Detecting haplotype-specific transcript variation in long reads with FLAIR2.

BACKGROUND: RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously. RESULTS: We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis. CONCLUSIONS: Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Detecting haplotype-specific transcript variation in long reads with FLAIR2.

Background: RNA-Seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme which mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung ADC cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, short read RNA-Seq has limited the community's ability to interrogate both forms of RNA variation simultaneously. Results: We employed long-read technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We have developed a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generated nanopore data with high sequence accuracy of H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We applied our workflow to identify key inosine-isoform associations to help clarify the prominence of ADAR in tumorigenesis. Conclusions: Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

Full-length transcript alterations in human bronchial epithelial cells with U2AF1 S34F mutations.

U2AF1 is one of the most recurrently mutated splicing factors in lung adenocarcinoma and has been shown to cause transcriptome-wide pre-mRNA splicing alterations; however, the full-length altered mRNA isoforms associated with the mutation are largely unknown. To better understand the impact U2AF1 has on full-length isoform fate and function, we conducted high-throughput long-read cDNA sequencing from isogenic human bronchial epithelial cells with and without a U2AF1 S34F mutation. We identified 49,366 multi-exon transcript isoforms, more than half of which did not match GENCODE or short-read-assembled isoforms. We found 198 transcript isoforms with significant expression and usage changes relative to WT, only 68% of which were assembled by short reads. Expression of isoforms from immune-related genes is largely down-regulated in mutant cells and without observed splicing changes. Finally, we reveal that isoforms likely targeted by nonsense-mediated decay are down-regulated in U2AF1 S34F cells, suggesting that isoform changes may alter the translational output of those affected genes. Altogether, our work provides a resource of full-length isoforms associated with U2AF1 S34F in lung cells.

Suitability of transiently expressed antibodies for clinical studies: product quality consistency at different production scales.

Transgenic human monoclonal antibodies derived from humanized mice against different epitopes of the Middle East respiratory syndrome coronavirus (MERS-CoV), and chimeric llama-human bispecific heavy chain-only antibodies targeting the Rift Valley fever virus (RVFV), were produced using a CHO-based transient expression system. Two lead candidates were assessed for each model virus before selecting and progressing one lead molecule. MERS-7.7G6 was used as the model antibody to demonstrate batch-to-batch process consistency and, together with RVFV-107-104, were scaled up to 200 L. Consistent expression titers were obtained in different batches at a 5 L scale for MERS-7.7G6. Although lower expression levels were observed for MERS-7.7G6 and RVFV-107-104 during scale up to 200 L, product quality attributes were consistent at different scales and in different batches. In addition to this, peptide mapping data suggested no detectable sequence variants for any of these candidates. Functional assays demonstrated comparable neutralizing activity for MERS-7.7G6 and RVFV-107-104 generated at different production scales. Similarly, MERS-7.7G6 batches generated at different scales were shown to provide comparable protection in mouse models. Our study demonstrates that a CHO-based transient expression process is capable of generating consistent product quality at different production scales and thereby supports the potential of using transient gene expression to accelerate the manufacturing of early clinical material.

SRSF1 governs progenitor-specific alternative splicing to maintain adult epithelial tissue homeostasis and renewal.

Alternative splicing generates distinct mRNA variants and is essential for development, homeostasis, and renewal. Proteins of the serine/arginine (SR)-rich splicing factor family are major splicing regulators that are broadly required for organ development as well as cell and organism viability. However, how these proteins support adult organ function remains largely unknown. Here, we used the continuously growing mouse incisor as a model to dissect the functions of the prototypical SR family protein SRSF1 during tissue homeostasis and renewal. We identified an SRSF1-governed alternative splicing network that is specifically required for dental proliferation and survival of progenitors but dispensable for the viability of differentiated cells. We also observed a similar progenitor-specific role of SRSF1 in the small intestinal epithelium, indicating a conserved function of SRSF1 across adult epithelial tissues. Thus, our findings define a regulatory mechanism by which SRSF1 specifically controls progenitor-specific alternative splicing events to support adult tissue homeostasis and renewal.

Twelve tips for surgeons to maximise medical student learning in the operating theatre.

Theatre-based learning is an essential component of undergraduate surgical education and offers a wide range of learning opportunities. However, studies have demonstrated that medical students have not always benefited from this holistic learning environment due to many reasons, including intimidation, hierarchies within the surgical environment and fear of making mistakes. The lead surgical educator's approach is an important influence on the experience and learning of their medical students. These twelve tips are aimed at surgical educators with undergraduate teaching responsibilities. This guidance is based upon evidence from literature and established theories of teaching and learning, supplemented by qualitative interviews with surgeons and medical students. The resulting tips were checked and refined by surgical teaching fellows. These learner-centred tips provide guidance on thorough induction, managing mutual expectations and approaches that optimise teaching and learning in the operating theatre. They are designed to support surgical educators in improving their students' engagement and learning experiences in this setting.

Zoonoses Anticipation and Preparedness Initiative, stakeholders conference, February 4 & 5, 2021.

The Zoonoses Anticipation and Preparedness Initiative (ZAPI) was set up to prepare for future outbreaks and to develop and implement new technologies to accelerate development and manufacturing of vaccines and monoclonal antibodies. To be able to achieve surge capacity, an easy deployment and production at multiple sites is needed. This requires a straightforward manufacturing system with a limited number of steps in upstream and downstream processes, a minimum number of in vitro Quality Control assays, and robust and consistent platforms. Three viruses were selected as prototypes: Middle East Respiratory Syndrome (MERS) coronavirus, Rift Valley fever virus, and Schmallenberg virus. Selected antibodies against the viral surface antigens were manufactured by transient gene expression in Chinese Hamster Ovary (CHO) cells, scaling up to 200 L. For vaccine production, viral antigens were fused to multimeric protein scaffold particles using the SpyCatcher/SpyTag system. In vivo models demonstrated the efficacy of both antibodies and vaccines. The final step in speeding up vaccine (and antibody) development is the regulatory appraisal of new platform technologies. Towards this end, within ZAPI, a Platform Master File (PfMF) was developed, as part of a licensing dossier, to facilitate and accelerate the scientific assessment by avoiding repeated discussion of already accepted platforms. The veterinary PfMF was accepted, whereas the human PfMF is currently under review by the European Medicines Agency, aiming for publication of the guideline by January 2022.

Referral patterns for catheter-directed thrombolysis for iliofemoral deep venous thrombosis.

OBJECTIVES: Post thrombotic syndrome (PTS) is a serious complication of deep venous thromboses (DVTs). PTS occurs more frequently and severely following iliofemoral DVT compared to distal DVTs. Catheter directed thrombolysis (CDT) of iliofemoral DVTs may reduce PTS incidence and severity.We aimed to determine the rate of iliofemoral DVT within our institution, their subsequent management, and compliance with NICE guidelines. METHODS: Retrospective review of all DVTs diagnosed over a 3-year period was conducted. Cases of iliofemoral DVT were identified using ICD-10 codes from patient notes, and radiology reports of Duplex scans. Further details were retrieved, such as patient demographics and referrals to vascular services. NICE guidance was applied to determine if patients would have been suitable for CDT. A survey was sent to clinicians within medicine to identify awareness of CDT and local guidelines for iliofemoral DVT management. RESULTS: 225 patients with lower limb DVTs were identified. Of these, 96 were radiographically confirmed as iliofemoral DVTs. The median age was 77. 67.7% of iliofemoral DVTs affected the left leg. Right leg DVTs made up 30.2% and 2.1% were bilateral DVTs. Of the 96 iliofemoral DVTs, 21 were deemed eligible for CDT. Only 3 patients (14.3%) were referred to vascular services, and 3 received thrombolysis.From our survey, 95.5% of respondents suggested anticoagulation alone as management for iliofemoral DVT. Only one respondent recommended referral to vascular services. There was a knowledge deficiency regarding venous anatomy, including superficial versus deep veins. CONCLUSIONS: CDT and other mechanochemical procedures have been shown to improve outcomes of patients post-iliofemoral DVT, however a lack of awareness regarding CDT as a management option results in under-referral to vascular services. We suggest closer relations between vascular services and their "tributary" DVT clinics, development of guidelines and robust care pathways in the management of iliofemoral DVT.

COVID-19: experience and development of simulation for training in a London District General Hospital.

The world is facing an unprecedented crisis in the form of the coronavirus disease-2019 (COVID-19) pandemic. Clinicians and their working environments are under considerable pressures that have not previously been encountered. Consequently, clinicians have had to change their practice significantly to enable safe care for their patients, whilst ensuring their own safety. The majority of COVID-19 simulation to date has been either virtual or in-situ, with the aim of training specific departments. With this in mind, as the Hillingdon Hospital Education Team, we developed a simulation that would provide generic training on COVID-19 for staff across our Trust in various departments and roles. Our aim was to teach staff how to manage patients whilst protecting themselves during this pandemic.

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns.

While splicing changes caused by somatic mutations in SF3B1 are known, identifying full-length isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

Author Correction: Nanopore native RNA sequencing of a human poly(A) transcriptome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nanopore native RNA sequencing of a human poly(A) transcriptome.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform-Like Purification Process.

Single chain variable fragment-IgGs (scFv-IgG) are a class of bispecific antibodies consisting of two single chain variable fragments (scFv) that are fused to an intact IgG molecule. A common trend observed for expression of scFv-IgGs in mammalian cell culture is a higher level of aggregates (10%-30%) compared to mAbs, which results in lower purification yields in order to meet product quality targets. Furthermore, the high aggregate levels also pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g., cation exchange chromatography [CEX]) for aggregate removal. Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP-TFF) and calcium phosphate precipitation were evaluated at the bench scale as means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process. The two most promising process variants, namely Protein A pH gradient elution followed by calcium phosphate precipitation were evaluated at pilot scale, demonstrating comparable performance. Implementing Protein A chromatography with gradient elution and/or calcium phosphate precipitation removed a sufficient portion of the aggregate burden prior to the CEX polishing step, enabling CEX to be operated robustly under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15% to 23% in the condition media, levels were reduced to between 2% and 3% at the end of the CEX step. The overall yield for the optimal process was 71%. Results of this work suggest an improved three-column mAb platform-like purification process for purification of high aggregate scFv-IgG bispecific antibodies is feasible. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2720, 2019.

Airway persistence by the emerging multi-azole-resistant Rasamsonia argillacea complex in cystic fibrosis.

Infections caused by Rasamsonia argillacea complex have been reported in various clinical settings. Cystic fibrosis (CF) is one of the main underlying conditions. An observational cohort study of CF patients with Rasamsonia in respiratory samples was conducted. Eight isolates from 6 patients were identified as R. argillacea complex and tested for antifungal susceptibility. All isolates had high MICs to voriconazole and posaconazole and low MECs to echinocandins. Four patients experienced lung function decline in the year preceding first Rasamsonia isolation. This continued in the year following first isolation in 3 out of 4 cases. Antifungal therapy was initiated in 2 patients, to which only one exhibited a clinical response. Three out of 6 patients died within 3 years of isolating Rasamsonia. Genotyping suggests that similar genotypes of Rasamsonia can persist in CF airways. Consistent with other fungi in CF, the clinical impact of airway colonisation by Rasamsonia is variable. In certain patients, Rasamsonia may be able to drive clinical decline. In others, though a clear impact on lung function may be difficult to determine, the appearance of Rasamsonia acts as a marker of disease severity. In others it does not appear to have an obvious clinical impact on disease progression.

A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus.

The Drosophila Genome Nexus is a population genomic resource that provides D. melanogaster genomes from multiple sources. To facilitate comparisons across data sets, genomes are aligned using a common reference alignment pipeline which involves two rounds of mapping. Regions of residual heterozygosity, identity-by-descent, and recent population admixture are annotated to enable data filtering based on the user's needs. Here, we present a significant expansion of the Drosophila Genome Nexus, which brings the current data object to a total of 1,121 wild-derived genomes. New additions include 305 previously unpublished genomes from inbred lines representing six population samples in Egypt, Ethiopia, France, and South Africa, along with another 193 genomes added from recently-published data sets. We also provide an aligned D. simulans genome to facilitate divergence comparisons. This improved resource will broaden the range of population genomic questions that can addressed from multi-population allele frequencies and haplotypes in this model species. The larger set of genomes will also enhance the discovery of functionally relevant natural variation that exists within and between populations.

Tidal day organic and inorganic material flux of ponds in the Liberty Island freshwater tidal wetland.

The loss of inorganic and organic material export and habitat produced by freshwater tidal wetlands is hypothesized to be an important contributing factor to the long-term decline in fishery production in San Francisco Estuary. However, due to the absence of freshwater tidal wetlands in the estuary, there is little information on the export of inorganic and organic carbon, nutrient or phytoplankton community biomass and the associated mechanisms. A single-day study was conducted to assess the potential contribution of two small vegetated ponds and one large open-water pond to the inorganic and organic material flux within the freshwater tidal wetland Liberty Island in San Francisco Estuary. The study consisted of an intensive tidal day (25.5 h) sampling program that measured the flux of inorganic and organic material at three ponds using continuous monitoring of flow, chlorophyll a, turbidity and salt combined with discrete measurements of phytoplankton community carbon, total and dissolved organic carbon and nutrient concentration at 1.5 h intervals. Vegetated ponds had greater material concentrations than the open water pond and, despite their small area, contributed up to 81% of the organic and 61% of the inorganic material flux of the wetland. Exchange between ponds was important to wetland flux. The small vegetated pond in the interior of the wetland contributed as much as 72-87% of the total organic carbon and chlorophyll a and 10% of the diatom flux of the wetland. Export of inorganic and organic material from the small vegetated ponds was facilitated by small-scale topography and tidal asymmetry that produced a 40% greater material export on ebb tide. The small vegetated ponds contrasted with the large open water pond, which imported 29-96% of the inorganic and 4-81% of the organic material into the wetland from the adjacent river. This study identified small vegetated ponds as an important source of inorganic and organic material to the wetland and the importance of small scale physical processes within ponds to material flux of the wetland.

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli.

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.