Risk Factors for Acute Asthma Exacerbations in Adults With Mild Asthma.

BACKGROUND: Although individuals with mild asthma account for 30% to 40% of acute asthma exacerbations (AAEs), relatively little attention has been paid to risk factors for AAEs in this population. OBJECTIVE: To identify risk factors associated with AAEs in patients with mild asthma. METHODS: This was a retrospective cohort study. We used administrative data from a large managed care organization to identify 199,010 adults aged 18 to 85 years who met study criteria for mild asthma between 2013 and 2018. An asthma-coded qualifying visit (index visit) was identified for each patient. We then used information at the index visit or from the year before the index visit to measure potential risk factors for AAEs in the subsequent year. An AAE was defined as either an asthma-coded hospitalization or emergency department visit, or an asthma-related systemic corticosteroid administration (intramuscular or intravenous) or oral corticosteroid dispensing. Poisson regression models with robust SEs were used to estimate the adjusted risk ratios for future AAEs. RESULTS: In the study cohort, mean age was 44 years and 64% were female; 6.5% had AAEs within 1 year after the index visit. In multivariate models, age, sex, race, ethnicity, smoking status, body mass index, prior acute asthma care, and a variety of comorbidities and other clinical characteristics were significant predictors for future AAE risk. CONCLUSION: Population-based disease management strategies for asthma should be expanded to include people with mild asthma in addition to those with moderate to severe disease.

Uncalled4 improves nanopore DNA and RNA modification detection via fast and accurate signal alignment.

Nanopore signal analysis enables detection of nucleotide modifications from native DNA and RNA sequencing, providing both accurate genetic/transcriptomic and epigenetic information without additional library preparation. Presently, only a limited set of modifications can be directly basecalled (e.g. 5-methylcytosine), while most others require exploratory methods that often begin with alignment of nanopore signal to a nucleotide reference. We present Uncalled4, a toolkit for nanopore signal alignment, analysis, and visualization. Uncalled4 features an efficient banded signal alignment algorithm, BAM signal alignment file format, statistics for comparing signal alignment methods, and a reproducible de novo training method for k-mer-based pore models, revealing potential errors in ONT's state-of-the-art DNA model. We apply Uncalled4 to RNA 6-methyladenine (m6A) detection in seven human cell lines, identifying 26% more modifications than Nanopolish using m6Anet, including in several genes where m6A has known implications in cancer. Uncalled4 is available open-source at github.com/skovaka/uncalled4.

Long-Read Sequencing Reveals Rapid Evolution of Immunity- and Cancer-Related Genes in Bats.

Bats are exceptional among mammals for their powered flight, extended lifespans, and robust immune systems and therefore have been of particular interest in comparative genomics. Using the Oxford Nanopore Technologies long-read platform, we sequenced the genomes of two bat species with key phylogenetic positions, the Jamaican fruit bat (Artibeus jamaicensis) and the Mesoamerican mustached bat (Pteronotus mesoamericanus), and carried out a comprehensive comparative genomic analysis with a diverse collection of bats and other mammals. The high-quality, long-read genome assemblies revealed a contraction of interferon (IFN)-α at the immunity-related type I IFN locus in bats, resulting in a shift in relative IFN-ω and IFN-α copy numbers. Contradicting previous hypotheses of constitutive expression of IFN-α being a feature of the bat immune system, three bat species lost all IFN-α genes. This shift to IFN-ω could contribute to the increased viral tolerance that has made bats a common reservoir for viruses that can be transmitted to humans. Antiviral genes stimulated by type I IFNs also showed evidence of rapid evolution, including a lineage-specific duplication of IFN-induced transmembrane genes and positive selection in IFIT2. In addition, 33 tumor suppressors and 6 DNA-repair genes showed signs of positive selection, perhaps contributing to increased longevity and reduced cancer rates in bats. The robust immune systems of bats rely on both bat-wide and lineage-specific evolution in the immune gene repertoire, suggesting diverse immune strategies. Our study provides new genomic resources for bats and sheds new light on the extraordinary molecular evolution in this critically important group of mammals.

Same-Cell Co-Occurrence of RAS Hotspot and BRAF V600E Mutations in Treatment-Naive Colorectal Cancer.

PURPOSE: Mitogen-activated protein kinase pathway-activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell. MATERIALS AND METHODS: We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival. RESULTS: Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/KRAS G12C, 95%; KRAS G12D/NRAS G12V, 48%; BRAF V600E/KRAS G12D, 44%; and KRAS G12D/NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability-high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability-high. CONCLUSION: Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway.

Tobacco counseling in the setting of thyroid eye disease / Aconselhamento sobre o uso de tabaco no contexto de doenças oculares associadas à tireoide

ABSTRACT Purpose: To collect data on the rate and efficacy of tobacco counseling sessions delivered by ophthalmologists under the setting of patients with thyroid eye disease. Methods: We analyzed the electronic medical records of a digital cohort of patients who visited ophthalmologists at the University of Pennsylvania Health System from 2012 to 2017 with reference to the International Classification of Disease (ICD) codes for Graves' disease, thyrotoxic exophthalmos, and/or thyroid eye disease. Tobacco histories were recorded at the first and last ophthalmology office visits or the most temporally proximal encounter in packs/day (ppd), and each ophthalmology visit note was analyzed to validate the occurrence of tobacco counseling. Results: A total of 435 patients met our study inclusion criteria, of which 72 (16.6%) were active smokers at the time of their first visit. Only 57 (79.2%) of these active smokers had recorded smoking burdens, 34 (59.6%) of which received at least one form of recorded tobacco counseling session. Nine (26.5%) of the subjects who received tobacco counseling and 1 (4.3%) of those who did not have a recorded counseling, quit smoking (risk difference of 22.1%; 95% CI, [1.7%, 39.1%]; p=0.04). In addition, 17 (50.0%) of the subjects who received counseling and 7 (30.4%) of those who did not have a recorded counseling, reduced their ppd consumption (risk difference of 19.6%; 95% CI [-6.3%, 41.3%]; p=0.18). Overall, 14 (25.5%) out of the 55 ophthalmologists who were active smokers had recorded evidence of tobacco counseling. Conclusions: Our cumulative results provide the consequence of both missed opportunities for tobacco counseling as well as its efficacy in the setting of thyroid eye disease.

RESUMO Objetivo: Fornecer informações sobre a ocorrência e a eficácia do aconselhamento sobre o uso de tabaco por oftalmologistas a pacientes com doenças oculares associadas à tireoide. Métodos: Analisamos os prontuários médicos eletrônicos de uma coorte digital de pacientes atendidos por oftalmologistas no Sistema de Saúde da Universidade da Pensilvânia entre o início de 2012 e o final de 2017 com os códigos da Classificação Internacional de Doenças (CID) para a doença de Graves, exoftalmia tireotóxica ou doença ocular associada à tireoide. Os históricos de uso de tabaco foram registrados na primeira e na última visita ao consultório de Oftalmologia, ou na visita mais próxima no tempo. A quantidade de maços/dia (mpd) e todas as anotações feitas nas visitas ao consultório de Oftalmologia foram analisadas para aconselhamento sobre o uso de tabaco. Resultados: Um total de 435 indivíduos preencheram os critérios de inclusão, dos quais 72 (16,6%) estavam fumando ativamente no momento do primeiro encontro. Apenas 57 (79,2%) desses indivíduos que fumam ativamente registraram queixas relacionadas ao tabagismo, sendo que 34 (59,6%) deles receberam alguma forma de aconselhamento sobre o uso de tabaco. Ao todo, 9 (26,5%) indivíduos dentre os que receberam aconselhamento sobre tabaco e 1 (4,3%) que não teve aconselhamento registrado pararam de fumar (diferença de risco de 22,1%; IC 95%, [1,7%, 39,1%]; p=0,04). Dentre aqueles que receberam aconselhamento, 17 (50,0%) reduziram seus mpd, além de 7 (30,4%) daqueles que não tiveram aconselhamento (diferença de risco de 19,6%; IC 95% [-6,3%, 41,3%]; p=0,18). No geral, 14 (25,5%) dos 55 oftalmologistas que tiveram um paciente fumante ativo registraram evidências de aconselhamento sobre o uso de tabaco. Conclusões: Os resultados deste estudo revelam tanto as oportunidades perdidas de aconselhamento sobre o uso do tabaco quanto a eficácia do aconselhamento no contexto de doenças oculares associadas à tireoide.

Tobacco counseling in the setting of thyroid eye disease.

PURPOSE: To collect data on the rate and efficacy of tobacco counseling sessions delivered by ophthalmologists under the setting of patients with thyroid eye disease. METHODS: We analyzed the electronic medical records of a digital cohort of patients who visited ophthalmologists at the University of Pennsylvania Health System from 2012 to 2017 with reference to the International Classification of Disease (ICD) codes for Graves' disease, thyrotoxic exophthalmos, and/or thyroid eye disease. Tobacco histories were recorded at the first and last ophthalmology office visits or the most temporally proximal encounter in packs/day (ppd), and each ophthalmology visit note was analyzed to validate the occurrence of tobacco counseling. RESULTS: A total of 435 patients met our study inclusion criteria, of which 72 (16.6%) were active smokers at the time of their first visit. Only 57 (79.2%) of these active smokers had recorded smoking burdens, 34 (59.6%) of which received at least one form of recorded tobacco counseling session. Nine (26.5%) of the subjects who received tobacco counseling and 1 (4.3%) of those who did not have a recorded counseling, quit smoking (risk difference of 22.1%; 95% CI, [1.7%, 39.1%]; p=0.04). In addition, 17 (50.0%) of the subjects who received counseling and 7 (30.4%) of those who did not have a recorded counseling, reduced their ppd consumption (risk difference of 19.6%; 95% CI [-6.3%, 41.3%]; p=0.18). Overall, 14 (25.5%) out of the 55 ophthalmologists who were active smokers had recorded evidence of tobacco counseling. CONCLUSIONS: Our cumulative results provide the consequence of both missed opportunities for tobacco counseling as well as its efficacy in the setting of thyroid eye disease.

Multimodal Imaging of Waldenstrom Macroglobulinemia-Associated Hyperviscosity-Related Retinopathy Treated with Plasmapheresis.

While plasmapheresis is well known to significantly improve both retinal findings and systemic manifestations associated with Waldenstrom macroglobulinemia, few reports exist documenting changes in optical coherence tomography angiography (OCT-A). The authors present a case of a patient with Waldenstrom macroglobulinemia who had resolution of white-centered peripheral retinal lesions and parafoveal outer nuclear layer hyperreflective material following plasmapheresis. Applying image analysis software to before and after OCT-A images, the authors were able to show an objective decrease in retinal capillary and large vessel density following plasmapheresis. This technique can be used to guide treatment and surveillance for patients with hyperviscosity-related retinopathy.

High resolution copy number inference in cancer using short-molecule nanopore sequencing.

Genome copy number is an important source of genetic variation in health and disease. In cancer, Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms, limiting CNA inference accuracy. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that short-molecule nanopore sequencing reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for short-molecule nanopore sequencing with applications in research and medicine, which includes but is not limited to, CNAs.

Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED.

Conventional targeted sequencing methods eliminate many of the benefits of nanopore sequencing, such as the ability to accurately detect structural variants or epigenetic modifications. The ReadUntil method allows nanopore devices to selectively eject reads from pores in real time, which could enable purely computational targeted sequencing. However, this requires rapid identification of on-target reads while most mapping methods require computationally intensive basecalling. We present UNCALLED ( https://github.com/skovaka/UNCALLED ), an open source mapper that rapidly matches streaming of nanopore current signals to a reference sequence. UNCALLED probabilistically considers k-mers that could be represented by the signal and then prunes the candidates based on the reference encoded within a Ferragina-Manzini index. We used UNCALLED to deplete sequencing of known bacterial genomes within a metagenomics community, enriching the remaining species 4.46-fold. UNCALLED also enriched 148 human genes associated with hereditary cancers to 29.6× coverage using one MinION flowcell, enabling accurate detection of single-nucleotide polymorphisms, insertions and deletions, structural variants and methylation in these genes.

Clonal Hematopoiesis Before, During, and After Human Spaceflight.

Clonal hematopoiesis (CH) occurs when blood cells harboring an advantageous mutation propagate faster than others. These mutations confer a risk for hematological cancers and cardiovascular disease. Here, we analyze CH in blood samples from a pair of twin astronauts over 4 years in bulk and fractionated cell populations using a targeted CH panel, linked-read whole-genome sequencing, and deep RNA sequencing. We show CH with distinct mutational profiles and increasing allelic fraction that includes a high-risk, TET2 clone in one subject and two DNMT3A mutations on distinct alleles in the other twin. These astronauts exhibit CH almost two decades prior to the mean age at which it is typically detected and show larger shifts in clone size than age-matched controls or radiotherapy patients, based on a longitudinal cohort of 157 cancer patients. As such, longitudinal monitoring of CH may serve as an important metric for overall cancer and cardiovascular risk in astronauts.

Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing.

Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of the disease and its progression. We performed whole-genome sequencing of the SKBR3 breast cancer cell line and patient-derived tumor and normal organoids from two breast cancer patients using Illumina/10x Genomics, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT) sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings show that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long reads even at relatively low coverage (25×-30×). Furthermore, we integrated SV and CNV data into a unifying karyotype-graph structure to present a more accurate representation of the mutated cancer genomes. We find hundreds of variants within known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.

GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes.

An important assessment prior to genome assembly and related analyses is genome profiling, where the k-mer frequencies within raw sequencing reads are analyzed to estimate major genome characteristics such as size, heterozygosity, and repetitiveness. Here we introduce GenomeScope 2.0 (https://github.com/tbenavi1/genomescope2.0), which applies combinatorial theory to establish a detailed mathematical model of how k-mer frequencies are distributed in heterozygous and polyploid genomes. We describe and evaluate a practical implementation of the polyploid-aware mixture model that quickly and accurately infers genome properties across thousands of simulated and several real datasets spanning a broad range of complexity. We also present a method called Smudgeplot (https://github.com/KamilSJaron/smudgeplot) to visualize and estimate the ploidy and genome structure of a genome by analyzing heterozygous k-mer pairs. We successfully apply the approach to systems of known variable ploidy levels in the Meloidogyne genus and the extreme case of octoploid Fragaria × ananassa.

Recovering rearranged cancer chromosomes from karyotype graphs.

BACKGROUND: Many cancer genomes are extensively rearranged with highly aberrant chromosomal karyotypes. Structural and copy number variations in cancer genomes can be determined via abnormal mapping of sequenced reads to the reference genome. Recently it became possible to reconcile both of these types of large-scale variations into a karyotype graph representation of the rearranged cancer genomes. Such a representation, however, does not directly describe the linear and/or circular structure of the underlying rearranged cancer chromosomes, thus limiting possible analysis of cancer genomes somatic evolutionary process as well as functional genomic changes brought by the large-scale genome rearrangements. RESULTS: Here we address the aforementioned limitation by introducing a novel methodological framework for recovering rearranged cancer chromosomes from karyotype graphs. For a cancer karyotype graph we formulate an Eulerian Decomposition Problem (EDP) of finding a collection of linear and/or circular rearranged cancer chromosomes that are determined by the graph. We derive and prove computational complexities for several variations of the EDP. We then demonstrate that Eulerian decomposition of the cancer karyotype graphs is not always unique and present the Consistent Contig Covering Problem (CCCP) of recovering unambiguous cancer contigs from the cancer karyotype graph, and describe a novel algorithm CCR capable of solving CCCP in polynomial time. We apply CCR on a prostate cancer dataset and demonstrate that it is capable of consistently recovering large cancer contigs even when underlying cancer genomes are highly rearranged. CONCLUSIONS: CCR can recover rearranged cancer contigs from karyotype graphs thereby addressing existing limitation in inferring chromosomal structures of rearranged cancer genomes and advancing our understanding of both patient/cancer-specific as well as the overall genetic instability in cancer.

Assembly of the 373k gene space of the polyploid sugarcane genome reveals reservoirs of functional diversity in the world's leading biomass crop.

BACKGROUND: Sugarcane cultivars are polyploid interspecific hybrids of giant genomes, typically with 10-13 sets of chromosomes from 2 Saccharum species. The ploidy, hybridity, and size of the genome, estimated to have >10 Gb, pose a challenge for sequencing. RESULTS: Here we present a gene space assembly of SP80-3280, including 373,869 putative genes and their potential regulatory regions. The alignment of single-copy genes in diploid grasses to the putative genes indicates that we could resolve 2-6 (up to 15) putative homo(eo)logs that are 99.1% identical within their coding sequences. Dissimilarities increase in their regulatory regions, and gene promoter analysis shows differences in regulatory elements within gene families that are expressed in a species-specific manner. We exemplify these differences for sucrose synthase (SuSy) and phenylalanine ammonia-lyase (PAL), 2 gene families central to carbon partitioning. SP80-3280 has particular regulatory elements involved in sucrose synthesis not found in the ancestor Saccharum spontaneum. PAL regulatory elements are found in co-expressed genes related to fiber synthesis within gene networks defined during plant growth and maturation. Comparison with sorghum reveals predominantly bi-allelic variations in sugarcane, consistent with the formation of 2 "subgenomes" after their divergence ∼3.8-4.6 million years ago and reveals single-nucleotide variants that may underlie their differences. CONCLUSIONS: This assembly represents a large step towards a whole-genome assembly of a commercial sugarcane cultivar. It includes a rich diversity of genes and homo(eo)logous resolution for a representative fraction of the gene space, relevant to improve biomass and food production.

Samovar: Single-Sample Mosaic Single-Nucleotide Variant Calling with Linked Reads.

Linked-read sequencing enables greatly improves haplotype assembly over standard paired-end analysis. The detection of mosaic single-nucleotide variants benefits from haplotype assembly when the model is informed by the mapping between constituent reads and linked reads. Samovar evaluates haplotype-discordant reads identified through linked-read sequencing, thus enabling phasing and mosaic variant detection across the entire genome. Samovar trains a random forest model to score candidate sites using a dataset that considers read quality, phasing, and linked-read characteristics. Samovar calls mosaic single-nucleotide variants (SNVs) within a single sample with accuracy comparable with what previously required trios or matched tumor/normal pairs and outperforms single-sample mosaic variant callers at minor allele frequency 5%-50% with at least 30X coverage. Samovar finds somatic variants in both tumor and normal whole-genome sequencing from 13 pediatric cancer cases that can be corroborated with high recall with whole exome sequencing. Samovar is available open-source at https://github.com/cdarby/samovar under the MIT license.

Evaluation of EGFR inhibitor-mediated acneiform skin toxicity within the double-blind randomized EVITA trial: A thorough gender-specific analysis using the WoMo score.

Acne-like skin reactions frequently occur in patients undergoing treatment with drugs inhibiting the epidermal growth factor receptor. Recently, the effects of vitamin K1 containing cream (Reconval K1) as prophylactic skin treatment in addition to doxycycline were explored in a double-blind randomized phase II trial (EVITA) in patients with metastatic colorectal cancer receiving cetuximab. EVITA demonstrated a trend towards less severe skin rash in Reconval K1-treated patients using the tripartite WoMo skin reaction grading score as a thorough tool for quantification of drug related skin reactions. This gender-specific analysis of the EVITA trial evaluated the application of the WoMo score for assessment of epidermal growth factor receptor (EGFR)-related skin toxicities according to treatment arm and gender. To show the robustness of results parametric and non-parametric statistical analyses were conducted. All three parts of the WoMo score independently demonstrated the superiority of the treatment arm (Reconval K1) regarding a significant reduction in acneiform skin reactions in women. Men did not benefit from Reconval K1 cream at any time point in none of the WoMo score analyses. The treatment effect in women was confirmed by the use of skin rash categories based on the final WoMo overall score and mixed effect longitudinal multiple linear regression analysis. The WoMo score represents a sensitive tool for studies exploiting treatments against EGFR mediated acne-like skin rash. Part C of the WoMo score seems to be sufficient for quantification of drug related skin toxicities in further studies. Standard WoMo skin reaction score values for future studies are provided.

Hypo-osmotic-like stress underlies general cellular defects of aneuploidy.

Aneuploidy, which refers to unbalanced chromosome numbers, represents a class of genetic variation that is associated with cancer, birth defects and eukaryotic micro-organisms1-4. Whereas it is known that each aneuploid chromosome stoichiometry can give rise to a distinct pattern of gene expression and phenotypic profile4,5, it remains a fundamental question as to whether there are common cellular defects that are associated with aneuploidy. Here we show the existence in budding yeast of a common aneuploidy gene-expression signature that is suggestive of hypo-osmotic stress, using a strategy that enables the observation of common transcriptome changes of aneuploidy by averaging out karyotype-specific dosage effects in aneuploid yeast-cell populations with random and diverse chromosome stoichiometry. Consistently, aneuploid yeast exhibited increased plasma-membrane stress that led to impaired endocytosis, and this defect was also observed in aneuploid human cells. Thermodynamic modelling showed that hypo-osmotic-like stress is a general outcome of the proteome imbalance that is caused by aneuploidy, and also predicted a relationship between ploidy and cell size that was observed in yeast and aneuploid cancer cells. A genome-wide screen uncovered a general dependency of aneuploid cells on a pathway of ubiquitin-mediated endocytic recycling of nutrient transporters. Loss of this pathway, coupled with the endocytic defect inherent to aneuploidy, leads to a marked alteration of intracellular nutrient homeostasis.

Novel circular RNA circNF1 acts as a molecular sponge, promoting gastric cancer by absorbing miR-16.

Circular RNAs (circRNAs) are a new class of RNA involved in multiple human malignancies. However, limited information exists regarding the involvement of circRNAs in gastric carcinoma (GC). Therefore, we sought to identify novel circRNAs, their functions and mechanisms in gastric carcinogenesis. We analyzed next-generation RNA sequencing data from GC tissues and cell lines, identifying 75,201 candidate circRNAs. Among these, we focused on one novel circRNA, circNF1 , which was upregulated in GC tissues and cell lines. Loss- and gain-of-function studies demonstrated that circNF1 significantly promotes cell proliferation. Furthermore, luciferase reporter assays showed that circNF1 binds to miR-16, thereby derepressing its downstream target mRNAs, MAP7 and AKT3. Targeted silencing or overexpression of circNF1 had no effect on levels of its linear RNA counterpart, NF1. Taken together, these results suggest that circNF1 acts as a novel oncogenic circRNA in GC by functioning as a miR-16 sponge.

Parrot Genomes and the Evolution of Heightened Longevity and Cognition.

Parrots are one of the most distinct and intriguing groups of birds, with highly expanded brains [1], highly developed cognitive [2] and vocal communication [3] skills, and a long lifespan compared to other similar-sized birds [4]. Yet the genetic basis of these traits remains largely unidentified. To address this question, we have generated a high-coverage, annotated assembly of the genome of the blue-fronted Amazon (Amazona aestiva) and carried out extensive comparative analyses with 30 other avian species, including 4 additional parrots. We identified several genomic features unique to parrots, including parrot-specific novel genes and parrot-specific modifications to coding and regulatory sequences of existing genes. We also discovered genomic features under strong selection in parrots and other long-lived birds, including genes previously associated with lifespan determination as well as several hundred new candidate genes. These genes support a range of cellular functions, including telomerase activity; DNA damage repair; control of cell proliferation, cancer, and immunity; and anti-oxidative mechanisms. We also identified brain-expressed, parrot-specific paralogs with known functions in neural development or vocal-learning brain circuits. Intriguingly, parrot-specific changes in conserved regulatory sequences were overwhelmingly associated with genes that are linked to cognitive abilities and have undergone similar selection in the human lineage, suggesting convergent evolution. These findings bring novel insights into the genetics and evolution of longevity and cognition, as well as provide novel targets for exploring the mechanistic basis of these traits.

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line.

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged, although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available, with nearly 20,000 variants present, most of which were missed by short-read sequencing. Surrounding the important ERBB2 oncogene (also known as HER2), we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the genome and sheds new light on the complex mechanisms involved in cancer genome evolution.