Paired-end small RNA sequencing reveals a possible overestimation in isomiR sequence repertoire previously reported from conventional single read data analysis
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ABSTRACT:
PROVIDER: PRJNA649535 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12344552_1.fastq.gz | Fastqsanger.gz | |||
| SRR12344552_2.fastq.gz | Fastqsanger.gz | |||
| SRR12344553_1.fastq.gz | Fastqsanger.gz | |||
| SRR12344553_2.fastq.gz | Fastqsanger.gz | |||
| SRR12344554_1.fastq.gz | Fastqsanger.gz |
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Project description:Next generation sequencing has allowed the discovery of miRNA isoforms, termed isomiRs. Some isomiRs are derived from imprecise processing of pre-miRNA precursors, leading to length variants. Additional variability is introduced by non-templated addition of bases at the ends or editing of internal bases, resulting in base differences relative to the template DNA sequence. We hypothesized that some component of the isomiR variation reported so far could be due to systematic technical noise. We have developed the XICRA pipeline to analyze small RNA sequencing data at the miRNA,. We exploited its ability to use single or merged reads to compare miRNA isomiR results derived from paired-end (PE) reads with those from single reads (SR) to address whether detectable sequence differences relative to canonical miRNAs found in isomiRs are true biological variations or the result of errors in sequencing. We have detected non-negligible systematic differences between single and PE data which primarily affect putative internally edited isomiRs, and at a much smaller frequency terminal length changing isomiRs. This is relevant for the identification of true isomiRs in small RNA sequencing datasets. . We conclude that potential artifacts derived from sequencing errors and/or data processing could result in an overestimation of abundance and diversity of miRNA isoforms. Efforts in annotating the isomiRnome should take this into account.
2021-03-24 | GSE155370 | GEO
Project description:BackgroundNext generation sequencing has allowed the discovery of miRNA isoforms, termed isomiRs. Some isomiRs are derived from imprecise processing of pre-miRNA precursors, leading to length variants. Additional variability is introduced by non-templated addition of bases at the ends or editing of internal bases, resulting in base differences relative to the template DNA sequence. We hypothesized that some component of the isomiR variation reported so far could be due to systematic technical noise and not real.ResultsWe have developed the XICRA pipeline to analyze small RNA sequencing data at the isomiR level. We exploited its ability to use single or merged reads to compare isomiR results derived from paired-end (PE) reads with those from single reads (SR) to address whether detectable sequence differences relative to canonical miRNAs found in isomiRs are true biological variations or the result of errors in sequencing. We have detected non-negligible systematic differences between SR and PE data which primarily affect putative internally edited isomiRs, and at a much smaller frequency terminal length changing isomiRs. This is relevant for the identification of true isomiRs in small RNA sequencing datasets.ConclusionsWe conclude that potential artifacts derived from sequencing errors and/or data processing could result in an overestimation of abundance and diversity of miRNA isoforms. Efforts in annotating the isomiRnome should take this into account.
| S-EPMC8077951 | biostudies-literature
Project description:In the recent past, the impact of isomiR expression changes in cancer has gained increasing interest. Here, we stably overexpress pre-miR-1307, a pre-miRNA which is frequently upregulated in cancer, and assess the effect on isomiR expression. While we see robust and significant upregulation of the canonical form miR-1307-3p|0 and its abundant 5'isomiR miR-1307-3p|1, as well as of the canonical miR from the 5p arm (miR-1307-5p|0), the overexpression of pre-miR-1307 did not lead to global alterations in isomiR expression patterns. These data validate the suitability of our model cell line for the analysis of the effects of joint upregulation of miR-1307-3p|0, -3p|1 and -5p|0.
2024-01-11 | GSE227354 | GEO
Project description:miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, so-called 5âisomiRs exhibit a shifted 5â end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5âisomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5âisomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. MiRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5âisomiR-140-3p were highly expressed in patients compared to normal breast tissue. In the current work, we present the functional characterization of 5âisomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa miR 140-3p, overexpression of the 5âisomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5âisomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5âismoiR-140-3p overexpression was found to cause a decrease in cell migration in MCF10A cells. We identified three novel direct target genes of the 5â isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5âisomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. In summary, this work presents evidence that there is a functional synergy between the canonical hsa-miR-140-3p and the newly identified 5âisomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration. With this array, we aimed to address the question which genes are regulated by either of the two forms of the miRNA. Samples were measured in one biological replicate of cells transfected with mimic-ctrl1 and mimic-ctrl2 (Dharmacon) as control samples and two biological replicates of cells transfected with hsa-miR-140-3p and 5'isomiR-140-3p (Exiqon) in 30nM concentration using Lipofectamin 2000 as transfection reagent.
2015-10-31 | E-GEOD-74539 | biostudies-arrayexpress
Project description:miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, so-called 5’isomiRs exhibit a shifted 5’ end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5’isomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5’isomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. MiRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5’isomiR-140-3p were highly expressed in patients compared to normal breast tissue. In the current work, we present the functional characterization of 5’isomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa miR 140-3p, overexpression of the 5’isomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5’isomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5’ismoiR-140-3p overexpression was found to cause a decrease in cell migration in MCF10A cells. We identified three novel direct target genes of the 5’ isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5’isomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. In summary, this work presents evidence that there is a functional synergy between the canonical hsa-miR-140-3p and the newly identified 5’isomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration. With this array, we aimed to address the question which genes are regulated by either of the two forms of the miRNA.
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Project description:Identification of 5'isomiR in HCC patients.
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Project description:The Cancer Genome Atlas (TCGA) Isoform Expression Quantification Data is the largest ressource of isomiR level sequenced cancer data publicly available. Since the datasets were built up over years and through different contributing institutions, it is not free of batch effects. We evaluated different batch correction approaches to remove batch effects in the data, details of the best performing algorithm and batch variables are included in the supplementary file. Additionally, annotation of the chromosomal end position of each isomiR feature was corrected by the offset of 1 to account for exclusive annotation.
2021-02-17 | GSE164767 | GEO
Project description:Previously Enterobase - NICD, A. Smith. Salmonella
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Project description:microRNAs (miRNAs) play an important role as key regulators controlling the post-transcriptional events in plants across development, abiotic and biotic stress, tissue polarity and also in defining the evolutionary basis of the origin of the post-transcriptional machinery. Identifying patterns of regulated and co-regulated small RNAs, in particular miRNAs and their sequence variants with the availability of next generation sequencing approaches has widely demonstrated the role of miRNAs and their temporal regulation in maintaining plant development and their response to stress conditions. Although the role of canonical miRNAs has been widely explored and functional diversity is revealed, those works for isomiRs are still limited and urgent to be carried out across plants. This relative lack of information with respect to isomiRs might be attributed to the non-availability of large-scale detection of isomiRs across wide plant species. In the present research, we addressed this by developing Plant isomiR Atlas, which provides large-scale detection of isomiRs across 23 plant species utilizing 677 smallRNAs datasets and reveals a total of 98,374 templated and non-templated isomiRs from 6,167 precursors. Plant isomiR Atlas provides several visualization features such as species specific isomiRs, isomiRs and canonical miRNAs overlap, terminal modification classifications, target identification using psRNATarget and TargetFinder and also canonical miRNAs:target interactions. Plant isomiR Atlas will play a key role in understanding the regulatory nature of miRNAome and will accelerate to understand the functional role of isomiRs. Plant isomiR Atlas is available at www.mcr.org.in/isomir. One Sentence Summary Plant isomiR Atlas will play a key role in understanding the regulatory nature of miRNAome and will accelerate the understanding and diversity of functional targets of plants isomiRs.
| S-EPMC6349829 | biostudies-literature
Project description:MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.
2014-03-28 | GSE56300 | GEO