Name: Peak Calling and Annotation in SCRAP for Small Noncoding RNA: Target RNA Interaction Studies
Uploaded: 2023-12-01T14:40:00
Description: Watch this Scientific Journal Video about Peak Calling and Annotation in SCRAP for Small Noncoding RNA: Target RNA Interaction Studies at JoVE.com

peak calling and annotation in scrap for small noncoding rna: target rna interaction studies

In vivo RNA: RNA-Interaktionen mit der SCRAP Bioinformatics Pipeline

Small noncoding RNAs are highly studied for their post-transcriptional roles in coordinating expression from suites of genes in diverse processes such as differentiation and development, signal processing, and disease1,2,3. The ability to accurately determine the target transcripts of gene-regulatory small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), is of importance to studies of RNA biology at both basic and translational levels. Bioinformatic algorithms that exploit anticipated complementarity between the miRNA seed sequence and its potential targets have been frequently used for the prediction of miRNA:target RNA interactions. While these bioinformatic algorithms have been successful, they also can harbor both false positive and false negative results, as has been reviewed elsewhere4,5,6. Recently, several biochemical approaches have been designed and implemented that allow unambiguous and semiquantitative determination of in vivo sncRNA:target RNA interactions by in vivo crosslinking and ensuing incorporation of a ligation step to physically attach the sncRNA to its target to form a single chimeric RNA4,5,7,8,9,10. Subsequent preparation of sequencing libraries from the chimeric RNAs allows assessment of the sncRNA:target RNA interactions by computational processing of the sequencing data. This video provides a tutorial for installing and using a computational pipeline termed small chimeric RNA analysis pipeline (SCRAP), which is designed to allow robust and reproducible analysis of sncRNA:target RNA interactions from chimeric RNA sequencing libraries6.
A goal of this tutorial is to assist investigators in avoiding excessive reliance on purely predictive bioinformatic algorithms by lowering barriers to the analysis of data generated through biochemical approaches providing chimeric molecular readouts of sncRNA:target RNA interactions. This tutorial provides practical steps and tips to guide entry-level computational scientists through the use of a pipeline, SCRAP, developed for analyzing chimeric RNA sequencing data, which can be generated by several existing biochemical protocols, including crosslinking, ligation, and sequencing of hybrids (CLASH) and covalent ligation of endogenous Argonaute-bound RNAs- crosslinking and immunoprecipitation (CLEAR-CLIP)7,9.
The use of SCRAP offers several advantages for the analysis of chimeric RNA sequencing data, compared to other computational pipelines6. One salient advantage is its extensive annotation and the incorporation of call-outs to well-supported and routinely updated bioinformatic scripts within the pipeline, in comparison to alternative pipelines that often rely on custom and/or unsupported scripts for steps in the pipeline. This feature lends stability to SCRAP, making it more worthwhile for researchers to familiarize themselves with the pipeline and to incorporate its use into their workflow. SCRAP has also been demonstrated to outperform alternative pipelines in calling peaks of sncRNA:target RNA interactions and to have cross-platform functionality, as detailed in a prior publication6.
By the end of this tutorial, users will be able to (i) know platform requirements for SCRAP and install SCRAP pipelines, (ii) install reference genomes and set up command line parameters for SCRAP, and (iii) understand peak calling criteria and perform peak calling and peak annotation.
This video will describe in practical detail how researchers studying RNA biology may install and optimally use the computational pipeline, SCRAP, to analyze sncRNA interactions with target RNAs, such as messenger RNAs, in chimeric RNA-sequencing data obtained through one of the discussed biochemical approaches to sequencing library preparation.
SCRAP is a command line utility. Generally, following the guide below, the user will need to (i) download and install SCRAP (https://github.com/Meffert-Lab/SCRAP), (ii) Install reference genomes and run SCRAP, and (iii) perform peak calling and annotation.
Further details of the computational steps in this procedure can be found at&#160;https://github.com/Meffert-Lab/SCRAP. This article will provide the setup and background information to allow investigators with entry-level computational skills to install, optimize, and use SCRAP on chimeric RNA sequencing library datasets.

Autor im Rampenlicht: Eine computergestützte Pipeline zur Analyse chimärer nichtkodierender RNA-Ziel-RNA-Interaktionen in Hochdurchsatz-Sequenzierungsdaten 

Computergestütztes Analyse-Tutorial für chimäre kleine nichtkodierende RNA: Ziel-RNA-Sequenzierungsbibliotheken

This protocol covers the installation and practical steps for the use of a computational pipeline designed to analyze high throughput sequencing data from chimeric noncoding RNA-target RNA-interactions. The molecular strategy to form chimeric RNAs is a relatively recent development that has advantages when compared to prior biochromatic prediction tools in unambiguously understanding the genome-wide noncoding RNA-target RNA-interaction landscape. Sequencing data generated from the high throughput sequencing of the chimeric RNAs can present an analysis challenge for new adopters of this strategy.We have established an open source cross-platform computational pipeline that is highly annotated to allow entry-level users to analyze data from their chimeric noncoding RNA-target RNA sequencing experiments. In future experiments, the Mefford lab plans to use computational analysis by scrap on dataset generated from our ongoing investigations into post transcriptional regulation in the mammalian nervous system, as well as to maintain an update scrap on our GitHub.

Watch this Scientific Journal Video about Peak Calling and Annotation in SCRAP for Small Noncoding RNA: Target RNA Interaction Studies at JoVE.com

Peak Calling and Annotation in SCRAP for Small Noncoding RNA: Target RNA Interaction Studies  

Peak-Calling und Annotation in SCRAP für kleine nicht-kodierende RNA: Target-RNA-Interaktionsstudien

Verwenden Sie nach dem Ausführen von SCRAP den angegebenen Skriptbefehl, um Peak-Aufrufe durchzuführen. Listen Sie die vollständigen Pfade zu dem Verzeichnis auf, das die Beispielordner und die Adapterdatei enthält. Definieren Sie dann die Kriterien für den Peak-Aufruf, einschließlich der Mindestanzahl von Sequenzierungslesevorgängen und der Anzahl der unterschiedlichen Sequenzierungsbibliotheken, die erforderlich sind, damit ein Peak erkannt wird.Geben Sie an, ob sncRNAs derselben Familie zu den Peaks beitragen müssen, was für miRNAs relevant ist, die Seed-Sequenzen teilen und an überlappende Genziele binden können. Geben Sie dann den vollständigen Pfad zum Referenzverzeichnis, die Abkürzung der MIR-Basis und die Abkürzung des Referenzgenoms an. Führen Sie nach dem Peak-Aufruf das Peak-Annotationsskript aus.Geben Sie den vollständigen Pfad zu den resultierenden Peaks an. Bett vom Peak-Aufruf bis zum Referenzverzeichnis und der gewünschten Art für die Annotation. Verwenden Sie samtools merge, um alle gewünschten BAM-Dateien zusammenzuführen, um eine kombinierte Visualisierung zu erleichtern.Verwenden Sie dann die samtools-Sortierung für die zeilenweise Sortierung der zusammengeführten. BAM-Datei. Indizieren Sie die sortierten.BAM-Datei unter Verwendung des samtools-Index, die eine binäre Indexdatei im samtools-Format generiert, die für genomische Visualisierungstools erforderlich ist. Öffnen Sie abschließend im integrativen Genomik-Viewer die sortierte. bam und indiziert.nach Datei. Die Anwendung einer modifizierten Version von SCRAP auf zuvor veröffentlichte Sequenzierungsdatensätze zeigte eine Abnahme der miRNA-Wechselwirkungen mit Intron-Regionen. Die Reduktion wurde nach der Isolierung von Interaktionen mit hoher Konfidenz durch Peak-Anrufe mit SCRAP beobachtet.

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JoVE Journal

Biology

Peak Calling and Annotation in SCRAP for Small Noncoding RNA: Target RNA Interaction Studies

Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries

An understanding of the in vivo gene regulatory interactions of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs), with their target RNAs has ...