scholarly journals Nanopore sequencing of full-length circRNAs in human and mouse brains reveals circRNA-specific exon usage and intron retention

2019 ◽  
Author(s):  
Karim Rahimi ◽  
Morten T. Venø ◽  
Daniel M. Dupont ◽  
Jørgen Kjems
Keyword(s):  
Mouse Brain ◽  
Intron Retention ◽  
Circular Rna ◽  
Nanopore Sequencing ◽  
Internal Exon ◽  
Long Read ◽  
Enrichment Protocol ◽  
Exon Usage ◽  
Non Coding Rnas ◽  
Human And Mouse

AbstractCircular RNA (circRNA) is a poorly understood class of non-coding RNAs, some of which have been shown to be functional important for cell proliferation and development. CircRNAs mainly derive from back splicing events of coding mRNAs, making it difficult to distinguish the internal exon composition of circRNA from the linearly spliced mRNA. To examine the global exon composition of circRNAs, we performed long-read sequencing of single molecules using nanopore technology for human and mouse brain-derived RNA. By applying an optimized circRNA enrichment protocol prior to sequencing, we were able to detect 7,834 and 10,975 circRNAs in human and mouse brain, respectively, of which 2,945 and 7,052 are not currently found in circBase. Alternative splicing was more prevalent in circRNAs than in linear spliced transcripts, and notably >200 not previously annotated exons were used in circRNAs. This suggests that properties associated with circRNA- specific features, e.g. the unusual back-splicing step during biogenesis, increased stability and /or their lack of translation, alter the general exon usage at steady state. We conclude that the nanopore sequencing technology provides a fast and reliable method to map the specific exon composition of circRNA.

scholarly journals Nanopore sequencing of brain-derived full-length circRNAs reveals circRNA-specific exon usage, intron retention and microexons

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Karim Rahimi ◽  
Morten T. Venø ◽  
Daniel M. Dupont ◽  
Jørgen Kjems
Keyword(s):  
Intron Retention ◽  
Circular Rna ◽  
Nanopore Sequencing ◽  
Nonsense Mediated Decay ◽  
Functional Roles ◽  
Cellular Processes ◽  
Long Read ◽  
Exon Usage ◽  
Non Coding Rnas ◽  
Human And Mouse

AbstractCircular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.

scholarly journals Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
V. Vern Lee ◽  
Louise M. Judd ◽  
Aaron R. Jex ◽  
Kathryn E. Holt ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Intron Retention ◽  
Transcript Level ◽  
Full Length ◽  
Nanopore Sequencing ◽  
Content Type ◽  
Sequencing Platform ◽  
Sequencing Studies ◽  
Long Read

ABSTRACT Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AU content of Plasmodium RNA, but also the limitations of short-read sequencing in deciphering complex splicing events. In this study, we utilized the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or nearly full-length transcripts with comparable quantification to Illumina sequencing. By comparing these data with available gene models, we find widespread alternative splicing, particularly intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This study highlights a strategy in using long-read sequencing to understand splicing events at the whole-transcript level and has implications in the future interpretation of transcriptome sequencing studies. IMPORTANCE We have used a novel nanopore sequencing technology to directly analyze parasite transcriptomes. The very long reads of this technology reveal the full-length genes of the parasites that cause malaria and toxoplasmosis. Gene transcripts must be processed in a process called splicing before they can be translated to protein. Our analysis reveals that these parasites very frequently only partially process their gene products, in a manner that departs dramatically from their human hosts.

scholarly journals MBRS-47. RAPID MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA BASED ON DNA METHYLATION BY NANOPORE SEQUENCING

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Julien Masliah-Planchon ◽  
Elodie Girard ◽  
Philipp Euskirchen ◽  
Christine Bourneix ◽  
Delphine Lequin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Nanopore Sequencing ◽  
Molecular Subgroup ◽  
Group Assignment ◽  
Group 4 ◽  
Methylation Assay ◽  
Tumor Group ◽  
Long Read ◽  
Group 3

Abstract Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

scholarly journals Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq

2021 ◽  
Author(s):  
Martin Philpott ◽  
Jonathan Watson ◽  
Anjan Thakurta ◽  
Tom Brown ◽  
Tom Brown ◽  
...  
Keyword(s):  
Single Cell ◽  
Error Detection ◽  
Single Cells ◽  
Fusion Transcript ◽  
Building Blocks ◽  
Myeloma Cell ◽  
Nanopore Sequencing ◽  
Long Read ◽  
Unique Molecular Identifier ◽  
Transcript Detection

AbstractHere we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

scholarly journals Prenatal Alcohol Exposure Results in Sex-Specific Alterations in Circular RNA Expression in the Developing Mouse Brain

2020 ◽  
Vol 14 ◽  
Author(s):  
Praveen Paudel ◽  
Caroline Pierotti ◽  
Evelyn Lozano ◽  
Stephen K. Amoah ◽  
Amy S. Gardiner ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Prenatal Alcohol Exposure ◽  
Alcohol Exposure ◽  
Circular Rna ◽  
Rna Expression ◽  
Prenatal Alcohol ◽  
Developing Mouse Brain

Abstract 110: Carotid Plaque Instability is Associated with an Increase in the Serum Ratio of circularRNA-284 to microRNA-221

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Hernan Bazan ◽  
Ashton Brooks ◽  
Daniel Lightell ◽  
T. Cooper Woods
Keyword(s):  
Ischemic Stroke ◽  
Sensitivity And Specificity ◽  
Carotid Plaque ◽  
Plaque Rupture ◽  
Serum Levels ◽  
Circular Rna ◽  
Cerebrovascular Event ◽  
Asymptomatic Group ◽  
Plaque Instability ◽  
Non Coding Rnas

Introduction: Atherosclerotic cap thinning and plaque instability occur as a result of a decrease in vascular smooth muscle cell proliferation, which is partly regulated by alterations in the expression of non-coding RNAs in the arterial wall. We recently reported that miR-221 expression in the carotid plaque shoulder is reduced immediately following a carotid-related ischemic cerebrovascular event and returns to normal levels after seven days. We hypothesized that changes in the expression of non-coding RNAs within carotid plaques are reflected in the serum of asymptomatic and acutely symptomatic patients with carotid disease. Methods: Serum levels of microRNA (miR) -221 and a circular RNA with potential binding sites for miR-221 (circR-284), were measured using real-time polymerase chain reaction in 41 patients undergoing carotid endarterectomy. Patients were grouped into those who were asymptomatic and those with an acute ischemic cerebrovascular event within the previous 5 days (urgent). Results: miR-221 was significantly lower (0.25 ± 0.11 vs. 1.00 ± 0.31, p = 0.01) while circR-284 was significantly elevated (2.96 ± 1.16 vs. 1.00 ± 0.37, p = 0.06) in the serum of the urgent compared to the asymptomatic group. Serum levels of these RNAs alone did not exhibit favorable sensitivity and specificity for use as a biomarker indicative of carotid-related ischemic stroke. The ratio of serum circR-284:miR-221, however, was significantly elevated in the urgent group [11.7 ± 0.48 vs. 1.0 ± 0.6, p = 0.0002 (Figure, A)]. Furthermore, receiver operator curve analysis of circR-284:miR-221 ratio demonstrated favorable sensitivity and specificity (Figure, B) for detecting carotid plaque rupture and ischemic stroke. Conclusions: Increases in the ratio of serum circR-284:miR-221 has potential as a diagnostic biomarker of carotid-related ischemic stroke. This data also supports the use and development of functionally related pairs of circulating non-coding RNAs as biomarkers.

scholarly journals How Long Are Long Tandem Repeats? A Challenge for Current Methods of Whole-Genome Sequence Assembly: The Case of Satellites in Caenorhabditis elegans

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 500
Author(s):  
Juan A. Subirana ◽  
Xavier Messeguer
Keyword(s):  
Caenorhabditis Elegans ◽  
Sanger Sequencing ◽  
Tandem Repeats ◽  
Whole Genome Sequence ◽  
Nanopore Sequencing ◽  
Original Sequence ◽  
Genome Sequence Assembly ◽  
Long Read ◽  
Genomic Regions ◽  
Caenorhabditis Elegans Genome

Repetitive genome regions have been difficult to sequence, mainly because of the comparatively small size of the fragments used in assembly. Satellites or tandem repeats are very abundant in nematodes and offer an excellent playground to evaluate different assembly methods. Here, we compare the structure of satellites found in three different assemblies of the Caenorhabditis elegans genome: the original sequence obtained by Sanger sequencing, an assembly based on PacBio technology, and an assembly using Nanopore sequencing reads. In general, satellites were found in equivalent genomic regions, but the new long-read methods (PacBio and Nanopore) tended to result in longer assembled satellites. Important differences exist between the assemblies resulting from the two long-read technologies, such as the sizes of long satellites. Our results also suggest that the lengths of some annotated genes with internal repeats which were assembled using Sanger sequencing are likely to be incorrect.

scholarly journals The effective function of circular RNA in colorectal cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mandana Ameli-Mojarad ◽  
Melika Ameli-Mojarad ◽  
Mahrooyeh Hadizadeh ◽  
Chris Young ◽  
Hosna Babini ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Circular Rna ◽  
Circular Rnas ◽  
Colorectal Tumorigenesis ◽  
Non Coding Rnas ◽  
Mirna Sponges ◽  
Mechanisms Of Development

AbstractColorectal cancer (CRC) is the 3rd most common type of cancer worldwide. Late detection plays role in one-third of annual mortality due to CRC. Therefore, it is essential to find a precise and optimal diagnostic and prognostic biomarker for the identification and treatment of colorectal tumorigenesis. Covalently closed, circular RNAs (circRNAs) are a class of non-coding RNAs, which can have the same function as microRNA (miRNA) sponges, as regulators of splicing and transcription, and as interactors with RNA-binding proteins (RBPs). Therefore, circRNAs have been investigated as specific targets for diagnostic and prognostic detection of CRC. These non-coding RNAs are also linked to metastasis, proliferation, differentiation, migration, angiogenesis, apoptosis, and drug resistance, illustrating the importance of understanding their involvement in the molecular mechanisms of development and progression of CRC. In this review, we present a detailed summary of recent findings relating to the dysregulation of circRNAs and their potential role in CRC.

scholarly journals High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

2021 ◽  
Author(s):  
Karlijn Doorenspleet ◽  
Lara Jansen ◽  
Saskia Oosterbroek ◽  
Oscar Bos ◽  
Pauline Kamermans ◽  
...  
Keyword(s):  
High Resolution ◽  
North Sea ◽  
Fish Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Nanopore Sequencing ◽  
Nature Restoration ◽  
Oxford Nanopore ◽  
Field Samples ◽  
Long Read

To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessments are vital. DNA based techniques and especially environmental DNA (eDNA) metabarcoding from seawater is becoming a powerful monitoring tool. However, current approaches are based on genetic target regions of <500 nucleotides, which offer limited taxonomic resolution. This study aims to develop and validate a long read nanopore sequencing method for eDNA that enables improved identification of fish species. We designed a universal primer pair targeting a 2kb region covering the 12S and 16S rRNA genes of fish mitochondria. eDNA was amplified and sequenced using the Oxford Nanopore MiniON. Sequence data was processed using the new pipeline Decona, and accurate consensus identities of above 99.9% were retrieved. The primer set efficiency was tested with eDNA from a 3.000.000 L zoo aquarium with 31 species of bony fish and elasmobranchs. Over 55% of the species present were identified on species level and over 75% on genus level. Next, our long read eDNA metabarcoding approach was applied to North Sea eDNA field samples collected at ship wreck sites, the Gemini Offshore Wind Farm, the Borkum Reef Grounds and a bare sand bottom. Here, location specific fish and vertebrate communities were obtained. Incomplete reference databases still form a major bottleneck in further developing high resolution long read metabarcoding. Yet, the method has great potential for rapid and accurate fish species monitoring in marine field studies.

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