A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome

Ettwiller, Laurence; Buswell, John A.; Yigit, Erbay; Schildkraut, Ira

doi:10.1101/034785

Cited by 31 publications

(86 citation statements)

References 28 publications

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“…We tested the ToNER tool for annotating transcript 5′ ends using Cappable-seq data [4]. The program output of frequency distributions of the number of mapped read starts per position of the two compared libraries reveal a lower complexity of enriched libraries, which are largely consistent with the effective enrichment of RNA 5′ end fragments employed in this method (S1 Fig).…”

Section: Resultsmentioning

confidence: 75%

“…tRNA are also resistant to exonuclease. Recently, the Cappable-seq method was described as an alternative transcript 5′ end mapping technique, which employs a more efficient enrichment method [4]. In Cappable-seq, a biotinylated guanosine cap is added specifically to the 5′ triphosphate end of mRNA using vaccinia virus capping enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…Although data of unenriched background are available for Cappable-seq, no algorithm has been specifically developed for analysis of Cappable-seq data that utilizes the control data for statistical modeling. In [4], candidate enriched positions were identified using a threshold of normalized read counts from the enriched Cappable-seq dataset, and no statistical modeling of enrichment was used to assess the confidence of annotations. In theory, the tools developed for dRNA-seq analysis could be applied for Cappable-seq data; however, the more pronounced bias in read count distribution in Cappable-seq enriched data (owing to the greater enrichment efficiency) compared with dRNA-seq could affect algorithm performance.…”

Section: Introductionmentioning

confidence: 99%

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ToNER: A tool for identifying nucleotide enrichment signals in feature-enriched RNA-seq data

et al. 2017

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BackgroundBiochemical methods are available for enriching 5′ ends of RNAs in prokaryotes, which are employed in the differential RNA-seq (dRNA-seq) and the more recent Cappable-seq protocols. Computational methods are needed to locate RNA 5′ ends from these data by statistical analysis of the enrichment. Although statistical-based analysis methods have been developed for dRNA-seq, they may not be suitable for Cappable-seq data. The more efficient enrichment method employed in Cappable-seq compared with dRNA-seq could affect data distribution and thus algorithm performance.ResultsWe present Transformation of Nucleotide Enrichment Ratios (ToNER), a tool for statistical modeling of enrichment from RNA-seq data obtained from enriched and unenriched libraries. The tool calculates nucleotide enrichment scores and determines the global transformation for fitting to the normal distribution using the Box-Cox procedure. From the transformed distribution, sites of significant enrichment are identified. To increase power of detection, meta-analysis across experimental replicates is offered. We tested the tool on Cappable-seq and dRNA-seq data for identifying Escherichia coli transcript 5′ ends and compared the results with those from the TSSAR tool, which is designed for analyzing dRNA-seq data. When combining results across Cappable-seq replicates, ToNER detects more known transcript 5′ ends than TSSAR. In general, the transcript 5′ ends detected by ToNER but not TSSAR occur in regions which cannot be locally modeled by TSSAR.ConclusionToNER uses a simple yet robust statistical modeling approach, which can be used for detecting RNA 5′ends from Cappable-seq data, in particular when combining information from experimental replicates. The ToNER tool could potentially be applied for analyzing other RNA-seq datasets in which enrichment for other structural features of RNA is employed. The program is freely available for download at ToNER webpage (http://www4a.biotec.or.th/GI/tools/toner) and GitHub repository (https://github.com/PavitaKae/ToNER).

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Section: Resultsmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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ToNER: A tool for identifying nucleotide enrichment signals in feature-enriched RNA-seq data

et al. 2017

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“…Interestingly, some of those methods rely on the same biochemical approaches as those developed for radioactive labeling. For example, it has been recently reported that VCE apart from GTP accepts certain GTP analogs as substrates, including those functionalized at the ribose moiety [67][68][69]. Taking advantage of this, VCE has been independently used to transfer a biotinylated [69] or anthraniloylated [68] GMP moiety to the 5 0 end of RNA from a GTP analog appropriately functionalized at the ribose moiety.…”

Section: Enzymatic Labeling Beyond Radioactive Phosphatesmentioning

confidence: 99%

“…For example, it has been recently reported that VCE apart from GTP accepts certain GTP analogs as substrates, including those functionalized at the ribose moiety [67][68][69]. Taking advantage of this, VCE has been independently used to transfer a biotinylated [69] or anthraniloylated [68] GMP moiety to the 5 0 end of RNA from a GTP analog appropriately functionalized at the ribose moiety. Notably, the label structure is of great importance for success in this approach, since a similar reaction using GTP carrying a manthraniloyl dye failed to produce capped transcripts [68].…”

Section: Enzymatic Labeling Beyond Radioactive Phosphatesmentioning

confidence: 99%

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

Warmiński

Sikorski

Kowalska

et al. 2017

Phosphate Labeling and Sensing in Chemical Biology

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The cap is a natural modification present at the 5 0 ends of eukaryotic messenger RNA (mRNA), which because of its unique structural features, mediates essential biological functions during the process of gene expression. The core structural feature of the mRNA cap is an N7-methylguanosine moiety linked by a 5 0 -5 0 triphosphate chain to the first transcribed nucleotide. Interestingly, other RNA 5 0 end modifications structurally and functionally resembling the m 7 G cap have been discovered in different RNA types and in different organisms. All these structures contain the 'inverted' 5 0 -5 0 oligophosphate bridge, which is necessary for interaction with specific proteins and also serves as a cleavage site for phosphohydrolases regulating RNA turnover. Therefore, cap analogs containing oligophosphate chain modifications or carrying spectroscopic labels attached to phosphate moieties serve as attractive molecular tools for studies on RNA metabolism and modification of natural RNA properties. Here, we review chemical, enzymatic, and chemoenzymatic approaches that enable preparation of modified cap structures and RNAs carrying such structures, with emphasis on phosphate-modified mRNA cap analogs and their potential applications.M. Warminski and P. J. Sikorski have contributed equally to this work.

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Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs

Desgranges

Caldelari

Marzi

et al. 2020

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs.

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A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome

Cited by 31 publications

References 28 publications

ToNER: A tool for identifying nucleotide enrichment signals in feature-enriched RNA-seq data

ToNER: A tool for identifying nucleotide enrichment signals in feature-enriched RNA-seq data

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs

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