RNA-seq-2023

This is my RNA-seq analysis update library

author:zhang jian date:2023.6.19 version:1.1v e-mail:[email protected]

this is my RNA-seq analysis protocol (SHELL + R)

THIS LIBRARY INCLUDE 2 PART

1.RNA-SEQ UPSTEAM ANALYSIS 1) RAW DATA QC scripts； 2）raw DATA QC clean scripts; 3) clean data QC scripts; 4) remove MT-RNA scripts(option); 5) mapping to genome by (HISAT2 or STAR); 6) count by featurecount.

2.RNA-SEQ DOWNSTEAM ANALYSIS 1) general DEG analysis R scripts； 2）GO KEGG GSEA general R scripts; 3) other analysis scripts

need run env

shell env softaqre:

1) fastqc
2) multiqc
3) cutadapt
4) hisat2
5) STAR
6) featureCounts

R package

crayon,praise,progress,DESeq2,dbplyr,FactoMineR,plyr,stringr,Hmisc,biomaRt,ggridges,latex2exp,ggpubr,ggthemes,ggrepel,rtracklayer,ggridges,RColorBrewer,pheatmap,dplyr.plotly,htmlwidgetsshiny,reshape2,clusterProfiler,topGO,dbplyr,pathview,DOSE,org.Mm.eg.db,tidyr,dplyr,forcats,ggplot2,ggpubr,enrichplot,cowplot,stringr,RColorBrewer

RNA-SEQ UPSTEAM ANALYSIS

UPSTEAM ANALYSIS SCRIPTS

# 1. downloda data form paper or seq company 
  you can use RNA-SEQ-UPSTEAM folder STEP1.2.Dowmload_data_form_EMBI-EBI.sh or STEP1.1.Dowmload_data_huawei_OBS.sh scripts 

# 2.create 1.raw_data folder & move you fq.gz file in this folder

# 3.use FASTQC & MULTIQC QC raw-data
  you can use RNA-SEQ-UPSTEAM folder STEP2.1.Data_1st_qc_by_fastqc.sh scripts
  
# 4.use cutadapter clean data
  you can use RNA-SEQ-UPSTEAM folder STEP2.2.Cutadapt_raw_data_by_cutadapt_1.0V.sh & STEP2.3.Readapter_data_qc_by_fastqc.sh remove low quality read and adapter & qc clean data result
  
# 5.use cutadapt remove 5' 15bp read (option)
 you can use RNA-SEQ-UPSTEAM folder STEP2.4.Cutadapt_option_rm_5_15bp_by_cutadapt_1.0V.sh & STEP2.5.Rm5_15bp_fastqc_report.sh
 
# 6.remove MT GENOME read if you qc result have MT-RNA-READ YOU should use down scripts remove mt-rna
  RNA-SEQ-UPSTEAM folder  STEP3.1.Build_mt-RNA-index.sh & STEP3.2.Remove_mapping_mt-RNA_hisat2.sh
  (PS:for reference genome file you can change & download for NCBI OR ENSMBL)
  
# 7.build index & mapping by HISAT2 OR STAR
  YOU can use RNA-SEQ-UPSTEAM folder include mapping or build tag scripts to mapping read to genome
  # NOTE: IF YOU BUILDING LIBRARY IS Strand-specific library preparation,you should use include dutp tag scripts
  
# 8.featurecount 
  YOU can use RNA-SEQ-UPSTEAM folder include featurecount to count featurecount 
  # NOTE: IF YOU BUILDING LIBRARY IS Strand-specific library preparation,you should use include dutp tag scripts
  # NOTE: YOU CAN USE 3 level to count RNA-seq featurecount

UPSTEAM ANALYSIS SCRIPTS

# NOTE YOU CAN CREATE SCRIPTS IN ROOT DIR
# AFTER RUN ABOVE SCRIPTS YOU CAN GET DOWN RESLUT FOLDER
1.raw_data       3.rm_adapter  6.mapping_result  7.count_exon_dutp  
2.fastqc_report  4.rm_15bp     7.count_cds_dutp  7.count_gene_dutp  scripts

DOWNSTEAM ANALYSIS SCRIPTS

# WHERE YOU FINISH UPSTEAM ANALYSIS, YOU NEED CREATE 7.sample_infor FOLDER
# 7.sample_infor FOLDER INCLUDE SAMPLE INFOR CSV FILE

7.sample_infor sample_infor.csv condition example

group_name	condition	type	patch	lane
MTco3-1_L1_Q0061W0004	MTco3	PE	1	Lane1
MTco3-2_L1_Q0061W0156	MTco3	PE	1	Lane1
MTco3-3_L1_Q0061W0159	MTco3	PE	1	Lane1
WT-1_L1_Q0060W0004	WT	PE	1	Lane1
WT-2_L1_Q0060W0156	WT	PE	1	Lane1
WT-3_L1_Q0060W0159	WT	PE	1	Lane1

# NOTE: featurecount result file = sample_infor.csv group_name
MTco3-1_L1_Q0061W0004_exon.read.count.tsv  MTco3-3_L1_Q0061W0159_exon.read.count.tsv  WT-2_L1_Q0060W0156_exon.read.count.tsv
MTco3-2_L1_Q0061W0156_exon.read.count.tsv  WT-1_L1_Q0060W0004_exon.read.count.tsv     WT-3_L1_Q0060W0159_exon.read.count.tsv

DEG SCRIPTS

Rscript 1.RNA-seq_deg_rat.r  <root-dir> <festurecount-result-dir> <sample-infor.csv path>

enrichment scripts

# this root-dir must include DEG RESULT folder
Rscript 2.RNA-seq_KEGG_GO_GSEA_rat.r <root-dir>