryo-EM structures using neural networks	299
SpaGCN: Integrating gene expression, spatial location and histology to identify spatial domains and spatially variable genes by graph convolutional network	286
CellRank for directed single-cell fate mapping	284
Spatial omics and multiplexed imaging to explore cancer biology	280
Deep learning and alignment of spatially resolved single-cell transcriptomes with Tangram	279
Next-generation GRAB sensors for monitoring dopaminergic activity in vivo	278

TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines	269
Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells	257
Deciphering molecular interactions by proximity labeling	255
Philosopher: a versatile toolkit for shotgun proteomics data analysis	252
Joint probabilistic modeling of single-cell multi-omic data with totalVI	243
Improvement of cryo-EM maps by density modification	233
Cellpose 2.0: how to train your own model	231
Souporcell: robust clustering of single-cell RNA-seq data by genotype without reference genotypes	227
An optimized acetylcholine sensor for monitoring in vivo cholinergic activity	213
Low-N protein engineering with data-efficient deep learning	212
Evaluation and development of deep neural networks for image super-resolution in optical microscopy	209
The emerging landscape of single-molecule protein sequencing technologies	198
SLEAP: A deep learning system for multi-animal pose tracking	197
DeepSTORM3D: dense 3D localization microscopy and PSF design by deep learning	192
Critical assessment of protein intrinsic disorder prediction	187
High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing	184
Programmable RNA editing with compact CRISPR–Cas13 systems from uncultivated microbes	183
Screening for functional circular RNAs using the CRISPR–Cas13 system	176
SpaceM reveals metabolic states of single cells	170
Spatially resolved transcriptomics adds a new dimension to genomics	169
Joint profiling of histone modifications and transcriptome in single cells from mouse brain	167
Multi-animal pose estimation, identification and tracking with DeepLabCut	164
The power of imaging to understand extracellular vesicle biology in vivo	162
A general method to optimize and functionalize red-shifted rhodamine dyes	156
Exosome detection via the ultrafast-isolation system: EXODUS	153
Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging	153
Meltome atlas—thermal proteome stability across the tree of life	152
Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing	148
A comparison of microfluidic methods for high-throughput cell deformability measurements	148
Predicting 3D genome folding from DNA sequence with Akita	144
Protein structure predictions to atomic accuracy with AlphaFold	144
Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research	144
AlphaFill: enriching AlphaFold models with ligands and cofactors	142
SNT: a unifying toolbox for quantification of neuronal anatomy	141
Benchmarking spatial and single-cell transcriptomics integration methods for transcript distribution prediction and cell type deconvolution	139
Defining the carrier proteome limit for single-cell proteomics	138
Critical Assessment of Metagenome Interpretation: the second round of challenges	136
An expanded palette of dopamine sensors for multiplex imaging in vivo	135
Targeted Perturb-seq enables genome-scale genetic screens in single cells	134
Haplotype-aware variant calling with PEPPER-Margin-DeepVariant enables high accuracy in nanopore long-reads	133
Fast and comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco	132
The triumphs and limitations of computational methods for scRNA-seq	132
Cumulus provides cloud-based data analysis for large-scale single-cell and single-nucleus RNA-seq	131
TRUST4: immune repertoire reconstruction from bulk and single-cell RNA-seq data	131
Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing	129
High-throughput and single-cell T cell receptor sequencing technologies	129
Spatially resolved single-cell genomics and transcriptomics by imaging	129
Ultra-high-throughput single-cell RNA sequencing and perturbation screening with combinatorial fluidic indexing	124
Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals	123
Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels	123

Deep learning enables fast and dense single-molecule localization with high accuracy	122
DeepImageJ: A user-friendly environment to run deep learning models in ImageJ	121
Real-time volumetric reconstruction of biological dynamics with light-field microscopy and deep learning	120
QSIPrep: an integrative platform for preprocessing and reconstructing diffusion MRI data	119
Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT	118
Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures	118
An efficient KRAB domain for CRISPRi applications in human cells	117
Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types	116
LIVECell—A large-scale dataset for label-free live cell segmentation	116
The SEIRS model for infectious disease dynamics	114
Intelligent image-based deformation-assisted cell sorting with molecular specificity	111
Miniature two-photon microscopy for enlarged field-of-view, multi-plane and long-term brain imaging	111
Systematic evaluation of chromosome conformation capture assays	110
Geometric deep learning enables 3D kinematic profiling across species and environments	110
L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma	110
Metabolite discovery through global annotation of untargeted metabolomics data	110
Imaging intact human organs with local resolution of cellular structures using hierarchical phase-contrast tomography	109
SMAP: a modular super-resolution microscopy analysis platform for SMLM data	109
Combining native and ‘omics’ mass spectrometry to identify endogenous ligands bound to membrane proteins	108
Single-molecule FRET imaging of GPCR dimers in living cells	107
DeepC: predicting 3D genome folding using megabase-scale transfer learning	106
Outbreak.info genomic reports: scalable and dynamic surveillance of SARS-CoV-2 variants and mutations	105
The impact of AlphaFold2 one year on	105
A systematic evaluation of the design and context dependencies of massively parallel reporter assays	105
Simultaneous cortex-wide fluorescence Ca2+ imaging and whole-brain fMRI	104
A compact Cascade–Cas3 system for targeted genome engineering	103
DOME: recommendations for supervised machine learning validation in biology	102
Deep learning-based mixed-dimensional Gaussian mixture model for characterizing variability in cryo-EM	101
A practical guide to cancer subclonal reconstruction from DNA sequencing	101
METLIN MS2 molecular standards database: a broad chemical and biological resource	101
Massively parallel and time-resolved RNA sequencing in single cells with scNT-seq	100
A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects	99
Deep learning-based point-scanning super-resolution imaging	97
Reproducibility standards for machine learning in the life sciences	97
Lineage recording in human cerebral organoids	97
3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing	97
Genetic tool development in marine protists: emerging model organisms for experimental cell biology	96
MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging	96
gEAR: Gene Expression Analysis Resource portal for community-driven, multi-omic data exploration	95
Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes	94
FlyWire: online community for whole-brain connectomics	94
The ENIGMA Toolbox: multiscale neural contextualization of multisite neuroimaging datasets	94
O-Pair Search with MetaMorpheus for O-glycopeptide characterization	94
Time-resolved cryo-EM using Spotiton	92
DeepLC can predict retention times for peptides that carry as-yet unseen modifications	92
Click-ExM enables expansion microscopy for all biomolecules	91
High-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy	91
US-align: universal structure alignments of proteins, nucleic acids, and macromolecular complexes	89
ZipSeq: barcoding for real-time mapping of single cell transcriptomes	89
DeepCell Kiosk: scaling deep learning–enabled cellular image analysis with Kubernetes	89
GPCRmd uncovers the dynamics of the 3D-GPCRome	88
Putative cell type discovery from single-cell gene expression data	88
Plasmonic scattering imaging of single proteins and binding kinetics	87
Deep learning improves macromolecule identification in 3D cellular cryo-electron tomograms	86
Three-photon head-mounted microscope for imaging deep cortical layers in freely moving rats	85
Live-cell super-resolved PAINT imaging of piconewton cellular traction forces	84
MARS: discovering novel cell types across heterogeneous single-cell experiments	83
CRISPR-assisted detection of RNA–protein interactions in living cells	83
Understudied proteins: opportunities and challenges for functional proteomics	83
Discovering multiple types of DNA methylation from bacteria and microbiome using nanopore sequencing	83
neuromaps: structural and functional interpretation of brain maps	81
Systematic investigation of cytokine signaling activity at the tissue and single-cell levels	80
Long-read mapping to repetitive reference sequences using Winnowmap2	80
Reproducible, scalable, and shareable analysis pipelines with bioinformatics workflow managers	79
Scanless two-photon excitation with temporal focusing	79
Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM	78
Precise, fast and comprehensive analysis of intact glycopeptides and modified glycans with pGlyco3	78
OME-NGFF: a next-generation file format for expanding bioimaging data-access strategies	78
Optimizing imaging speed and excitation intensity for single-molecule localization microscopy	77
ReDU: a framework to find and reanalyze public mass spectrometry data	77
Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes	77
SEAM is a spatial single nuclear metabolomics method for dissecting tissue microenvironment	77
High-definition imaging using line-illumination modulation microscopy	75
Automatic detection of synaptic partners in a whole-brain Drosophila electron microscopy data set	75
Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles	74
Deep learning-enhanced light-field imaging with continuous validation	74
Human organoid models to study SARS-CoV-2 infection	73
StrucGP: de novo structural sequencing of site-specific N-glycan on glycoproteins using a modularization strategy	73
Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge	73
A ‘Build and Retrieve’ methodology to simultaneously solve cryo-EM structures of membrane proteins	72

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells	72
Spectral entropy outperforms MS/MS dot product similarity for small-molecule compound identification	72
Method of the year: long-read sequencing	71
T cell antigen discovery	71
Optogenetic control of gene expression in plants in the presence of ambient white light	71
The GlyCosmos Portal: a unified and comprehensive web resource for the glycosciences	71
Modeling infectious epidemics	71
High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy	70
Alignment and integration of spatial transcriptomics data	70
Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis	70
Emu: species-level microbial community profiling of full-length 16S rRNA Oxford Nanopore sequencing data	70
Deep learning and protein structure modeling	70
Genome-scale deconvolution of RNA structure ensembles	69
Reinforcing neuron extraction and spike inference in calcium imaging using deep self-supervised denoising	69
Method of the Year 2020: spatially resolved transcriptomics	69
CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning	68
Spatially resolved isotope tracing reveals tissue metabolic activity	68
Genome-wide detection of enhancer-hijacking events from chromatin interaction data in rearranged genomes	68
Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines	68
SCENIC+: single-cell multiomic inference of enhancers and gene regulatory networks	68
Joint single-cell measurements of nuclear proteins and RNA in vivo	68
Supervised enhancer prediction with epigenetic pattern recognition and targeted validation	68
Mid-infrared metabolic imaging with vibrational probes	67
Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval	67
MassIVE.quant: a community resource of quantitative mass spectrometry–based proteomics datasets	67
High-sensitivity and high-specificity biomechanical imaging by stimulated Brillouin scattering microscopy	66
Engineered HaloTag variants for fluorescence lifetime multiplexing	66
Efficient targeted insertion of large DNA fragments without DNA donors	66
Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ	65
Omnipose: a high-precision morphology-independent solution for bacterial cell segmentation	65
Unsupervised removal of systematic background noise from droplet-based single-cell experiments using CellBender	65
Highly specific multiplexed RNA imaging in tissues with split-FISH	64
ScanNet: an interpretable geometric deep learning model for structure-based protein binding site prediction	64
REMBI: Recommended Metadata for Biological Images—enabling reuse of microscopy data in biology	64
Best practices and tools for reporting reproducible fluorescence microscopy methods	64
Deep three-photon imaging of the brain in intact adult zebrafish	63
Challenges in benchmarking metagenomic profilers	62
Identifying temporal and spatial patterns of variation from multimodal data using MEFISTO	62
Spatially resolved transcriptomics in neuroscience	61
Screening cell–cell communication in spatial transcriptomics via collective optimal transport	61
Mechanistic modeling of chromatin folding to understand function	61
Understanding the invisible hands of sample preparation for cryo-EM	61
DaXi—high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy	61
Quantum computing at the frontiers of biological sciences	61
Single-cell proteomics enabled by next-generation sequencing or mass spectrometry	60
Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors	59
Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome	59
Best practice standards for circular RNA research	58
Directed evolution of adeno-associated virus for efficient gene delivery to microglia	58
Impact of phosphorylation on thermal stability of proteins	58
Removing independent noise in systems neuroscience data using DeepInterpolation	57
Improved AlphaFold modeling with implicit experimental information	57
Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions	57
Avoiding a replication crisis in deep-learning-based bioimage analysis	57
A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues	56
NEAT-seq: simultaneous profiling of intra-nuclear proteins, chromatin accessibility and gene expression in single cells	56
Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution	56
Transgenic mice for in vivo epigenome editing with CRISPR-based systems	55
Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers	54
Pycro-Manager: open-source software for customized and reproducible microscope control	54
Metagenome assembly of high-fidelity long reads with hifiasm-meta	54
CODA: quantitative 3D reconstruction of large tissues at cellular resolution	53
Miniaturized head-mounted microscope for whole-cortex mesoscale imaging in freely behaving mice	53
Single-cell metabolomics hits its stride	53
Automated segmentation and tracking of mitochondria in live-cell time-lapse images	53
Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging	53
Mapping the functional landscape of T cell receptor repertoires by single-T cell transcriptomics	52
Animal models in SARS-CoV-2 research	52
VDJdb in the pandemic era: a compendium of T cell receptors specific for SARS-CoV-2	52
Image-guided MALDI mass spectrometry for high-throughput single-organelle characterization	52
RefMet: a reference nomenclature for metabolomics	51
EthoLoop: automated closed-loop neuroethology in naturalistic environments	51
Real-time 3D movement correction for two-photon imaging in behaving animals	50
Detection of m6A from direct RNA sequencing using a multiple instance learning framework	50
Cell surface thermal proteome profiling tracks perturbations and drug targets on the plasma membrane	50
Parallel cryo electron tomography on in situ lamellae	49
Investigating higher-order interactions in single-cell data with scHOT	49
A genetically encoded sensor for in vivo imaging of orexin neuropeptides	49
Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging	48
Isotropic super-resolution light-sheet microscopy of dynamic intracellular structures at subsecond timescales	48
Homogeneous multifocal excitation for high-throughput super-resolution imaging	48
MSNovelist: de novo structure generation from mass spectra	48
Quantitative shotgun proteome analysis by direct infusion	48
Elasticizing tissues for reversible shape transformation and accelerated molecular labeling	48
Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions	48
Challenges and opportunities for the next generation of cardiovascular tissue engineering	47
Metabolomic profiling of single enlarged lysosomes	47
Universal Spectrum Identifier for mass spectra	47
Illumination guidelines for ultrafast pump–probe experiments by serial femtosecond crystallography	47
A pan-tissue DNA methylation atlas enables in silico decomposition of human tissue methylomes at cell-type resolution	47
3DFlex: determining structure and motion of flexible proteins from cryo-EM	46
Advances in nanopore direct RNA sequencing	46
An adaptive optics module for deep tissue multiphoton imaging in vivo	45
jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths	44
Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library	44