Biological replicates in RNA-seq

Question

[Mars]

After googling these questions for ages I have not found an answer so I assume it's extremely simple and I apologise in advance.

What do I do with biological replicates for RNA-seq? Do I just combine them? And how do I do what I need to do (which tools)?

I am completely new to RNA-seq analysis and I need to compare one condition to another. Specifically, I want to compare splicing in I have 2 developmental stages, 2 conditions and 3 replicates for each so I am very confused.

Thank you very much and apologies for the silly question.

[Matthias Zepper]

At which stage of the analysis are you? Do you have a count matrix or the raw sequencing reads?

[jv]

start here: https://bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html. Do not skip or overlook QC and exploratory data analysis.

Answer 1

[mark.ziemann]

For each sample you will have two fastq files, one corresponding to the forward read (R1) and reverse (R2). The general idea is to use an aligner to map the pair of reads to the reference transcriptome/genome so that the number of detections of each transcript/gene can be quantified. A good mapper is Kallisto (transcript-level), another is STAR (gene-level). The result of this process will be a table of counts for that sample. This process is repeated for each sample so that you have count tables for all samples. Then you can load all of this data into a statistical analysis environment like R, where you can use a program like DESeq2 to compare sample groups.