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

Workman, Rachael E.; Tang, Alison D.; Tang, Paul S.; Jain, Miten; Tyson, John R.; Zuzarte, Philip C.; Gilpatrick, Timothy; Razaghi, Roham; Quick, Joshua; Sadowski, Norah; Holmes, Nadine; Jesus, Jaqueline Góes de; Jones, Karen L.; Snutch, Terrance P.; Loman, Nicholas J.; Paten, Benedict; Loose, Matthew; Simpson, Jared T.; Olsen, Hugh E.; Brooks, Angela N.; Akeson, Mark; Timp, Winston

doi:10.1101/459529

Cited by 188 publications

(384 citation statements)

References 83 publications

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“…To compare whether our algorithm results in similar performance, we measured poly(A) tail lengths from Nanopolish and tailfindr on different barcoded eGFP molecules. Our analysis showed that both tools matched well in their estimated poly(A) tail lengths, as exemplified in Figure 1E for 40 and 100 nt poly(A) tail length (full comparison including analyses on published data set by Workman et al 2018 in Supplemental Fig. S2).…”

Section: Resultssupporting

confidence: 57%

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tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

Krause

Niazi

Labun

et al. 2019

RNA

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Polyadenylation at the 3 ′ ′ ′ ′ ′ -end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3 ′ ′ ′ ′ ′ -ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr's performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.

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Section: Resultssupporting

confidence: 57%

“…While this study was in progress, another tool estimating poly(A) tail lengths from ONT RNA data was developed (Workman et al 2018). Instead of estimating poly(A) tails from base-called data directly, this tool requires read FIGURE 1.…”

Section: Resultsmentioning

confidence: 99%

tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

Krause

Niazi

Labun

et al. 2019

RNA

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“…Conventional "RNA-seq" with short reads, which is rather inappropriately termed so since the RNA molecules are not directly sequenced (Hrdlickova, Toloue, & Tian, 2017), requires reverse transcription (RT) of the template RNA molecule into complementary DNA (cDNA), which is typically further amplified by PCR. The gene annotation process is simplified by the direct RNA sequencing, and it has therefore allowed the identification of more complex or novel transcript isoforms genome-wide (Byrne et al, 2017;Krizanovic, Echchiki, Roux, & Sikic, 2018), and the ability to differentiate transcript haplotypes as well as to identify 3′ poly (A) tail lengths (Workman et al, 2018). The gene annotation process is simplified by the direct RNA sequencing, and it has therefore allowed the identification of more complex or novel transcript isoforms genome-wide (Byrne et al, 2017;Krizanovic, Echchiki, Roux, & Sikic, 2018), and the ability to differentiate transcript haplotypes as well as to identify 3′ poly (A) tail lengths (Workman et al, 2018).…”

Section: Tr Anscrip Tome Analys Is and D Irec T Rna S Equen Cingmentioning

confidence: 99%

“…Since the presence of modified bases results in altered ionic current signal from the unmodified base as it passes through the pore, nanopore sequencing is able to detect the modifications without any additional sample preparation. A comprehensive study of the human poly (A) transcriptome using direct RNA sequencing has recently been reported, including the detection of these modifications as well as A-to-I RNA editing (Workman et al, 2018). For RNA modification, N6-methyladenosine (m6A) or 5-mC is the most common internal modifications in mRNA that are implicated in various RNA metabolism and regulations, and nanopore sequencing can identify these modifications (Garalde et al, 2018).…”

Section: Tr Anscrip Tome Analys Is and D Irec T Rna S Equen Cingmentioning

confidence: 99%

Nanopore sequencing: Review of potential applications in functional genomics

2019

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Molecular biology has been led by various measurement technologies, and increased throughput has developed omics analysis. The development of massively parallel sequencing technology has enabled access to fundamental molecular data and revealed genomic and transcriptomic signatures. Nanopore sequencers have driven such evolution to the next stage. Oxford Nanopore Technologies Inc. provides a new type of single molecule sequencer using protein nanopore that realizes direct sequencing without DNA synthesizing or amplification. This nanopore sequencer can sequence an ultra‐long read limited by the input nucleotide length, or can determine DNA/RNA modifications. Recently, many fields such as medicine, epidemiology, ecology, and education have benefited from this technology. In this review, we explain the features and functions of the nanopore sequencer, introduce various situations where it has been used as a critical technology, and expected future applications.

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“…Furthermore, viral transcriptomes have been investigated using nanopore sequencing of cDNA (Moldován et al , 2018aTombácz et al 2017), being subject to bias from reverse transcription and amplification. Other studies used DRS to study the human poly(A) transcriptome (Workman et al 2018) and the transcriptome of DNA viruses such as HSV (Depledge et al 2018). Furthermore, the genome of influenza A virus has been completely sequenced in its original form using DRS (Keller et al 2018).…”

mentioning

confidence: 99%

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

et al. 2019

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Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

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

Cited by 188 publications

References 83 publications

tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

Nanopore sequencing: Review of potential applications in functional genomics

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

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