Understanding RNA conformational dynamics is essential to understand its roles in complex biological processes. While computational methods have revolutionized the prediction of static 3D RNA structures, predicting local flexibility directly from structure remains a significant challenge. We developed DeepRMSF, a deep learning-based method that leverages atomic-level descriptions of RNA to predict vibrational flexibility given a tertiary structure. Trained on MD-derived root-mean-square fluctuations(RMSF), DeepRMSF was benchmarked on 371 nonredundant RNAs, with 311 RNAs used for five-fold cross-validation (PCC = 0.7219-0.7464) and 60 RNAs as an independent test set (PCC = 0.734), ensuring minimal sequence/structural similarity between sets. DeepRMSF predicts the local flexibility of medium-sized RNAs (~75 nucleotides) in ~8.2 s, achieving >3000-fold speed-up over MD simulations while maintaining strong extrapolative accuracy. Rather than replacing MD, DeepRMSF offers a scalable and practical alternative for transcriptome-scale screening of RNA flexibility, facilitating studies on RNA structure-dynamics-function relationships and supporting computational modeling in RNA biology.

SUMMARY: Cryo-electron microscopy (cryo-EM) visualization transforms high-resolution 3D density volumes into intuitive representations, playing a vital role in structural biology. However, the increasing scale of cryo-EM data poses challenges for interactive visualization, including storage, transmission, and exploration. To address this, we propose a hybrid quantized implicit neural representation (INR) method that compresses cryo-EM volumes while supporting efficient on-demand access. To evaluate its effectiveness, we benchmark our approach against traditional compression techniques and one classic INR compressor, assessing both compression efficiency and visual quality. Beyond standard metrics, we examine performance on key cryo-EM tasks, including overall structure identification, secondary structure recognition, and fine-chain inspection. Our results demonstrate that the quantized INR achieves superior storage efficiency and task-relevant fidelity, and we provide an interactive tool and guidelines to assist users in selecting optimal compression strategies. AVAILABILITY: To facilitate future research, we provide our quantized neural representation approach and interactive tool available at Zenodo (https://doi.org/10.5281/zenodo.19688284) and GitHub (https://github.com/ChiefMoo/Lossy-Cryo-EM-Compression).

MOTIVATION: Serial section electron microscopy (ssEM) is essential for studying biological cell structures at nanometer resolution. However, supporting film folding (SFF) degradation frequently occurs during sample preparation, causing structural distortions and information loss that severely impair downstream analyses such as 3D reconstruction and neuron segmentation. RESULTS: We propose RegInpaint, a novel recovery framework that jointly addresses deformation correction and missing-information restoration caused by SFF degradation. RegInpaint formulates SFF recovery as a joint problem of 3D elastic registration and image inpainting, providing a generalizable solution for ssEM restoration. Experiments on four EM datasets show that RegInpaint consistently outperforms existing methods in image restoration quality and significantly improves neuron segmentation accuracy. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://github.com/zhangzhenbang2021/RegInpaint.git.

MOTIVATION: With the advancement of cryo-electron microscopy (cryo-EM) into the atomic resolution era, accurate Cα atom modeling has become essential for macromolecular structure determination. However, existing evaluation systems overly rely on full-atom metrics and lack a dedicated, comprehensive benchmark for assessing Cα prediction modules within automated modeling tools. RESULTS: To address this gap, we establish a rigorous benchmark to evaluate the Cα prediction performance of four prominent deep learning-based methods (ModelAngelo, DeepMainMast, EModelX, and CryoAtom) across multiple dimensions. We construct a diverse dataset covering a wide range of resolutions (1-8 Å), molecular weights, and noise levels. A novel evaluation framework is introduced, incorporating multi-threshold RMSD-based metrics (1-3 Å) alongside advanced point-cloud similarity measures (Chamfer Distance, Earth Mover's Distance) for quantitative and nuanced assessment. Our results reveal that method performance is highly dependent on the chosen evaluation criteria and intrinsic data characteristics. ModelAngelo excels under loose thresholds with high-quality data but shows sensitivity to resolution degradation; CryoAtom demonstrates notable computational efficiency, however, its completeness-oriented design leads to a certain loss of precision; EModelX demonstrates balanced generalization across varied conditions; DeepMainMast achieves high localization accuracy under stringent criteria but incurs a high computational cost. AVAILABILITY AND IMPLEMENTATION: This work provides a reproducible, Cα-centric evaluation framework to guide method development and advance automated cryo-EM structure determination. The source code for the benchmark and evaluation metrics is freely available at https://github.com/zhtianz/Benchmarking\_CA.

Series Section Electron Microscopy (ssEM) is a crucial technique for visualizing three-dimensional (3D) biological structures, which involves collecting electron microscopy images from a series of biological sections along the z-axis and reconstructing the 3D structure. 3D registration is an essential step in ssEM, designed to eliminate axial misalignment and nonlinear distortions introduced during sample sectioning. A significant challenge in 3D registration is eliminating nonlinear distortions while preserving natural deformations. In this paper, we present a new formulation of the 3D registration problem from a frequency domain perspective and propose a Gaussian filtering-based 3D registration method, which defines 3D registration as a superposition problem of high-frequency and low-frequency components. We extend the concept of a one-dimensional Gaussian filter to three-dimensional image stacks and integrate it with optical flow networks to consolidate the deformation field within the receptive field. Extensive experiments demonstrate that our method can successfully decouple nonlinear distortions and natural deformations in the frequency domain, proving superior to existing methods in rapidly and accurately eliminating nonlinear distortions and restoring biological structures, and has the potential to be extended to large datasets.

Accurate parameter estimation is paramount for high-fidelity three-dimensional (3D) reconstruction in cryo-electron tomography (Cryo-ET), a pivotal technique for visualizing the intricate structures of biological macromolecules and cellular components in their near-native state. The alignment of the image sequence acquired at varying tilt angles constitutes a critical bottleneck in this process, directly governing reconstruction accuracy. While bundle adjustment (BA) serves as the computational cornerstone for parameter refinement, its conventional implementation via the Levenberg-Marquardt (L-M) algorithm exhibits critical limitations: sensitivity to initialization causes slow convergence and oscillatory behavior in noisy Cryo-ET datasets. To address this limitation and enhance parameter optimization robustness, this paper introduces a novel algorithm, leveraging principles from optimal control theory to directly optimize the nonlinear BA objective function. The optimal control principle (OCP) method demonstrates superior convergence characteristics compared to the L- M algorithm, effectively mitigating oscillations and achieving faster convergence rates. Extensive experiments on both synthetic and real-world Cryo-ET datasets validate the algorithm's performance. Results demonstrate that the OCP consistently achieves faster convergence than the L-M algorithm. Furthermore, incorporating a bisection-based update procedure for the OCP's control weight matrix significantly enhances its performance, particularly under conditions of poor initialization. These findings establish the OCP as a powerful tool that can substantially improve the efficiency and robustness of parameter optimization, thereby accelerating high-resolution 3D reconstructions in Cryo-ET.

Series Section Electron Microscopy (ssEM) has emerged as a pivotal technology for deciphering nanoscale biological architectures. Three-dimensional (3D) registration is a critical step in ssEM, tasked with rectifying axial misalignments and nonlinear distortions introduced during serial sectioning. The core scientific challenge lies in achieving distortion mitigation without erasing the natural morphological deformations of biological tissues, thereby enabling faithful reconstruction of 3D ultrastructural organization. In this study, we present a paradigm-shifting optimization framework that rethinks 3D registration through the lens of manifold trajectory optimization. We propose the first continuous trajectory dynamics formulation for 3D registration and introduce a novel optimization strategy. Specifically, we introduce a dual optimization objective that inherently balances global trajectory smoothness with local structural preservation, while developing a solver that combines Gauss-Seidel iteration with Neural ODEs to systematically integrate biophysical priors with data-driven deformation compensation. A key strength of our method is its fully unsupervised training, which avoids reliance on ground truth and suits ssEM scenarios where annotations are difficult to obtain. Extensive experiments on multiple datasets spanning diverse tissue types demonstrate our method's superior performance in structural restoration accuracy and cross-tissue robustness.

Few-shot learning is a crucial approach for macromolecule classification of the cryo-electron tomography (Cryo-ET) subvolumes, enabling rapid adaptation to novel tasks with a small support set of labeled data. However, existing few-shot classification methods for macromolecules in Cryo-ET consider only marginal distributions and overlook joint distributions, failing to capture feature dependencies fully. To address this issue, we propose a method for macromolecular few-shot classification using deep Brownian Distance Covariance (BDC). Our method models the joint distribution within a transfer learning framework, enhancing the modeling capabilities. We insert the BDC module after the feature extractor and only train the feature extractor during the training phase. Then, we enhance the model's generalization capability with self-distillation techniques. In the adaptation phase, we fine-tune the classifier with minimal labeled data. We conduct experiments on publicly available SHREC datasets and a small-scale synthetic dataset to evaluate our method. Results show that our method improves the classification capabilities by introducing the joint distribution.

Automatic single particle picking is a critical step in the data processing pipeline of cryo-electron microscopy structure reconstruction. In recent years, several deep learning-based algorithms have been developed, demonstrating their potential to solve this challenge. However, current methods highly depend on manually labeled training data, which is labor-intensive and prone to biases especially for high-noise and low-contrast micrographs, resulting in suboptimal precision and recall. To address these problems, we propose UPicker, a semi-supervised transformer-based particle-picking method with a two-stage training process: unsupervised pretraining and supervised fine-tuning. During the unsupervised pretraining, an Adaptive Laplacian of Gaussian region proposal generator is proposed to obtain pseudo-labels from unlabeled data for initial feature learning. For the supervised fine-tuning, UPicker only needs a small amount of labeled data to achieve high accuracy in particle picking. To further enhance model performance, UPicker employs a contrastive denoising training strategy to reduce redundant detections and accelerate convergence, along with a hybrid data augmentation strategy to deal with limited labeled data. Comprehensive experiments on both simulated and experimental datasets demonstrate that UPicker outperforms state-of-the-art particle-picking methods in terms of accuracy and robustness while requiring fewer labeled data than other transformer-based models. Furthermore, ablation studies demonstrate the effectiveness and necessity of each component of UPicker. The source code and data are available at https://github.com/JachyLikeCoding/UPicker.

MOTIVATION: Cryo-electron tomography (cryo-ET) has revolutionized our ability to observe structures from the subcellular to the atomic level in their native states. Achieving high-resolution reconstruction involves collecting tilt series at different angles and subsequently backprojecting them into 3D space or iteratively reconstructing them to build a 3D volume of the specimen. However, the intricate computational demands of tomographic reconstruction pose significant challenges, requiring extensive calculation times that hinder efficiency, especially with large and complex datasets. RESULTS: We present TiltRec, an open-source toolkit that leverages the parallel capabilities of Central Processing Units and Graphics Processing Units to enhance tomographic reconstruction. TiltRec implements six classical tomographic reconstruction algorithms, utilizing optimized parallel computation strategies and advanced memory management techniques. Performance evaluations across multiple datasets of varying sizes demonstrate that TiltRec significantly improves efficiency, reducing computational times while maintaining reconstruction resolution. SUMMARY: TiltRec effectively addresses the computational challenges associated with cryo-ET reconstruction by fully exploiting parallel acceleration. As an open-source tool, TiltRec not only facilitates extensive applications by the research community but also supports further algorithm modifications and extensions, enabling the continued development of novel algorithms. AVAILABILITY AND IMPLEMENTATION: The source code, documentation, and sample data can be downloaded at https://github.com/icthrm/TiltRec.

MOTIVATION: With the rapid advancements in Cryo-Electron Microscopy (Cryo-EM), an increasing number of high-resolution 3D density maps are being made publicly available, highlighting the urgent need for efficient structure similarity retrieval. Exploring map similarity at various levels is critical for fully utilizing these valuable resources. Our previously proposed CryoAlign can provide more accurate density map alignment while maintaining a low failure rate. However, CryoAlign only offers a method for aligning density maps, with low efficiency in local alignment, and has not yet been applied to the retrieval of Cryo-EM density maps. RESULTS: We have developed an alignment-based retrieval tool to perform both global and local retrieval. Our approach adopts parallel-accelerated CryoAlign for high-precision 3D alignment and transforms density maps into point clouds for efficient retrieval and storage. Additionally, a multi-dimension scoring function is introduced to accurately assess structural similarities between superimposed density maps. To demonstrate its applicability, we conducted thorough testing across different retrieval tasks, such as global, local or hybrid similarity retrieval. Our tool achieves up to a 7-fold speedup while supporting precise local alignments. Comprehensive experiments demonstrate that even when one density map is entirely contained within another, our tool performs exceptionally well in high-resolution density map retrieval. It provides researchers with an efficient and accurate solution for density map similarity search. AVAILABILITY AND IMPLEMENTATION: The source code, documentation, and sample data can be downloaded at https://github.com/JokerL2/CryoAlign2.

MOTIVATION: Cryogenic electron tomography (cryo-ET) enables high-resolution 3D reconstruction of biological samples, with accurate subtomogram classification critical for structural analysis. However, current subtomogram classification methods often struggle with out-of-distribution (OOD) data issue, causing misclassification and mismatched structures. RESULTS: To solve this problem, we propose a unified subtomogram classification framework that incorporates OOD detection to distinguish unknown (OOD) from known (in-distribution, ID) classes and predict labels for ID data, thereby enhancing existing subtomogram classification methods. Within this framework, we develop a noise-robust classification method that integrates a 3D discrete wavelet transform-based encoder to reduce high-frequency noise and extract robust features. Additionally, we incorporate a Mahalanobis distance-based OOD detector with a reliable metric for 3D subtomograms and introduce an adaptive classifier that adjusts to accommodate datasets of varying scales. The experimental and visualization results demonstrate that our noise-robust method improves subtomogram classification accuracy and effectively models features while enhancing OOD detection. AVAILABILITY AND IMPLEMENTATION: Our code is available at https://github.com/yxs1137/Subtomo-Classification-with-OOD.git. The real data used in this study can be accessed through CryoET Data Portal.

MOTIVATION: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss and cognitive decline. While graph convolutional networks (GCNs) have emerged as popular tools for AD diagnosis due to their ability to handle structural information and fuse multi-modal features, deep learning approaches face significant challenges including the requirement for large datasets and sensitivity to unbalanced label distributions in AD research. To address these limitations and enhance the flexibility of GCNs, we propose a graph convolutional network based on the meta-learning paradigm (ADMGCN) for early AD diagnosis. This approach incorporates weighting and dimensionality reduction to improve performance, storage, and training efficiency. By leveraging meta-learning, we sample subjects to create numerous label-balanced tasks, maximizing data utilization and mitigating the impact of label imbalance. Additionally, the meta-learning framework enables rapid adaptation to new tasks and facilitates independent testing of the GCN. RESULTS: Our model, ADMGCN, was extensively validated on the Alzheimer's Disease Neuroimaging Initiative datasets. It achieved a maximum accuracy of 73.7% in the multi-classification task for early AD diagnosis. In three binary classification tasks, the model also demonstrated strong performance, achieving accuracies of 92.8%, 88.0%, and 79.6%, respectively. These results confirm that the proposed method provides an effective approach and worthwhile support for the early diagnosis of Alzheimer's disease. AVAILABILITY AND IMPLEMENTATION: ADMGCN is freely available at https://github.com/WendySun16/ADMGCN.

Volume electron microscopy (vEM) is becoming a prominent technique in three-dimensional (3D) cellular visualization. vEM collects a series of two-dimensional (2D) images and reconstructs ultrastructures at the nanometer scale by rational axial interpolation between neighboring sections. However, section damage inevitably occurs in the sample preparation and imaging process, suffering from manual operational errors or occasional mechanical failures. The damaged regions present blurry and contaminated structure information, even local blank holes. Despite significant progress in single-image inpainting, it is still a great challenge to recover missing biological structures, that satisfy 3D structural continuity among sections. In this paper, we propose an optical flow-based serial section inpainting architecture to effectively combine the 3D structure information from neighboring sections and 2D image features from surrounding regions. We design a two-stage reference generation strategy to predict a rational and detailed intermediate state image from coarse to fine. Then, a GAN-based inpainting network is adopted to integrate all reference information and guide the restoration of missing structures, while ensuring consistent distribution of pixel values across the 2D image. Extensive experimental results well demonstrate the superiority of our method over existing inpainting tools. Our code is available at https://github.com/chengyr1999/FlowInpaint/.

MOTIVATION: The advancement of long-read RNA sequencing technologies leads to a bright future for transcriptome analysis, in which clustering long reads according to their gene family of origin is of great importance. However, existing de novo clustering algorithms require plenty of computing resources. RESULTS: We developed a new algorithm GeLuster for clustering long RNA-seq reads. Based on our tests on one simulated dataset and nine real datasets, GeLuster exhibited superior performance. On the tested Nanopore datasets it ran 2.9-17.5 times as fast as the second-fastest method with less than one-seventh of memory consumption, while achieving higher clustering accuracy. And on the PacBio data, GeLuster also had a similar performance. It sets the stage for large-scale transcriptome study in future. AVAILABILITY AND IMPLEMENTATION: GeLuster is freely available at https://github.com/yutingsdu/GeLuster.

MOTIVATION: Serial-section electron microscopy (ssEM) is a powerful technique for cellular visualization, especially for large-scale specimens. Limited by the field of view, a megapixel image of whole-specimen is regularly captured by stitching several overlapping images. However, suffering from distortion by manual operations, lens distortion or electron impact, simple rigid transformations are not adequate for perfect mosaic generation. Non-linear deformation usually causes 'ghosting' phenomenon, especially with high magnification. To date, existing microscope image processing tools provide mature rigid stitching methods but have no idea with local distortion correction. RESULTS: In this article, following the development of unsupervised deep learning, we present a multi-scale network to predict the dense deformation fields of image pairs in ssEM and blend these images into a clear and seamless montage. The model is composed of two pyramidal backbones, sharing parameters and interacting with a set of registration modules, in which the pyramidal architecture could effectively capture large deformation according to multi-scale decomposition. A novel 'intermediate-space solving' paradigm is adopted in our model to treat inputted images equally and ensure nearly perfect stitching of the overlapping regions. Combining with the existing rigid transformation method, our model further improves the accuracy of sequential image stitching. Extensive experimental results well demonstrate the superiority of our method over the other traditional methods. AVAILABILITY AND IMPLEMENTATION: The code is available at https://github.com/HeracleBT/ssEM_stitching. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Cryo-electron microscopy (cryo-EM) is a widely used technology for ultrastructure determination, which constructs the 3D structures of protein and macromolecular complex from a set of 2D micrographs. However, limited by the electron beam dose, the micrographs in cryo-EM generally suffer from the extremely low signal-to-noise ratio (SNR), which hampers the efficiency and effectiveness of downstream analysis. Especially, the noise in cryo-EM is not simple additive or multiplicative noise whose statistical characteristics are quite different from the ones in natural image, extremely shackling the performance of conventional denoising methods. RESULTS: Here, we introduce the Noise-Transfer2Clean (NT2C), a denoising deep neural network (DNN) for cryo-EM to enhance image contrast and restore specimen signal, whose main idea is to improve the denoising performance by correctly learning the noise distribution of cryo-EM images and transferring the statistical nature of noise into the denoiser. Especially, to cope with the complex noise model in cryo-EM, we design a contrast-guided noise and signal re-weighted algorithm to achieve clean-noisy data synthesis and data augmentation, making our method authentically achieve signal restoration based on noise's true properties. Our work verifies the feasibility of denoising based on mining the complex cryo-EM noise patterns directly from the noise patches. Comprehensive experimental results on simulated datasets and real datasets show that NT2C achieved a notable improvement in image denoising, especially in background noise removal, compared with the commonly used methods. Moreover, a case study on the real dataset demonstrates that NT2C can greatly alleviate the obstacles caused by the SNR to particle picking and simplify the identifying of particles. AVAILABILITYAND IMPLEMENTATION: The code is available at https://github.com/Lihongjia-ict/NoiseTransfer2Clean/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cryo-electron tomography, combined with subtomogram averaging (STA), can reveal three-dimensional (3D) macromolecule structures in the near-native state from cells and other biological samples. In STA, to get a high-resolution 3D view of macromolecule structures, diverse macromolecules captured by the cellular tomograms need to be accurately classified. However, due to the poor signal-to-noise-ratio (SNR) and severe ray artifacts in the tomogram, it remains a major challenge to classify macromolecules with high accuracy. In this paper, we propose a new convolutional neural network, named 3D-Dilated-DenseNet, to improve the performance of macromolecule classification. In 3D-Dilated-DenseNet, there are two key strategies to guarantee macromolecule classification accuracy: 1) Using dense connections to enhance feature map utilization (corresponding to the baseline 3D-C-DenseNet); 2) Adopting dilated convolution to enrich multi-level information in feature maps. We tested 3D-Dilated-DenseNet and 3D-C-DenseNet both on synthetic data and experimental data. The results show that, on synthetic data, compared with the state-of-the-art method in the SHREC contest (SHREC-CNN), both 3D-C-DenseNet and 3D-Dilated-DenseNet outperform SHREC-CNN. In particular, 3D-Dilated-DenseNet improves 0.393 of F1 metric on tiny-size macromolecules and 0.213 on small-size macromolecules. On experimental data, compared with 3D-C-DenseNet, 3D-Dilated-DenseNet can increase classification performance by 2.1 percent.

Scanning transmission electron microscopy (STEM) is a powerful technique in high-resolution atomic imaging of materials. Decreasing scanning time and reducing electron beam exposure with an acceptable signal-to-noise ratio are two popular research aspects when applying STEM to beam-sensitive materials. Specifically, partially sampling with fixed electron doses is one of the most important solutions, and then the lost information is restored by computational methods. Following successful applications of deep learning in image in-painting, we have developed an encoder-decoder network to reconstruct STEM images in extremely sparse sampling cases. In our model, we combine both local pixel information from convolution operators and global texture features, by applying specific filter operations on the frequency domain to acquire initial reconstruction and global structure prior. Our method can effectively restore texture structures and be robust in different sampling ratios with Poisson noise. A comprehensive study demonstrates that our method gains about 50% performance enhancement in comparison with the state-of-art methods. Code is available at https://github.com/icthrm/Sparse-Sampling-Reconstruction.

Cryo-Electron Tomography (cryo-ET) is a powerful tool for 3D cellular visualization. Due to instrumental limitations, cryo-ET images and their volumetric reconstruction suffer from extremely low signal-to-noise ratio. In this paper, we propose a novel end-to-end self-supervised learning model, the Sparsity Constrained Network (SC-Net), to restore volumetric image from single noisy data in cryo-ET. The proposed method only requires a single noisy data as training input and no ground-truth is needed in the whole training procedure. A new target function is proposed to preserve both local smoothness and detailed structure. Additionally, a novel procedure for the simulation of electron tomographic photographing is designed to help the evaluation of methods. Experiments are done on three simulated data and four real-world data. The results show that our method could produce a strong enhancement for a single very noisy cryo-ET volumetric data, which is much better than the state-of-the-art Noise2Void, and with a competitive performance comparing with Noise2Noise. Code is available at https://github.com/icthrm/SC-Net.

RNA-seq technology is widely employed in various research areas related to transcriptome analyses, and the identification of all the expressed transcripts from short sequencing reads presents a considerable computational challenge. In this study, we introduce TransRef, a new computational algorithm for accurate transcriptome assembly by redefining a novel graph model, the neo-splicing graph, and then iteratively applying a constrained dynamic programming to reconstruct all the expressed transcripts for each graph. When TransRef is utilized to analyze both real and simulated datasets, its performance is notably better than those of several state-of-the-art assemblers, including StringTie2, Cufflinks and Scallop. In particular, the performance of TransRef is notably strong in identifying novel transcripts and transcripts with low-expression levels, while the other assemblers are less effective.

MOTIVATION: Electron tomography (ET) has become an indispensable tool for structural biology studies. In ET, the tilt series alignment and the projection parameter calibration are the key steps toward high-resolution ultrastructure analysis. Usually, fiducial markers are embedded in the sample to aid the alignment. Despite the advances in developing algorithms to find correspondence of fiducial markers from different tilted micrographs, the error rate of the existing methods is still high such that manual correction has to be conducted. In addition, existing algorithms do not work well when the number of fiducial markers is high. RESULTS: In this article, we try to completely solve the fiducial marker correspondence problem. We propose to divide the workflow of fiducial marker correspondence into two stages: (i) initial transformation determination, and (ii) local correspondence refinement. In the first stage, we model the transform estimation as a correspondence pair inquiry and verification problem. The local geometric constraints and invariant features are used to reduce the complexity of the problem. In the second stage, we encode the geometric distribution of the fiducial markers by a weighted Gaussian mixture model and introduce drift parameters to correct the effects of beam-induced motion and sample deformation. Comprehensive experiments on real-world datasets demonstrate the robustness, efficiency and effectiveness of the proposed algorithm. Especially, the proposed two-stage algorithm is able to produce an accurate tracking within an average of ⩽ 100 ms per image, even for micrographs with hundreds of fiducial markers, which makes the real-time ET data processing possible. AVAILABILITY AND IMPLEMENTATION: The code is available at https://github.com/icthrm/auto-tilt-pair. Additionally, the detailed original figures demonstrated in the experiments can be accessed at https://rb.gy/6adtk4. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Electron tomography (ET) offers a unique capacity to image biological structures in situ. However, the resolution of ET reconstructed tomograms is not comparable to that of the single-particle cryo-EM. If many copies of the object of interest are present in the tomograms, their structures can be reconstructed in the tomogram, picked, aligned and averaged to increase the signal-to-noise ratio and improve the resolution, which is known as the subtomogram averaging. To date, the resolution improvement of the subtomogram averaging is still limited because each reconstructed subtomogram is of low reconstruction quality due to the missing wedge issue. RESULTS: In this article, we propose a novel computational model, the constrained reconstruction model (CRM), to better recover the information from the multiple subtomograms and compensate for the missing wedge issue in each of them. CRM is supposed to produce a refined reconstruction in the final turn of subtomogram averaging after alignment, instead of directly taking the average. We first formulate the averaging method and our CRM as linear systems, and prove that the solution space of CRM is no larger, and in practice much smaller, than that of the averaging method. We then propose a sparse Kaczmarz algorithm to solve the formulated CRM, and further extend the solution to the simultaneous algebraic reconstruction technique (SART). Experimental results demonstrate that CRM can significantly alleviate the missing wedge issue and improve the final reconstruction quality. In addition, our model is robust to the number of images in each tilt series, the tilt range and the noise level. AVAILABILITY AND IMPLEMENTATION: The codes of CRM-SIRT and CRM-SART are available at https://github.com/icthrm/CRM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Genome diagnostics have gradually become a prevailing routine for human healthcare. With the advances in understanding the causal genes for many human diseases, targeted sequencing provides a rapid, cost-efficient and focused option for clinical applications, such as single nucleotide polymorphism (SNP) detection and haplotype classification, in a specific genomic region. Although nanopore sequencing offers a perfect tool for targeted sequencing because of its mobility, PCR-freeness and long read properties, it poses a challenging computational problem of how to efficiently and accurately search and map genomic subsequences of interest in a pool of nanopore reads (or raw signals). Due to its relatively low sequencing accuracy, there is no reliable solution to this problem, especially at low sequencing coverage. RESULTS: Here, we propose a brand new signal-based subsequence inquiry pipeline as well as two novel algorithms to tackle this problem. The proposed algorithms follow the principle of subsequence dynamic time warping and directly operate on the electrical current signals, without loss of information in base-calling. Therefore, the proposed algorithms can serve as a tool for sequence inquiry in targeted sequencing. Two novel criteria are offered for the consequent signal quality analysis and data classification. Comprehensive experiments on real-world nanopore datasets show the efficiency and effectiveness of the proposed algorithms. We further demonstrate the potential applications of the proposed algorithms in two typical tasks in nanopore-based targeted sequencing: SNP detection under low sequencing coverage, and haplotype classification under low sequencing accuracy. AVAILABILITY AND IMPLEMENTATION: The project is accessible at https://github.com/icthrm/cwSDTWnano.git, and the presented bench data is available upon request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Abstract Background Electron tomography (ET) is an important technique for the study of complex biological structures and their functions. Electron tomography reconstructs the interior of a three-dimensional object from its projections at different orientations. However, due to the instrument limitation, the angular tilt range of the projections is limited within +70∘ to −70∘. The missing angle range is known as the missing wedge and will cause artifacts. Results In this paper, we proposed a novel algorithm, compressed sensing improved iterative reconstruction-reprojection (CSIIRR), which follows the schedule of improved iterative reconstruction-reprojection but further considers the sparsity of the biological ultra-structural content in specimen. The proposed algorithm keeps both the merits of the improved iterative reconstruction-reprojection (IIRR) and compressed sensing, resulting in an estimation of the electron tomography with faster execution speed and better reconstruction result. A comprehensive experiment has been carried out, in which CSIIRR was challenged on both simulated and real-world datasets as well as compared with a number of classical methods. The experimental results prove the effectiveness and efficiency of CSIIRR, and further show its advantages over the other methods. Conclusions The proposed algorithm has an obvious advance in the suppression of missing wedge effects and the restoration of missing information, which provides an option to the structural biologist for clear and accurate tomographic reconstruction.

MOTIVATION: Electron tomography (ET) is a widely used technology for 3D macro-molecular structure reconstruction. To obtain a satisfiable tomogram reconstruction, several key processes are involved, one of which is the calibration of projection parameters of the tilt series. Although fiducial marker-based alignment for tilt series has been well studied, marker-free alignment remains a challenge, which requires identifying and tracking the identical objects (landmarks) through different projections. However, the tracking of these landmarks is usually affected by the pixel density (intensity) change caused by the geometry difference in different views. The tracked landmarks will be used to determine the projection parameters. Meanwhile, different projection parameters will also affect the localization of landmarks. Currently, there is no alignment method that takes interrelationship between the projection parameters and the landmarks. RESULTS: Here, we propose a novel, joint method for marker-free alignment of tilt series in ET, by utilizing the information underlying the interrelationship between the projection model and the landmarks. The proposed method is the first joint solution that combines the extrinsic (track-based) alignment and the intrinsic (intensity-based) alignment, in which the localization of landmarks and projection parameters keep refining each other until convergence. This iterative approach makes our solution robust to different initial parameters and extreme geometric changes, which ensures a better reconstruction for marker-free ET. Comprehensive experimental results on three real datasets show that our new method achieved a significant improvement in alignment accuracy and reconstruction quality, compared to the state-of-the-art methods. AVAILABILITY AND IMPLEMENTATION: The main program is available at https://github.com/icthrm/joint-marker-free-alignment. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Motivation: Dual-axis electron tomography is an important 3 D macro-molecular structure reconstruction technology, which can reduce artifacts and suppress the effect of missing wedge. However, the fully automatic data process for dual-axis electron tomography still remains a challenge due to three difficulties: (i) how to track the mass of fiducial markers automatically; (ii) how to integrate the information from the two different tilt series; and (iii) how to cope with the inconsistency between the two different tilt series. Results: Here we develop a toolkit for fully automatic alignment of dual-axis electron tomography, with a simultaneous reconstruction procedure. The proposed toolkit and its workflow carries out the following solutions: (i) fully automatic detection and tracking of fiducial markers under large-field datasets; (ii) automatic combination of two different tilt series and global calibration of projection parameters; and (iii) inconsistency correction based on distortion correction parameters and the consequently simultaneous reconstruction. With all of these features, the presented toolkit can achieve accurate alignment and reconstruction simultaneously and conveniently under a single global coordinate system. Availability and implementation: The toolkit AuTom-dualx (alignment module dualxmauto and reconstruction module volrec_mltm) are accessible for general application at http://ear.ict.ac.cn, and the key source code is freely available under request. Supplementary information: Supplementary data are available at Bioinformatics online.

BACKGROUND: Cryo-electron microscopy (cryo-EM) has become a widely used tool for determining the structures of proteins and macromolecular complexes. To acquire the input for single-particle cryo-EM reconstruction, researchers must select hundreds of thousands of particles from micrographs. As the signal-to-noise ratio (SNR) of micrographs is extremely low, the performance of automated particle-selection methods is still unable to meet research requirements. To free researchers from this laborious work and to acquire a large number of high-quality particles, we propose an automated particle-selection method (PIXER) based on the idea of segmentation using a deep neural network. RESULTS: First, to accommodate low-SNR conditions, we convert micrographs into probability density maps using a segmentation network. These probability density maps indicate the likelihood that each pixel of a micrograph is part of a particle instead of just background noise. Particles selected from density maps have a more robust signal than do those directly selected from the original noisy micrographs. Second, at present, there is no segmentation-training dataset for cryo-EM. To enable our plan, we present an automated method to generate a training dataset for segmentation using real-world data. Third, we propose a grid-based, local-maximum method to locate the particles from the probability density maps. We tested our method on simulated and real-world experimental datasets and compared PIXER with the mainstream methods RELION, DeepEM and DeepPicker to demonstrate its performance. The results indicate that, as a fully automated method, PIXER can acquire results as good as the semi-automated methods RELION and DeepEM. CONCLUSION: To our knowledge, our work is the first to address the particle-selection problem using the segmentation network concept. As a fully automated particle-selection method, PIXER can free researchers from laborious particle-selection work. Based on the results of experiments, PIXER can acquire accurate results under low-SNR conditions within minutes.

Motivation: Long-reads, point-of-care and polymerase chain reaction-free are the promises brought by nanopore sequencing. Among various steps in nanopore data analysis, the end-to-end mapping between the raw electrical current signal sequence and the reference expected signal sequence serves as the key building block to signal labeling, and the following signal visualization, variant identification and methylation detection. One of the classic algorithms to solve the signal mapping problem is the dynamic time warping (DTW). However, the ultra-long nanopore sequencing and an order of magnitude difference in the sampling speed complexify the scenario and make the classical DTW infeasible to solve the problem. Results: Here, we propose a novel multi-level DTW algorithm, continuous wavelet DTW (cwDTW), based on continuous wavelet transforms with different scales of the two signal sequences. Our algorithm starts from low-resolution wavelet transforms of the two sequences, such that the transformed sequences are short and have similar sampling rates. Then the peaks and nadirs of the transformed sequences are extracted to form feature sequences with similar lengths, which can be easily mapped by the original DTW. Our algorithm then recursively projects the warping path from a lower-resolution level to a higher-resolution one by building a context-dependent boundary and enabling a constrained search for the warping path in the latter. Comprehensive experiments on two real nanopore datasets on human and on Pandoraea pnomenusa demonstrate the efficiency and effectiveness of the proposed algorithm. In particular, cwDTW can gain remarkable acceleration with tiny loss of the alignment accuracy. On the real nanopore datasets, cwDTW can finish an alignment task in few seconds, which is about 3000 times faster than the original DTW. By successfully applying cwDTW on the tasks of signal labeling and ultra-long sequence comparison, we further demonstrate the power and applicability of cwDTW. Availability and implementation: Our program is available at https://github.com/realbigws/cwDTW. Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Automatic alignment, especially fiducial marker-based alignment, has become increasingly important due to the high demand of subtomogram averaging and the rapid development of large-field electron microscopy. Among the alignment steps, fiducial marker tracking is a crucial one that determines the quality of the final alignment. Yet, it is still a challenging problem to track the fiducial markers accurately and effectively in a fully automatic manner. Results: In this paper, we propose a robust and efficient scheme for fiducial marker tracking. Firstly, we theoretically prove the upper bound of the transformation deviation of aligning the positions of fiducial markers on two micrographs by affine transformation. Secondly, we design an automatic algorithm based on the Gaussian mixture model to accelerate the procedure of fiducial marker tracking. Thirdly, we propose a divide-and-conquer strategy against lens distortions to ensure the reliability of our scheme. To our knowledge, this is the first attempt that theoretically relates the projection model with the tracking model. The real-world experimental results further support our theoretical bound and demonstrate the effectiveness of our algorithm. This work facilitates the fully automatic tracking for datasets with a massive number of fiducial markers. Availability and implementation: The C/C ++ source code that implements the fast fiducial marker tracking is available at https://github.com/icthrm/gmm-marker-tracking. Markerauto 1.6 version or later (also integrated in the AuTom platform at http://ear.ict.ac.cn/) offers a complete implementation for fast alignment, in which fast fiducial marker tracking is available by the '-t' option. Contact: [email protected]. Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Oxford Nanopore sequencing is a rapidly developed sequencing technology in recent years. To keep pace with the explosion of the downstream data analytical tools, a versatile Nanopore sequencing simulator is needed to complement the experimental data as well as to benchmark those newly developed tools. However, all the currently available simulators are based on simple statistics of the produced reads, which have difficulty in capturing the complex nature of the Nanopore sequencing procedure, the main task of which is the generation of raw electrical current signals. Results: Here we propose a deep learning based simulator, DeepSimulator, to mimic the entire pipeline of Nanopore sequencing. Starting from a given reference genome or assembled contigs, we simulate the electrical current signals by a context-dependent deep learning model, followed by a base-calling procedure to yield simulated reads. This workflow mimics the sequencing procedure more naturally. The thorough experiments performed across four species show that the signals generated by our context-dependent model are more similar to the experimentally obtained signals than the ones generated by the official context-independent pore model. In terms of the simulated reads, we provide a parameter interface to users so that they can obtain the reads with different accuracies ranging from 83 to 97%. The reads generated by the default parameter have almost the same properties as the real data. Two case studies demonstrate the application of DeepSimulator to benefit the development of tools in de novo assembly and in low coverage SNP detection. Availability and implementation: The software can be accessed freely at: https://github.com/lykaust15/DeepSimulator. Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Super-resolution fluorescence microscopy with a resolution beyond the diffraction limit of light, has become an indispensable tool to directly visualize biological structures in living cells at a nanometer-scale resolution. Despite advances in high-density super-resolution fluorescent techniques, existing methods still have bottlenecks, including extremely long execution time, artificial thinning and thickening of structures, and lack of ability to capture latent structures. Results: Here, we propose a novel deep learning guided Bayesian inference (DLBI) approach, for the time-series analysis of high-density fluorescent images. Our method combines the strength of deep learning and statistical inference, where deep learning captures the underlying distribution of the fluorophores that are consistent with the observed time-series fluorescent images by exploring local features and correlation along time-axis, and statistical inference further refines the ultrastructure extracted by deep learning and endues physical meaning to the final image. In particular, our method contains three main components. The first one is a simulator that takes a high-resolution image as the input, and simulates time-series low-resolution fluorescent images based on experimentally calibrated parameters, which provides supervised training data to the deep learning model. The second one is a multi-scale deep learning module to capture both spatial information in each input low-resolution image as well as temporal information among the time-series images. And the third one is a Bayesian inference module that takes the image from the deep learning module as the initial localization of fluorophores and removes artifacts by statistical inference. Comprehensive experimental results on both real and simulated datasets demonstrate that our method provides more accurate and realistic local patch and large-field reconstruction than the state-of-the-art method, the 3B analysis, while our method is more than two orders of magnitude faster. Availability and implementation: The main program is available at https://github.com/lykaust15/DLBI. Supplementary information: Supplementary data are available at Bioinformatics online.