Gadji-M/RNA-Seq_Uganda

RNA-seq analysis pipeline to detect major differentially expressed genes associated to pyrethroid resistance escalation in An. funestus in Uganda # RNA-Seq_Uganda RNA-seq analysis pipeline to detect major differentially expressed genes associated to pyrethroid resistance escalation in An. funestus in Uganda Contact: gadji.mahamat@crid-cam.net / gadji.mahamat@fasciences-uy1.cm ##### Hey there 👋 😁 We got some new stuff to analyse and come out with DE genes associated to pyrethroid resistance escalation in Anopheles funestus in Uganda. We are describing here a simple pipeline we used to analyse our RNA-Seq data. ## Contents 1. #### Quality Control of RNA-Seq data 2. #### Alignment and statistics 3. #### Quantification of gene expression 4. #### Differential Gene Expression Analysis (DGE) 5. #### Functional Enrichment Analysis 6. #### Pathway Analysis 7. #### Visualisation and Interpretation 8. #### Validation Requirements: - FastQC - MultiQC - fastp - STAR, HISAT2 or TopHat - FeatureCounts, HTSeq or StringTie - Samtools 1.13 or latest - Bedtools v2.30.0 - Picard tool - Varscan - freebayes v1.3.6 - awk 5.1.0 - SnpEff 5.1d - R 4.2.3 or latest - DEseq2, EdgeR or Limma-voom - Enrichr, DAVID or g\:Profiler - Ingenuity Pathway Analysis (IPA), KEGG or Reactome, - Intergrative Genomic Viewer (IGV) Before beginning the analysis, please install the above packages and clone this repository in your PC using: git clone github.com ## Quality Control of RNA-Seq data Here, we applied the script `Fastq_Quality_check.sh` previously design in github.com to quality check our data. Please have a look and follow the instructions to run the command. ### Trimming data using fastp based on QC reports Here, we'll use `fastp` to trim reads from RNA-Seq data: `fastp -i Path/to/read1 R1_1.fq.gz -I Path/to/read2 R2_2.fq.gz -o Path/to/output/read1 trimmed_R1_1.fq.gz -O Path/to/output/read2 trimmed_R2_2.fq.gz -a read1_adapter_Seq --adapter_sequence_r2 read2_adapter_seq -l 25 -j Path/to/json {sample}.fastp.json -h Path/to/html/ {sample}.fastp.html -w threads` Note …