spaceladymehwish / step-by-step-rnaseq-analysis

Step by Step RNASeq analysis

RNA-seq analysis involves several steps that ensure the quality and accuracy of the data. Here is a step-by-step guide to RNA-seq analysis:

1. Experiment Planning Determine the method of RNA purification. Decide on the read depth needed. Choose the sequencing platform. Ensure a reference genome is available. Assess the quality of RNA. Decide if enrichment or barcoding is necessary. Determine the number of biological and technical replicates. Decide on single-end or paired-end sequencing. Determine the read length. Decide if strand-specific information is required.
2. RNA Extraction and Conversion Extract RNA from the sample. Convert RNA into complementary DNA (cDNA) using reverse transcription.
3. Library Preparation Fragment the cDNA. Ligate adapters. Ligate indexes. Perform quality control checks.
4. Sequencing Use the prepared library for sequencing on the chosen platform.
5. Alignment Align the RNA-seq reads to the reference genome using a splice-aware aligner like STAR.
6. Quality Control Perform pre-alignment quality control (QC) checks. Post-alignment QC checks. Visualize the data. Quantify transcripts. Visualize tracks against the reference genome.
7. Data Analysis Use tools like DESeq2 for differential gene expression analysis. Perform gene enrichment analyses. Identify differentially expressed genes (DEGs). Visualize the data using plots like volcano plots and heatmaps.
8. Interpretation Ensure the results make biological sense. Be cautious of automated tools and their limitations. Verify the results by cross-checking with other methods.
9. Visualization and Reporting Use tools like IGV for visual inspection. Generate reports and visualizations to present the findings.
10. Validation Validate the results by verifying the expression levels of known genes. Validate the results by comparing with other methods or datasets. These steps ensure that RNA-seq data is accurately analyzed and interpreted to gain insights into gene expression and biological processes.