RNA-seq

Updated by Hongjiang on 09/17/2022

Bulk RNA-seq is a common sequencing technique for transcriptome analysis.

mRNA were enriched using ploy-T tails and reverse transcripted to cDNA.

Upstream analysis

Upstream means the QC & mapping of raw sequcening data.

fastp

seqkit

STAR

Salmon

Salmon documentation

Salmon Github

Salmon paper (2017,NBT)

Salmon is a novel, fast, accuarte sofeware published recently. It's developed for TPM/counts calculation.

To install Salmon using conda:

conda install salmon

Salmon uses transcriptome FASTA for analysis. You can download cdna files directly from ensembl.org.

If you have access to the genome FASTA and GTF used for alignment consider generating a transcriptome FASTA using a command like:

gffread -w output.fa -g genome.fa genome.gtf

you can find the gffread utility at (http://ccb.jhu.edu/software/stringtie/gff.shtml), which could be installed by conda install gffread.

Rsem

Downstream analysis

edgeR

edgeR Users Guide

使用ggplot2和ggrepel包做火山图

ClusterProfiler

bioconductor

Github

Manual

Install

BiocManager::install("clusterProfiler")
BiocManager::install("pathview")
BiocManager::install("org.Hs.eg.db") # Homo sapiens

org.xx.eg.db

You can search org.xx.eg.db on Bioconductor, below are some frequently used ones.

BiocManager::install("org.Hs.eg.db") # Homo sapiens
BiocManager::install("org.Mm.eg.db") # Mus musculus
BiocManager::install("org.Rn.eg.db") # Rattus norvegicus