Simulation of nanopore sequencing signal data with tunable parameters.

In silico simulation of high-throughput sequencing data is a technique used widely in the genomics field. However, there is currently a lack of effective tools for creating simulated data from nanopore sequencing devices, which measure DNA or RNA molecules in the form of time-series current signal data. Here, we introduce Squigulator, a fast and simple tool for simulation of realistic nanopore signal data. Squigulator takes a reference genome, a transcriptome, or read sequences, and generates corresponding raw nanopore signal data. This is compatible with basecalling software from Oxford Nanopore Technologies (ONT) and other third-party tools, thereby providing a useful substrate for development, testing, debugging, validation, and optimization at every stage of a nanopore analysis workflow. The user may generate data with preset parameters emulating specific ONT protocols or noise-free "ideal" data, or they may deterministically modify a range of experimental variables and/or noise parameters to shape the data to their needs. We present a brief example of Squigulator's use, creating simulated data to model the degree to which different parameters impact the accuracy of ONT basecalling and downstream variant detection. This analysis reveals new insights into the nature of ONT data and basecalling algorithms. We provide Squigulator as an open-source tool for the nanopore community.

Streamlining remote nanopore data access with slow5curl.

BACKGROUND: As adoption of nanopore sequencing technology continues to advance, the need to maintain large volumes of raw current signal data for reanalysis with updated algorithms is a growing challenge. Here we introduce slow5curl, a software package designed to streamline nanopore data sharing, accessibility, and reanalysis. RESULTS: Slow5curl allows a user to fetch a specified read or group of reads from a raw nanopore dataset stored on a remote server, such as a public data repository, without downloading the entire file. Slow5curl uses an index to quickly fetch specific reads from a large dataset in SLOW5/BLOW5 format and highly parallelized data access requests to maximize download speeds. Using all public nanopore data from the Human Pangenome Reference Consortium (>22 TB), we demonstrate how slow5curl can be used to quickly fetch and reanalyze raw signal reads corresponding to a set of target genes from each individual in large cohort dataset (n = 91), minimizing the time, egress costs, and local storage requirements for their reanalysis. CONCLUSIONS: We provide slow5curl as a free, open-source package that will reduce frictions in data sharing for the nanopore community: https://github.com/BonsonW/slow5curl.

A Safe Framework for Quantitative In Vivo Human Evaluation of Image Guidance.

Goal: We present a new framework for in vivo image guidance evaluation and provide a case study on robotic partial nephrectomy. Methods: This framework (called the "bystander protocol") involves two surgeons, one who solely performs the therapeutic process without image guidance, and another who solely periodically collects data to evaluate image guidance. This isolates the evaluation from the therapy, so that in-development image guidance systems can be tested without risk of negatively impacting the standard of care. We provide a case study applying this protocol in clinical cases during robotic partial nephrectomy surgery. Results: The bystander protocol was performed successfully in 6 patient cases. We find average lesion centroid localization error with our IGS system to be 6.5 mm in vivo compared to our prior result of 3.0 mm in phantoms. Conclusions: The bystander protocol is a safe, effective method for testing in-development image guidance systems in human subjects.

CAR+ and CAR- T cells share a differentiation trajectory into an NK-like subset after CD19 CAR T cell infusion in patients with B cell malignancies.

Chimeric antigen receptor (CAR) T cell therapy is effective in treating B cell malignancies, but factors influencing the persistence of functional CAR+ T cells, such as product composition, patients' lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, here we conduct a single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR+ and CAR- T cells from patients from a CARTELL study (ACTRN12617001579381) who received a donor-derived 4-1BB CAR product targeting CD19. Following infusion, CAR+ T cells and CAR- T cells shows similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. We validate these findings in 31 patients with large B cell lymphoma treated with CD19 CAR T therapy. For these patients, we identify using longitudinal mass-cytometry data an association between NK-like subsets and clinical outcomes at 6 months with both CAR+ and CAR- T cells. These results suggest that non-CAR-derived signals can provide information about patients' immune recovery and be used as correlate of clinically relevant parameters.

HIVepsilon-seq-scalable characterization of intact persistent proviral HIV reservoirs in women.

IMPORTANCE: The lack of a reliable method to accurately detect when replication-competent HIV has been cleared is a major challenge in developing a cure. This study introduces a new approach called the HIVepsilon-seq (HIVε-seq) assay, which uses long-read sequencing technology and bioinformatics to scrutinize the HIV genome at the nucleotide level, distinguishing between defective and intact HIV. This study included 30 participants on antiretroviral therapy, including 17 women, and was able to discriminate between defective and genetically intact viruses at the single DNA strand level. The HIVε-seq assay is an improvement over previous methods, as it requires minimal sample, less specialized lab equipment, and offers a shorter turnaround time. The HIVε-seq assay offers a promising new tool for researchers to measure the intact HIV reservoir, advancing efforts towards finding a cure for this devastating disease.

Unified Robot and Inertial Sensor Self-Calibration.

Robots and inertial measurement units (IMUs) are typically calibrated independently. IMUs are placed in purpose-built, expensive automated test rigs. Robot poses are typically measured using highly accurate (and thus expensive) tracking systems. In this paper, we present a quick, easy, and inexpensive new approach to calibrate both simultaneously, simply by attaching the IMU anywhere on the robot's end effector and moving the robot continuously through space. Our approach provides a fast and inexpensive alternative to both robot and IMU calibration, without any external measurement systems. We accomplish this using continuous-time batch estimation, providing statistically optimal solutions. Under Gaussian assumptions, we show that this becomes a nonlinear least squares problem and analyze the structure of the associated Jacobian. Our methods are validated both numerically and experimentally and compared to standard individual robot and IMU calibration methods.

Accelerated nanopore basecalling with SLOW5 data format.

MOTIVATION: Nanopore sequencing is emerging as a key pillar in the genomic technology landscape but computational constraints limiting its scalability remain to be overcome. The translation of raw current signal data into DNA or RNA sequence reads, known as 'basecalling', is a major friction in any nanopore sequencing workflow. Here, we exploit the advantages of the recently developed signal data format 'SLOW5' to streamline and accelerate nanopore basecalling on high-performance computing (HPC) and cloud environments. RESULTS: SLOW5 permits highly efficient sequential data access, eliminating a potential analysis bottleneck. To take advantage of this, we introduce Buttery-eel, an open-source wrapper for Oxford Nanopore's Guppy basecaller that enables SLOW5 data access, resulting in performance improvements that are essential for scalable, affordable basecalling. AVAILABILITY AND IMPLEMENTATION: Buttery-eel is available at https://github.com/Psy-Fer/buttery-eel.

Flexible and efficient handling of nanopore sequencing signal data with slow5tools.

Nanopore sequencing is being rapidly adopted in genomics. We recently developed SLOW5, a new file format with advantages for storage and analysis of raw signal data from nanopore experiments. Here we introduce slow5tools, an intuitive toolkit for handling nanopore data in SLOW5 format. Slow5tools enables lossless data conversion and a range of tools for interacting with SLOW5 files. Slow5tools uses multi-threading, multi-processing, and other engineering strategies to achieve fast data conversion and manipulation, including live FAST5-to-SLOW5 conversion during sequencing. We provide examples and benchmarking experiments to illustrate slow5tools usage, and describe the engineering principles underpinning its performance.

Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing.

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care.

Fast nanopore sequencing data analysis with SLOW5.

Nanopore sequencing depends on the FAST5 file format, which does not allow efficient parallel analysis. Here we introduce SLOW5, an alternative format engineered for efficient parallelization and acceleration of nanopore data analysis. Using the example of DNA methylation profiling of a human genome, analysis runtime is reduced from more than two weeks to approximately 10.5 h on a typical high-performance computer. SLOW5 is approximately 25% smaller than FAST5 and delivers consistent improvements on different computer architectures.

Patient-specific, touch-based registration during robotic, image-guided partial nephrectomy.

Image-guidance during partial nephrectomy enables navigation within the operative field alongside a 3-dimensional roadmap of renal anatomy generated from patient-specific imaging. Once a process is performed by the human mind, the technology will allow standardization of the task for the benefit of all patients undergoing robot-assisted partial nephrectomy. Any surgeon will be able to visualize the kidney and key subsurface landmarks in real-time within a 3-dimensional simulation, with the goals of improving operative efficiency, decreasing surgical complications, and improving oncologic outcomes. For similar purposes, image-guidance has already been adopted as a standard of care in other surgical fields; we are now at the brink of this in urology. This review summarizes touch-based approaches to image-guidance during partial nephrectomy, as the technology begins to enter in vivo human evaluation. The processes of segmentation, localization, registration, and re-registration are all described with seamless integration into the da Vinci surgical system; this will facilitate clinical adoption sooner.

InterARTIC: an interactive web application for whole-genome nanopore sequencing analysis of SARS-CoV-2 and other viruses.

MOTIVATION: InterARTIC is an interactive web application for the analysis of viral whole-genome sequencing (WGS) data generated on Oxford Nanopore Technologies (ONT) devices. A graphical interface enables users with no bioinformatics expertise to analyze WGS experiments and reconstruct consensus genome sequences from individual isolates of viruses, such as SARS-CoV-2. InterARTIC is intended to facilitate widespread adoption and standardization of ONT sequencing for viral surveillance and molecular epidemiology. RESULTS: We demonstrate the use of InterARTIC for the analysis of ONT viral WGS data from SARS-CoV-2 and Ebola virus, using a laptop computer or the internal computer on an ONT GridION sequencing device. We showcase the intuitive graphical interface, workflow customization capabilities and job-scheduling system that facilitate execution of small- and large-scale WGS projects on any common virus. AVAILABILITY AND IMPLEMENTATION: InterARTIC is a free, open-source web application implemented in Python that executes best-practice command line workflows from the ARTIC network. The application can be downloaded as a set of pre-compiled binaries that are compatible with all common Linux distributions, Windows with Linux subsystems, MacOSX and ARM systems. All code can be found on GitHub at https://github.com/Psy-Fer/interARTIC/ and documentation can be found at https://github.com/Psy-Fer/interARTIC/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome.

BACKGROUND: Basenjis are considered an ancient dog breed of central African origins that still live and hunt with tribesmen in the African Congo. Nicknamed the barkless dog, Basenjis possess unique phylogeny, geographical origins and traits, making their genome structure of great interest. The increasing number of available canid reference genomes allows us to examine the impact the choice of reference genome makes with regard to reference genome quality and breed relatedness. RESULTS: Here, we report two high quality de novo Basenji genome assemblies: a female, China (CanFam_Bas), and a male, Wags. We conduct pairwise comparisons and report structural variations between assembled genomes of three dog breeds: Basenji (CanFam_Bas), Boxer (CanFam3.1) and German Shepherd Dog (GSD) (CanFam_GSD). CanFam_Bas is superior to CanFam3.1 in terms of genome contiguity and comparable overall to the high quality CanFam_GSD assembly. By aligning short read data from 58 representative dog breeds to three reference genomes, we demonstrate how the choice of reference genome significantly impacts both read mapping and variant detection. CONCLUSIONS: The growing number of high-quality canid reference genomes means the choice of reference genome is an increasingly critical decision in subsequent canid variant analyses. The basal position of the Basenji makes it suitable for variant analysis for targeted applications of specific dog breeds. However, we believe more comprehensive analyses across the entire family of canids is more suited to a pangenome approach. Collectively this work highlights the importance the choice of reference genome makes in all variation studies.

Accuracy of Touch-Based Registration During Robotic Image-Guided Partial Nephrectomy Before and After Tumor Resection in Validated Phantoms.

Aim: Image-guided surgery (IGS) allows for accurate, real-time localization of subsurface critical structures during surgery. No prior IGS systems have described a feasible method of intraoperative reregistration after manipulation of the kidney during robotic partial nephrectomy (PN). We present a method for seamless reregistration during IGS and evaluate accuracy before and after tumor resection in two validated kidney phantoms. Materials and Methods: We performed robotic PN on two validated kidney phantoms-one with an endophytic tumor and one with an exophytic tumor-with our IGS system utilizing the da Vinci Xi robot. Intraoperatively, the kidney phantoms' surfaces were digitized with the da Vinci robotic manipulator via a touch-based method and registered to a three-dimensional segmented model created from cross-sectional CT imaging of the phantoms. Fiducial points were marked with a surgical marking pen and identified after the initial registration using the robotic manipulator. Segmented images were displayed via picture-in-picture in the surgeon console as tumor resection was performed. After resection, reregistration was performed by reidentifying the fiducial points. The accuracy of the initial registration and reregistration was compared. Results: The root mean square (RMS) averages of target registration error (TRE) were 2.53 and 4.88 mm for the endophytic and exophytic phantoms, respectively. IGS enabled resection along preplanned contours. Specifically, the RMS averages of the normal TRE over the entire resection surface were 0.75 and 2.15 mm for the endophytic and exophytic phantoms, respectively. Both tumors were resected with grossly negative margins. Point-based reregistration enabled instantaneous reregistration with minimal impact on RMS TRE compared with the initial registration (from 1.34 to 1.70 mm preresection and from 1.60 to 2.10 mm postresection). Conclusions: We present a novel and accurate registration and reregistration framework for use during IGS for PN with the da Vinci Xi surgical system. The technology is easily integrated into the surgical workflow and does not require additional hardware.

Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis.

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.

Molecular barcoding of native RNAs using nanopore sequencing and deep learning.

Nanopore sequencing enables direct measurement of RNA molecules without conversion to cDNA, thus opening the gates to a new era for RNA biology. However, the lack of molecular barcoding of direct RNA nanopore sequencing data sets severely affects the applicability of this technology to biological samples, where RNA availability is often limited. Here, we provide the first experimental protocol and associated algorithm to barcode and demultiplex direct RNA nanopore sequencing data sets. Specifically, we present a novel and robust approach to accurately classify raw nanopore signal data by transforming current intensities into images or arrays of pixels, followed by classification using a deep learning algorithm. We demonstrate the power of this strategy by developing the first experimental protocol for barcoding and demultiplexing direct RNA sequencing libraries. Our method, DeePlexiCon, can classify 93% of reads with 95.1% accuracy or 60% of reads with 99.9% accuracy. The availability of an efficient and simple multiplexing strategy for native RNA sequencing will improve the cost-effectiveness of this technology, as well as facilitate the analysis of lower-input biological samples. Overall, our work exemplifies the power, simplicity, and robustness of signal-to-image conversion for nanopore data analysis using deep learning.

Genopo: a nanopore sequencing analysis toolkit for portable Android devices.

The advent of portable nanopore sequencing devices has enabled DNA and RNA sequencing to be performed in the field or the clinic. However, advances in in situ genomics require parallel development of portable, offline solutions for the computational analysis of sequencing data. Here we introduce Genopo, a mobile toolkit for nanopore sequencing analysis. Genopo compacts popular bioinformatics tools to an Android application, enabling fully portable computation. To demonstrate its utility for in situ genome analysis, we use Genopo to determine the complete genome sequence of the human coronavirus SARS-CoV-2 in nine patient isolates sequenced on a nanopore device, with Genopo executing this workflow in less than 30 min per sample on a range of popular smartphones. We further show how Genopo can be used to profile DNA methylation in a human genome sample, illustrating a flexible, efficient architecture that is suitable to run many popular bioinformatics tools and accommodate small or large genomes. As the first ever smartphone application for nanopore sequencing analysis, Genopo enables the genomics community to harness this cheap, ubiquitous computational resource.

Comparing the accuracy of the da Vinci Xi and da Vinci Si for image guidance and automation.

BACKGROUND: Current laparoscopic surgical robots are teleoperated, which requires high fidelity differential motions but does not require absolute accuracy. Emerging applications, including image guidance and automation, require absolute accuracy. The absolute accuracy of the da Vinci Xi robot has not yet been characterized or compared to the Si system, which is now being phased out. This study compares the accuracy of the two. METHODS: We measure robot tip positions and encoder values assessing accuracy with and without robot calibration. RESULTS: The Si is accurate if the setup joints are not moved but loses accuracy otherwise. The Xi is always accurate. CONCLUSION: The Xi can achieve submillimetric average error. Calibration improves accuracy, but excellent baseline accuracy of the Xi means that calibration may not be needed for some applications. Importantly, the external tracking systems needed to account for setup joint error in the Si are no longer required with the Xi.