Introduction to Transcriptomics: What is RNA-Seq?
RNA sequencing (RNA-Seq), also known as whole-transcriptome sequencing, is a high-throughput sequencing technique used to analyze the RNA composition of a sample. This method provides insights into RNA abundance and composition within a cell and enables the identification and quantification of all RNA molecules present at a specific time point or under particular conditions, including mRNA, non-coding RNA, and small RNA.
RNA-Seq begins with RNA extraction and purification. Subsequently, reverse transcription is performed to convert RNA into complementary DNA (cDNA). To facilitate sequencing, the cDNA is fragmented into shorter sequences, which are then processed by a sequencing platform, typically generating millions to billions of short reads. These reads are then aligned to a reference genome or assembled de novo into transcripts.
A key advantage of RNA-Seq is its broad dynamic range, which allows for the detection of genes expressed at both low and high levels without requiring prior knowledge, such as predefined gene expression probes. This method enables the identification of novel transcripts, alternative splicing variants, and single nucleotide polymorphisms (SNPs) and can also be used to detect gene fusion events. By analyzing sequencing data across different samples, RNA-Seq facilitates the identification of differentially expressed genes, which is crucial for elucidating disease mechanisms and identifying potential drug targets.
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