Detection and mitigation of spurious antisense expression with RoSA

Mourão, Kira; Schurch, Nicholas J.; Lucoszek, Radek; Froussios, Kimon; Mackinnon, Katarzyna; Duc, Céline; Simpson, Gordon G.; Barton, Geoffrey J.

doi:10.12688/f1000research.18952.1

Cited by 16 publications

(9 citation statements)

References 39 publications

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“…RNA sequencing (RNAseq) is used to dissect transcriptome complexity: it involves copying RNA into complementary DNA (cDNA) with reverse transcriptases (RTs) and then sequencing the subsequent DNA copies. RNAseq reveals diverse features of transcriptomes, but limitations can include misidentification of 3′ ends through internal priming 3 , spurious antisense and splicing events produced by RT template switching 4, 5 , and the inability to detect all base modifications in the copying process 6 . The fragmentation of RNA prior to short-read sequencing makes it difficult to interpret the combination of authentic RNA processing events and remains an unsolved problem 7 .…”

Section: Introductionmentioning

confidence: 99%

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Parker

Knop

Sherwood

et al. 2019

Preprint

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21Understanding genome organization and gene regulation requires insight into RNA transcription, 22 processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a 23 wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA 24 methylation (m 6 A). Here we show that m 6 A can be mapped in full-length mRNAs transcriptome-wide 25 and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, 26 poly(A) site choice and poly(A) tail length. Loss of m 6 A from 3' untranslated regions is associated 27 with decreased relative transcript abundance and defective RNA 3′ end formation. A functional 28 consequence of disrupted m 6 A is a lengthening of the circadian period. We conclude that nanopore 29 direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length 30 single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying 31 this approach to less well-studied species could transform our understanding of what their genomes 32 encode. 33 34 misidentification of 3′ ends through internal priming 3 , spurious antisense and splicing events 46 produced by RT template switching 4,5 , and the inability to detect all base modifications in the 47 copying process 6 . The fragmentation of RNA prior to short-read sequencing makes it difficult to 48 interpret the combination of authentic RNA processing events and remains an unsolved problem 7 . 49We investigated whether long-read direct RNA sequencing (DRS) with nanopores 8 could 50 reveal the complexity of Arabidopsis mRNA processing and modifications. In nanopore DRS, the 51 protein pore (nanopore) sits in a membrane through which an electrical current is passed, and intact 52 RNA is fed through the nanopore by a motor protein 8 . Each RNA sequence within the nanopore 53 (5 bases) can be identified by the magnitude of signal it produces. Arabidopsis is a pathfinder model 54 in plant biology, and its genome annotation strongly influences the annotation and our 55 understanding of what other plant genomes encode. We applied nanopore DRS and Illumina RNAseq 56 to wild-type Arabidopsis (Col-0) and mutants defective in m 6 A 9 and exosome-mediated RNA decay 10 . 57We reveal m 6 A and combinations of RNA processing events (alternative patterns of 5′ capped 58 transcription start sites, splicing, 3′ polyadenylation and poly(A) tail length) in full-length Arabidopsis 59 mRNAs transcriptome-wide. 60 61 Results 62Nanopore DRS detects long, complex mRNAs and short, structured non-coding RNAs 63We purified poly (A)+ RNA from four biological replicates of 14-day-old Arabidopsis Col-0 seedlings. 64We incorporated synthetic External RNA Controls Consortium (ERCC) RNA Spike-In mixes into all 65 replicates 11,12 and carried out nanopore DRS. Illumina RNAseq was performed in parallel on similar 66 material. Using Guppy base-calling (Oxford Nanopore Technologies) and minimap2 alignment 67 software 13 , we identified around 1 mi...

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Section: Introductionmentioning

confidence: 99%

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Parker

Knop

Sherwood

et al. 2019

Preprint

Self Cite

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“…Cufflinks could generate up to 80% misassembled transcripts, including ~55% transcripts with false SJs. In addition, both Cufflinks and Scallop produced a high number of mono-exon antisense transcripts (mono-exon transcripts that overlap with a gene are in antisense), which is likely to be a result of mis-assembly from reads with incorrect strand information (Mourão et al, 2019). Also, Scallop generates 20,000 (50%) fewer transcripts compared with Stringtie.…”

Section: Evaluation Of Genome Quality and The Bias Caused By The Tran...mentioning

confidence: 99%

The value of genotype-specific reference for transcriptome analyses in barley

et al. 2022

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It is increasingly apparent that although different genotypes within a species share “core” genes, they also contain variable numbers of “specific” genes and different structures of “core” genes that are only present in a subset of individuals. Using a common reference genome may thus lead to a loss of genotype-specific information in the assembled Reference Transcript Dataset (RTD) and the generation of erroneous, incomplete or misleading transcriptomics analysis results. In this study, we assembled genotype-specific RTD (sRTD) and common reference–based RTD (cRTD) from RNA-seq data of cultivated Barke and Morex barley, respectively. Our quantitative evaluation showed that the sRTD has a significantly higher diversity of transcripts and alternative splicing events, whereas the cRTD missed 40% of transcripts present in the sRTD and it only has ∼70% accurate transcript assemblies. We found that the sRTD is more accurate for transcript quantification as well as differential expression analysis. However, gene-level quantification is less affected, which may be a reasonable compromise when a high-quality genotype-specific reference is not available.

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“…Numerous techniques and protocols produce strand-specific RNA-seq data, thus retaining the strand information. However, RT-PCR can generate various artefacts through, for example, reverse transcriptase template switching or mispriming, which can lead to erroneous assignment of antisense transcripts [65][66][67]. As cis-NATs are often weakly expressed relative to their cognate sense mRNAs, care must be exercised when the ratio of sense mRNA to cis-NAT expression level is very large (>1000) [2].…”

Section: Box 1 Detection Of Cis-natsmentioning

confidence: 99%

Making sense of the natural antisense transcript puzzle

Reis

Poirier

2021

Trends in Plant Science

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In plants, thousands of genes are associated with antisense transcription, which often produces noncoding RNAs. Although widespread, sense-antisense pairs have been implicated in a limited variety of functions in plants and are often thought to form extensive dsRNA stretches triggering gene silencing. In this opinion, we show that evidence does not support gene silencing as a major role for antisense transcription. In fact, it is more likely that antisense transcripts play diverse functions in gene regulation. We propose a general framework for the initial functional dissection of antisense transcripts, suggesting testable hypotheses relying on an experiment-based decision tree. By moving beyond the gene silencing paradigm, we argue that a broad and diverse role for natural antisense transcription will emerge. Natural antisense transcripts in plantsNatural antisense transcripts (NATs) (see Glossary) are widespread in eukaryotic genomes. The bulk of our current knowledge on the role of NATs in gene regulation is for cis-NATs (i.e., NATs and sense transcripts are transcribed from the same genomic locus), which are the focus of this work. Early work based on strand-specific RNA sequencing (RNA-seq) using short-read RNA sequencing of polyA+ mRNA, revealed that approximately 30% of loci of arabidopsis (Arabidopsis thaliana) are associated with cis-NATs [1]. More recent analysis of arabidopsis transcriptomics based on strand-specific RNA seq of either polyA+ mRNAs [2] or RNAs transcriptionally engaged with RNA Polymerase II (RNAPII) [3], reported 4300 to 5400 distinct cis-NATs, indicating that approximately 15-20% of arabidopsis loci possess a cis-NAT. Work in rice (Oryza sativum) using single-molecule long-read RNA sequencing reported that nearly 60% of loci are associated with a cis-NAT [4]. Similar high levels of genes associated with cis-NATs are also found in mice and humans [5].

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Detection and mitigation of spurious antisense expression with RoSA

Cited by 16 publications

References 39 publications

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

The value of genotype-specific reference for transcriptome analyses in barley

Making sense of the natural antisense transcript puzzle

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