Comprehensive short and long read sequencing analysis for the Gaucher and Parkinson's disease-associated GBA gene.
Ontology highlight
ABSTRACT:
SUBMITTER: Toffoli M
PROVIDER: S-EPMC9259685 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Inherited deficiency of acid ?-glucosidase (GCase) due to biallelic mutations in the GBA (glucosidase, ?, acid) gene causes the classic manifestations of Gaucher disease (GD) involving the viscera, the skeleton, and the lungs. Clinical observations point to immune defects in GD beyond the accumulation of activated macrophages engorged with lysosomal glucosylceramide. Here, we show a plethora of immune cell aberrations in mice in which the GBA gene is deleted conditionally in hematopoietic stem cells (HSCs). The thymus exhibited the earliest and most striking alterations reminiscent of impaired T-cell maturation, aberrant B-cell recruitment, enhanced antigen presentation, and impaired egress of mature thymocytes. These changes correlated strongly with disease severity. In contrast to the profound defects in the thymus, there were only limited cellular defects in peripheral lymphoid organs, mainly restricted to mice with severe disease. The cellular changes in GCase deficiency were accompanied by elevated T-helper (Th)1 and Th2 cytokines that also tracked with disease severity. Finally, the proliferation of GCase-deficient HSCs was inhibited significantly by both GL1 and Lyso-GL1, suggesting that the "supply" of early thymic progenitors from bone marrow may, in fact, be reduced in GBA deficiency. The results not only point to a fundamental role for GBA in immune regulation but also suggest that GBA mutations in GD may cause widespread immune dysregulation through the accumulation of substrates.
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Project description:Chrysanthemum morifolium is one of the most popular ornamental plants globally. Owing to its large and complex genome (around 10 Gb, segmental hexaploid), it has been difficult to obtain comprehensive transcriptome, which will promote to perform new breeding technique, such as genome editing, in C. morifolium. In this study, we used single-molecule real-time (SMRT) sequencing and RNA-seq technologies, combined them with an error-correcting process, and obtained high-coverage ray-floret transcriptome. The SMRT-seq data increased the ratio of long mRNAs containing complete open-reading frames, and the combined dataset provided a more complete transcriptomic data than those produced from either SMRT-seq or RNA-seq-derived transcripts. We finally obtained 'Sei Arabella' transcripts containing 928,645 non-redundant mRNA, which showed 96.6% Benchmarking Universal Single-Copy Orthologs (BUSCO) score. We also validated the reliability of the dataset by analyzing a mapping rate, annotation and transcript expression. Using the dataset, we searched anthocyanin biosynthesis gene orthologs and performed a qRT-PCR experiment to assess the usability of the dataset. The assessment of the dataset and the following analysis indicated that our dataset is reliable and useful for molecular biology. The combination of sequencing methods provided genetic information and a way to analyze the complicated C. morifolium transcriptome.
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Project description:Individuals with mutation in the lysosomal enzyme glucocerebrosidase (GBA) gene are at significantly high risk of developing Parkinson's disease with cognitive deficit. We examined whether visual short-term memory impairments, long associated with patients with Parkinson's disease, are also present in GBA-positive individuals-both with and without Parkinson's disease. Precision of visual working memory was measured using a serial order task in which participants observed four bars, each of a different colour and orientation, presented sequentially at screen centre. Afterwards, they were asked to adjust a coloured probe bar's orientation to match the orientation of the bar of the same colour in the sequence. An additional attentional 'filtering' condition tested patients' ability to selectively encode one of the four bars while ignoring the others. A sensorimotor task using the same stimuli controlled for perceptual and motor factors. There was a significant deficit in memory precision in GBA-positive individuals-with or without Parkinson's disease-as well as GBA-negative patients with Parkinson's disease, compared to healthy controls. Worst recall was observed in GBA-positive cases with Parkinson's disease. Although all groups were impaired in visual short-term memory, there was a double dissociation between sources of error associated with GBA mutation and Parkinson's disease. The deficit observed in GBA-positive individuals, regardless of whether they had Parkinson's disease, was explained by a systematic increase in interference from features of other items in memory: misbinding errors. In contrast, impairments in patients with Parkinson's disease, regardless of GBA status, was explained by increased random responses. Individuals who were GBA-positive and also had Parkinson's disease suffered from both types of error, demonstrating the worst performance. These findings provide evidence for dissociable signature deficits within the domain of visual short-term memory associated with GBA mutation and with Parkinson's disease. Identification of the specific pattern of cognitive impairment in GBA mutation versus Parkinson's disease is potentially important as it might help to identify individuals at risk of developing Parkinson's disease.
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Project description:Next-generation DNA sequencing has revolutionized the study of biology. However, the short read lengths of the dominant instruments complicate assembly of complex genomes and haplotype phasing of mixtures of similar sequences. Here we demonstrate a method to reconstruct the sequences of individual nucleic acid molecules up to 11.6 kilobases in length from short (150-bp) reads. We show that our method can construct 99.97%-accurate synthetic reads from bacterial, plant, and animal genomic samples, full-length mRNA sequences from human cancer cell lines, and individual HIV env gene variants from a mixture. The preparation of multiple samples can be multiplexed into a single tube, further reducing effort and cost relative to competing approaches. Our approach generates sequencing libraries in three days from less than one microgram of DNA in a single-tube format without custom equipment or specialized expertise.
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Project description:Parkinson's disease (PD) is the second most common degenerative disorder. Although the disease was described more than 200 years ago, its pathogenetic mechanisms have not yet been fully described. In recent years, the discovery of the association between mutations of the GBA gene (encoding for the lysosomal enzyme glucocerebrosidase) and PD facilitated a better understating of this disorder. GBA mutations are the most common genetic risk factor of the disease. However, mutations of this gene can be found in different phenotypes, such as Gaucher's disease (GD), PD, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behavior disorders (RBDs). Understanding the pathogenic role of this mutation and its different manifestations is crucial for geneticists and scientists to guide their research and to select proper cohorts of patients. Moreover, knowing the implications of the GBA mutation in the context of PD and the other associated phenotypes is also important for clinicians to properly counsel their patients and to implement their care. With the present review we aim to describe the genetic, clinical, and therapeutic features related to the mutation of the GBA gene.
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Project description:Application of Oxford Nanopore Technologies' long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to the high sequence error and small library sizes, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopore RNA-seq datasets with the goal of obtaining gene- and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs ('sequins') as well as a mouse neural stem cell dataset from samples with a null mutation of the epigenetic regulator Smchd1 was analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods for downstream analysis. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent samples. Overall, our work shows that transcriptomic analysis of long-read nanopore data using long-read specific preprocessing methods together with short-read differential expression methods and software that are already in wide use can yield meaningful results.
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Project description:Evaluation of short-read-only, long-read-only, and hybrid assembly approaches on metagenomic samples demonstrating how they affect gene and protein prediction which is relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic, and metaproteomic data to evaluate the metagenomic-based protein predictions.
2022-02-17 | PXD025505 | Pride
Project description:MotivationPercent Spliced-In (PSI) values are commonly used to report alternative pre-mRNA splicing (AS) changes. Previous PSI-detection tools were limited to specific AS events and were evaluated by in silico RNA-seq data. We developed PSI-Sigma, which uses a new PSI index, and we employed actual (non-simulated) RNA-seq data from spliced synthetic genes (RNA Sequins) to benchmark its performance (i.e. precision, recall, false positive rate and correlation) in comparison with three leading tools (rMATS, SUPPA2 and Whippet).ResultsPSI-Sigma outperformed these tools, especially in the case of AS events with multiple alternative exons and intron-retention events. We also briefly evaluated its performance in long-read RNA-seq analysis, by sequencing a mixture of human RNAs and RNA Sequins with nanopore long-read sequencers.Availability and implementationPSI-Sigma is implemented is available at https://github.com/wososa/PSI-Sigma.
| S-EPMC6901072 | biostudies-literature
Project description:IntroductionGenome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time.MethodsIn this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%.Results and discussionFor a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.
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Project description:Current therapies for Parkinson's disease (PD) are palliative, of which the levodopa/carbidopa therapy remains the primary choice but is unable to modulate the progression of neurodegeneration. Due to the complication of such a multifactorial disorder and significant limitations of the therapy, numerous genetic approaches have been proved effective in finding out genes and mechanisms implicated in this disease. Following the observation of a higher frequency of PD in Gaucher's disease (GD), a lysosomal storage condition, mutations of glycosylceramidase beta (GBA) encoding glucocerebrosidase (GCase) have been shown to be involved and have been explored in the context of PD. GBA mutations are the most common genetic risk factor of PD. Various studies have revealed the relationships between PD and GBA gene mutations, facilitating a better understanding of this disorder. Various hypotheses delineate that the pathological mutations of GBA minimize the enzymatic activity of GCase, which affects the proliferation and clearance of α-synuclein; this affects the lysosomal homeostasis, exacerbating the endoplasmic reticulum stress or encouraging the mitochondrial dysfunction. Identification of the pathological mechanisms underlying the GBA-associated parkinsonism (GBA + PD) advances our understanding of PD. This review based on current literature aims to elucidate various genetic and clinical characteristics correlated with GBA mutations and to identify the numerous pathological processes underlying GBA + PD. We also delineate the therapeutic strategies to interfere with the mutant GCase function for further improvement of the related α-synuclein-GCase crosstalks. Moreover, the various therapeutic approaches such as gene therapy, chaperone proteins, and histone deacetylase inhibitors for the treatment of GBA + PD are discussed.
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