The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read differential expression analysis tools

Dong, Xueyi; Tian, Luyi; Gouil, Quentin; Kariyawasam, Hasaru; Su, Shian; Paoli-Iseppi, Ricardo De; Prawer, Yair D J; Clark, Michael B.; Breslin, Kelsey; Iminitoff, Megan; Blewitt, Marnie E.; Law, Charity W.; Ritchie, Matthew E.

doi:10.1093/nargab/lqab028

Cited by 31 publications

(34 citation statements)

References 47 publications

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“…LRS can also be performed without any PCR or reverse transcription steps, reducing the amount of bias in expression quantification. These features allow for accurate quantification of both genes and isoforms, with results from LRS being comparable to or outperforming those from SRS (Oikonomopoulos et al, 2016;Garalde et al, 2018;Sessegolo et al, 2019;Soneson et al, 2019;Dong et al, 2020;Chen et al, 2021). Isoforms arising from genes with highly complex splicing patterns can be accurately detected with LRS, overcoming one of the major limitations of SRS (Clark et al, 2020;Chen et al, 2021).…”

Section: Progress and Applications Of Long-read Sequencing For Profiling Spliced Isoformsmentioning

confidence: 99%

“…Various computational programs have been developed to produce a set of high-confidence isoforms from LRS data such as FLAIR, FLAMES, SQANTI and TALON (Tardaguila et al, 2018;Wyman et al, 2019;Dong et al, 2020;Tang et al, 2020). These programs correct splice junctions within reads and collapse identical transcripts into a set of unique high-confidence isoforms.…”

Section: Progress and Applications Of Long-read Sequencing For Profiling Spliced Isoformsmentioning

confidence: 99%

“…This creates a trade-off between per-cell depth (important for isoform comparisons) and number of cells sequenced. FLT-Seq (Tian et al, 2020) is a recently developed method that subsamples 10-20% of the cells from a 10x Genomics experiment for nanopore sequencing, which combined with SR scRNA-Seq on all cells allows for an integrated analysis using the FLAMES package (Dong et al, 2020). SR scRNA-Seq identifies the cell barcodes and UMIs present, (similar to ScNaUmi-Seq) while also providing a broader view of the cell-types present.…”

Section: Long-read Sequencing For Single Cell Transcriptomicsmentioning

confidence: 99%

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Isoform Age - Splice Isoform Profiling Using Long-Read Technologies

Paoli-Iseppi

Gleeson

Clark

2021

Front. Mol. Biosci.

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Alternative splicing (AS) of RNA is a key mechanism that results in the expression of multiple transcript isoforms from single genes and leads to an increase in the complexity of both the transcriptome and proteome. Regulation of AS is critical for the correct functioning of many biological pathways, while disruption of AS can be directly pathogenic in diseases such as cancer or cause risk for complex disorders. Current short-read sequencing technologies achieve high read depth but are limited in their ability to resolve complex isoforms. In this review we examine how long-read sequencing (LRS) technologies can address this challenge by covering the entire RNA sequence in a single read and thereby distinguish isoform changes that could impact RNA regulation or protein function. Coupling LRS with technologies such as single cell sequencing, targeted sequencing and spatial transcriptomics is producing a rapidly expanding suite of technological approaches to profile alternative splicing at the isoform level with unprecedented detail. In addition, integrating LRS with genotype now allows the impact of genetic variation on isoform expression to be determined. Recent results demonstrate the potential of these techniques to elucidate the landscape of splicing, including in tissues such as the brain where AS is particularly prevalent. Finally, we also discuss how AS can impact protein function, potentially leading to novel therapeutic targets for a range of diseases.

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Section: Progress and Applications Of Long-read Sequencing For Profiling Spliced Isoformsmentioning

confidence: 99%

Section: Progress and Applications Of Long-read Sequencing For Profiling Spliced Isoformsmentioning

confidence: 99%

Section: Long-read Sequencing For Single Cell Transcriptomicsmentioning

confidence: 99%

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Isoform Age - Splice Isoform Profiling Using Long-Read Technologies

Paoli-Iseppi

Gleeson

Clark

2021

Front. Mol. Biosci.

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“…ONT sequencing is a bona fide single-molecule technique and hence offers the possibility to detect molecular heterogeneity in a transcriptome ( Workman et al 2019 ). Recently, the technology was exploited to sequence viral RNA genomes ( Keller et al 2018 ; Boldogko˝i et al 2019 ; Viehweger et al 2019 ; Wang et al 2021 ) to gain insights into viral and eukaryotic transcriptomes ( Tombácz et al 2019 ; Zhao et al 2019 ; Sahlin et al 2021 ) and to detect and quantify RNA isoforms in eukaryotes ( Byrne et al 2017 ; Workman et al 2019 ; Parker et al 2020 ; Dong et al 2021 ; Seki et al 2021 ). Essentially, the requirements, but also the possibilities in eukaryotes and prokaryotes, are the same ( Choi 2016 ), with a poly(A) tail being an essential prerequisite, which is required to capture the RNAs.…”

Section: Introductionmentioning

confidence: 99%

Nanopore sequencing of RNA and cDNA molecules in Escherichia coli

Grünberger

Ferreira-Cerca

Grohmann

2021

RNA

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High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely employed to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli, which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that (PCR)-cDNA-seq offers improved yield and accuracy compared to direct RNA sequencing. Notably, (PCR)-cDNA-seq is suitable for quantitative measurements and can be readily used for simultaneous and accurate detection of transcript 5'and 3' boundaries, analysis of transcriptional units and transcriptional heterogeneity. In summary, based on our comprehensive study, we show that Nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features. Thereby Nanopore RNA-seq holds the potential to become a valuable alternative method for RNA analysis in prokaryotes.

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“…Despite all these advantages, a major obstacle to the adoption of ONT long-read transcriptomics is the high error rate in both cDNA and direct RNA sequencing (Workman et al, 2019). One way to mitigate sequencing errors is by adopting a reference-based approach, and as mentioned earlier, wellcharacterized, species-speci c reference databases can go a long way in resolving transcript sequences and their expression levels, even to the level of isoforms (Dong et al, 2021;Sessegolo et al, n.d.;Workman et al, 2019). This approach with ONT has been documented in species with good quality reference databases such as humans (e.g., Soneson et al, 2019;Weirather et al, 2017;Workman et al, 2019), mice (Sessegolo et al, n.d.), cattle (e.g., Halstead et al, 2021), fruit ies (Bayega et al, 2018), viruses (e.g., Boldogkői et al, 2018), and well-studied plants (e.g., Cui et al, 2020;Wang et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Discovering Novel Genes in Non-Model Fly Accessory Glands Using De Novo Nanopore Transcriptomics

Mrinalini¹,

Puniamoorthy²

2021

Preprint

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BackgroundOxford Nanopore Technologies (ONT) long-read transcriptomes offer many advantages including long reads (>10kbp), end-to-end transcripts, structural variants, isoform-level resolution of genes and expression. However, uptake of ONT transcriptomics is still low, largely due to high error rates (2 to 13%) and reliance on reference databases that are unavailable for many non-model species. Additionally, bioinformatics tools and pipelines for de novo ONT transcriptomics are still in early stages of development. ResultsHere, we use de novo ONT GridION transcriptomics to discover novel genes from the male accessory glands (AG) of a widespread, non-model dung fly, Sepsis punctum. Insect AGs are of particular interest for this as they are hotspots for rapid evolution of novel reproductive genes, and they synthesize seminal fluid proteins that lack homology to any other known proteins. We implement a completely de novo ONT GridION transcriptome pipeline, incorporating quality-filtering and rigorous error-correction procedures, to characterize this novel gene set and to quantify their expression. Specifically, we compare these ONT genes and their expression against de novo lllumina HiSeq transcriptome data. We find 40 high-quality and high-confidence ONT genes that cross-verify against Illumina genes; twenty-six of which are novel and specific to S. punctum. Read count based expression quantification in ONT samples is highly congruent with Illumina’s Transcript per Million (TPM), both in overall pattern and within functional categories. Novel genes account for an average of 81% of total gene expression underscoring their functional importance in S. punctum AGs. Eighty percentage of these genes are secretory in nature, responsible for 74% total gene expression. Notably, median sequence similarities of ONT nucleotide and protein sequences match within-Illumina sequence similarities indicating that our de novo ONT transcriptome pipeline successfully mitigated sequencing errors. ConclusionsThis is the first study to adapt ONT transcriptomics for completely de novo characterization of novel genes in animals. Our study demonstrates that ONT long-reads, constituting a quarter of the number of bases sequenced at less than a third the cost of Illumina reads, can be a resource-friendly and cost-effective solution for end-to-end sequencing of unknown genes even in the absence of a reference database.

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The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read differential expression analysis tools

Cited by 31 publications

References 47 publications

Isoform Age - Splice Isoform Profiling Using Long-Read Technologies

Isoform Age - Splice Isoform Profiling Using Long-Read Technologies

Nanopore sequencing of RNA and cDNA molecules in Escherichia coli

Discovering Novel Genes in Non-Model Fly Accessory Glands Using De Novo Nanopore Transcriptomics

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