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RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome.
The binding of the ADAR2 enzyme to the RNA timestamp initiates the gradual conversion of adenosine to inosine molecules. Over time, these edits accumulate and are then read through RNA-seq. This technology allows us to glean cell-type specific temporal information associated with RNA-seq data, that until now, has not been accessible. [1]
Currently RNA-Seq relies on copying RNA molecules into cDNA molecules prior to sequencing; therefore, the subsequent platforms are the same for transcriptomic and genomic data. Consequently, the development of DNA sequencing technologies has been a defining feature of RNA-Seq. [ 78 ] [ 80 ] [ 81 ] Direct sequencing of RNA using nanopore ...
Normally, in a traditional RNA-seq, microarray, or SAGE experiment RNA is extracted from a biological sample such as cultured cells, and the RNA is analyzed using the chosen method. The data obtained from such an experiment corresponds to abundance of RNA under the given experimental conditions at the time of harvest.
Sequencing occurs as normal and the data can be analyzed as appropriate for its use. This basic snRNA-seq methodology is capable of profiling RNA from tissues that are preserved or cannot be dissociated, but it does not have high throughput capability due to its reliance on nuclei sorting by FACS. [3]
DESeq2 is a software package in the field of bioinformatics and computational biology for the statistical programming language R.It is primarily employed for the analysis of high-throughput RNA sequencing (RNA-seq) data to identify differentially expressed genes between different experimental conditions.
Normalisation of RNA-seq data accounts for cell to cell variation in the efficiencies of the cDNA library formation and sequencing. One method relies on the use of extrinsic RNA spike-ins (RNA sequences of known sequence and quantity) that are added in equal quantities to each cell lysate and used to normalise read count by the number of reads ...
The final step regards analysis of data and storage: after obtaining the sequencing reads, UMI and index sequences are automatically removed from the reads and their quality is analyzed by PHRED (software able to evaluate the quality of the sequencing process); reads can then be mapped or aligned to a reference genome in order to extract ...
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