子宫内膜癌的RNA editing数据分析

参考文献

[PMID: 27694136]
DRETools - F1000Research

数据下载

使用本实验室一个RNA-seq（GSE56087）和一个circRNA-seq（未发布）的数据作为discovery dataset 和 validation dataset。

RNA-seq数据下载

shell命令批量下载

#!/bin/sh
#调用prefetch下载SRA数据库的测序文件，并用pfastq-dump转化为fastq文件。
#需要在当前目录的download_list.txt文件中把SRA编号，每个一行事先准备好，具体格式参考example.txt
#建议使用NCBI的SRA RUN SELECTOR直接导出。
read  -p "请输入下载列表文件名:" downloadlist
read  -p "请选择单端(SE)，双端(PE)测序:" class
read  -p "请输入要使用的线程数:" thread
if [ $class = "SE" ]; then
    cat $downloadlist | while read line
  do
    echo $line 
    prefetch $line -o ./${line}.sra
    pfastq-dump --threads $thread --outdir ./ ./${line}.sra
  done
elif [ $class = "PE" ]; then
    cat $downloadlist | while read line
  do
    echo $line
    prefetch $line -o ./${line}.sra
    pfastq-dump --split-3 --threads $thread --outdir ./ ./${line}.sra 
  done
else echo "错误的输入！"
fi

相应的accession number

SRR1200850
SRR1200851
SRR1200852
SRR1200853
SRR1200854
SRR1200855
SRR1200856
SRR1200857
SRR1200860
SRR1200861
SRR1200858
SRR1200859
SRR1200862
SRR1200863
SRR1200864
SRR1200865
SRR1200866
SRR1200867
SRR1200868
SRR1200869
SRR1200870
SRR1200871
SRR1200872
SRR1200873
SRR1200874
SRR1200875
SRR1200876
SRR1200877
SRR1200878
SRR1200879
SRR1200880
SRR1200881
SRR1200882
SRR1200883
SRR1200884
SRR1200885

数据分析

主要使用RNAEditor软件分析，环境配置按照官网的配置，注意相应软件的版本，命令需要在RNAEditor目录下运行，并且要求不能在SSH界面下运行，会报错（无法画图），需要在图形界面下打开终端运行。
shell批量分析

for ((i=1200851;i<1200886;i++))
do
fastp -w 16 -i ~/RNASEQ/EC_lina/SRR${i}_1.fastq -I ~/RNASEQ/EC_lina/SRR${i}_2.fastq -o ~/RNASEQ/EC_lina/SRR${i}_1_fastpedited.fastq -O ~/RNASEQ/EC_lina/SRR${i}_2_fastpedited.fastq 
python RNAEditor.py -i ~/RNASEQ/EC_lina/SRR${i}_1_fastpedited.fastq ~/RNASEQ/EC_lina/SRR${i}_2_fastpedited.fastq -c configuration.txt
done

配置文件configuration.txt如下

#This file is used to configure the behaviour of RNAeditor

#Standard input files
refGenome = /home/zhou/rnaEditor_annotations/human/GRCH38/Homo_sapiens.GRCh38.dna.primary_assembly.fa
gtfFile = /home/zhou/rnaEditor_annotations/human/GRCH38/Homo_sapiens.GRCh38.83.gtf
dbSNP = /home/zhou/rnaEditor_annotations/human/GRCH38/dbSNP.vcf
hapmap = /home/zhou/rnaEditor_annotations/human/GRCH38/HAPMAP.vcf
omni = /home/zhou/rnaEditor_annotations/human/GRCH38/1000GenomeProject.vcf
esp = /home/zhou/rnaEditor_annotations/human/GRCH38/ESP_filtered
aluRegions = /home/zhou/rnaEditor_annotations/human/GRCH38/repeats.bed
output = default
sourceDir = /usr/local/bin/
maxDiff = 0.04
seedDiff = 2
standCall = 0
standEmit = 0
edgeDistance = 3
paired = True 
keepTemp = True
overwrite = False
threads = 23

开23个核，每个样本分析时间大概在十个小时左右。

结果解析

结果实例

image.png

VCF文件

VCF文件包含所有的编辑站点以供进一步分析.

image.png

GCF文件

GCF文件保存了每个编辑站点的附加信息，如基因名称、片段、总读数、编辑读数和编辑比。

image.png

summary文件

summary文件显示每个基因的RNA编辑所处基因位置（如3‘UTR，5‘UTR）的数量。

image.png

bed文件

Editing island的bed文件，可供后续可视化等分析。

image.png

打包除了bam，sam，sai这两种文件以外的所有结果

tar -cvf RNAEditor_1.tar ~/RNASEQ/EC_lina/rnaEditor/ --exclude=*bam --exclude=*sam --exclude=*sai
pigz RNAEditor_1.tar

circRNA-seq数据集的验证工作

shell批量分析

#!/bin/sh
#把fastq文件路径存入数组
c=0
for file in `find ~/RNASEQ/CircularRNA-seq/rawdata/ -name *fastq.gz -print`
do
  filelist[$c]=$file
  ((c++))
done

for ((i=0;i<${#filelist[@]};i=i+2))
    do
        tmp1=$(echo ${filelist[$i]}|sed 's/\.fastq\.gz/\_fastpedited\.fastq/g')
        tmp2=$(echo ${filelist[$i+1]}|sed 's/\.fastq\.gz/\_fastpedited\.fastq/g')
    #fastp预处理
        fastp -w 16 -i ${filelist[$i]} -I ${filelist[$i+1]} -o $tmp1 -O $tmp2 
        #pigz -d $tmp1
        #pigz -d $tmp2
        python RNAEditor.py -i $tmp1 $tmp2 -c configuration.txt
    done

configuration.txt不变

打包除了bam，sam，sai这两种文件以外的所有结果

tar -cvf RNAEditor_2.tar ~/RNASEQ/CircularRNA-seq/rawdata/Sample_ZYF-*/rnaEditor --exclude=*bam --exclude=*sam --exclude=*sai
pigz RNAEditor_2.tar

后续分析

DREtools差异分析

#Merge editing sites from multiple samples
dretools edsite-merge     --min-editing  3      --min-coverage 5      --min-samples  3      --vcf ./*/*.editingSites.vcf > DRE/consensus_sites.vcf
#Calculate sample-wise EPK
for i in `find ./SRR*/ -name *fastpedited.bam -print` ; 
do  
name=$(echo ${i}|sed 's/_1.*bam//'|sed 's/\.\///');  
dretools sample-epk --name $name --vcf ./DRE/consensus_sites.vcf --alignment $i > ./DRE/${name}.sample_epk.tsv; 
done
#Calculate site-wise EPK
for i in `find ./SRR*/ -name *fastpedited.bam -print` ; 
do 
name=$(echo ${i}|sed 's/_1.*bam//'|sed 's/\.\///');  
dretools edsite-epk --vcf ./DRE/consensus_sites.vcf --alignment $i > ./DRE/${name}.edsite_epk.tsv; 
done
#Find differentially edited editing sites
dretools edsite-diff                    --max-depth-cov 5.0                 --min-depth 2       \
--names normal,tumor \
--sites ./DRE/consensus_sites.vcf \
--sample-epk ./DRE/SRR1200850.sample_epk.tsv,./DRE/SRR1200851.sample_epk.tsv,./DRE/SRR1200854.sample_epk.tsv,./DRE/SRR1200855.sample_epk.tsv,./DRE/SRR1200858.sample_epk.tsv,./DRE/SRR1200859.sample_epk.tsv,./DRE/SRR1200862.sample_epk.tsv,./DRE/SRR1200863.sample_epk.tsv,./DRE/SRR1200866.sample_epk.tsv,./DRE/SRR1200867.sample_epk.tsv,./DRE/SRR1200872.sample_epk.tsv,./DRE/SRR1200873.sample_epk.tsv,./DRE/SRR1200874.sample_epk.tsv,./DRE/SRR1200875.sample_epk.tsv,./DRE/SRR1200878.sample_epk.tsv,./DRE/SRR1200879.sample_epk.tsv,./DRE/SRR1200882.sample_epk.tsv,./DRE/SRR1200883.sample_epk.tsv \
./DRE/SRR1200852.sample_epk.tsv,./DRE/SRR1200853.sample_epk.tsv,./DRE/SRR1200856.sample_epk.tsv,./DRE/SRR1200857.sample_epk.tsv,./DRE/SRR1200860.sample_epk.tsv,./DRE/SRR1200861.sample_epk.tsv,./DRE/SRR1200864.sample_epk.tsv,./DRE/SRR1200865.sample_epk.tsv,./DRE/SRR1200868.sample_epk.tsv,./DRE/SRR1200869.sample_epk.tsv,./DRE/SRR1200870.sample_epk.tsv,./DRE/SRR1200871.sample_epk.tsv,./DRE/SRR1200876.sample_epk.tsv,./DRE/SRR1200877.sample_epk.tsv,./DRE/SRR1200880.sample_epk.tsv,./DRE/SRR1200881.sample_epk.tsv,./DRE/SRR1200884.sample_epk.tsv,./DRE/SRR1200885.sample_epk.tsv  \
--site-epk ./DRE/SRR1200850.edsite_epk.tsv,./DRE/SRR1200851.edsite_epk.tsv,./DRE/SRR1200854.edsite_epk.tsv,./DRE/SRR1200855.edsite_epk.tsv,./DRE/SRR1200858.edsite_epk.tsv,./DRE/SRR1200859.edsite_epk.tsv,./DRE/SRR1200862.edsite_epk.tsv,./DRE/SRR1200863.edsite_epk.tsv,./DRE/SRR1200866.edsite_epk.tsv,./DRE/SRR1200867.edsite_epk.tsv,./DRE/SRR1200872.edsite_epk.tsv,./DRE/SRR1200873.edsite_epk.tsv,./DRE/SRR1200874.edsite_epk.tsv,./DRE/SRR1200875.edsite_epk.tsv,./DRE/SRR1200878.edsite_epk.tsv,./DRE/SRR1200879.edsite_epk.tsv,./DRE/SRR1200882.edsite_epk.tsv,./DRE/SRR1200883.edsite_epk.tsv \
./DRE/SRR1200852.edsite_epk.tsv,./DRE/SRR1200853.edsite_epk.tsv,./DRE/SRR1200856.edsite_epk.tsv,./DRE/SRR1200857.edsite_epk.tsv,./DRE/SRR1200860.edsite_epk.tsv,./DRE/SRR1200861.edsite_epk.tsv,./DRE/SRR1200864.edsite_epk.tsv,./DRE/SRR1200865.edsite_epk.tsv,./DRE/SRR1200868.edsite_epk.tsv,./DRE/SRR1200869.edsite_epk.tsv,./DRE/SRR1200870.edsite_epk.tsv,./DRE/SRR1200871.edsite_epk.tsv,./DRE/SRR1200876.edsite_epk.tsv,./DRE/SRR1200877.edsite_epk.tsv,./DRE/SRR1200880.edsite_epk.tsv,./DRE/SRR1200881.edsite_epk.tsv,./DRE/SRR1200884.edsite_epk.tsv,./DRE/SRR1200885.edsite_epk.tsv  \
> ./DRE/diff_sites.tsv
#Detect editing islands
dretools find-islands \
    --min-editing 3   \
    --min-coverage 5  \
    --min-length 20   \
    --min-points 5    \
    --epsilon 50      \
    --vcf ./*/*.editingSites.vcf  > ./DRE/islands.bed
#Calculate Island-EPK
for i in `find ./SRR*/ -name *fastpedited.bam -print` ; 
do  
name=$(echo ${i}|sed 's/_1.*bam//'|sed 's/\.\///');  
dretools region-epk --regions ./DRE/islands.bed --vcf ./DRE/consensus_sites.vcf --alignment $i > ./DRE/${name}.island_epk.tsv; 
done
#Find differentially edited islands
dretools region-diff         --regions  ./DRE/islands.bed           --min-area  1                 --min-depth 1       \
--names normal,tumor \
--sample-epk ./DRE/SRR1200850.sample_epk.tsv,./DRE/SRR1200851.sample_epk.tsv,./DRE/SRR1200854.sample_epk.tsv,./DRE/SRR1200855.sample_epk.tsv,./DRE/SRR1200858.sample_epk.tsv,./DRE/SRR1200859.sample_epk.tsv,./DRE/SRR1200862.sample_epk.tsv,./DRE/SRR1200863.sample_epk.tsv,./DRE/SRR1200866.sample_epk.tsv,./DRE/SRR1200867.sample_epk.tsv,./DRE/SRR1200872.sample_epk.tsv,./DRE/SRR1200873.sample_epk.tsv,./DRE/SRR1200874.sample_epk.tsv,./DRE/SRR1200875.sample_epk.tsv,./DRE/SRR1200878.sample_epk.tsv,./DRE/SRR1200879.sample_epk.tsv,./DRE/SRR1200882.sample_epk.tsv,./DRE/SRR1200883.sample_epk.tsv \
./DRE/SRR1200852.sample_epk.tsv,./DRE/SRR1200853.sample_epk.tsv,./DRE/SRR1200856.sample_epk.tsv,./DRE/SRR1200857.sample_epk.tsv,./DRE/SRR1200860.sample_epk.tsv,./DRE/SRR1200861.sample_epk.tsv,./DRE/SRR1200864.sample_epk.tsv,./DRE/SRR1200865.sample_epk.tsv,./DRE/SRR1200868.sample_epk.tsv,./DRE/SRR1200869.sample_epk.tsv,./DRE/SRR1200870.sample_epk.tsv,./DRE/SRR1200871.sample_epk.tsv,./DRE/SRR1200876.sample_epk.tsv,./DRE/SRR1200877.sample_epk.tsv,./DRE/SRR1200880.sample_epk.tsv,./DRE/SRR1200881.sample_epk.tsv,./DRE/SRR1200884.sample_epk.tsv,./DRE/SRR1200885.sample_epk.tsv  \
--region-epk  ./DRE/SRR1200850.island_epk.tsv,./DRE/SRR1200851.island_epk.tsv,./DRE/SRR1200854.island_epk.tsv,./DRE/SRR1200855.island_epk.tsv,./DRE/SRR1200858.island_epk.tsv,./DRE/SRR1200859.island_epk.tsv,./DRE/SRR1200862.island_epk.tsv,./DRE/SRR1200863.island_epk.tsv,./DRE/SRR1200866.island_epk.tsv,./DRE/SRR1200867.island_epk.tsv,./DRE/SRR1200872.island_epk.tsv,./DRE/SRR1200873.island_epk.tsv,./DRE/SRR1200874.island_epk.tsv,./DRE/SRR1200875.island_epk.tsv,./DRE/SRR1200878.island_epk.tsv,./DRE/SRR1200879.island_epk.tsv,./DRE/SRR1200882.island_epk.tsv,./DRE/SRR1200883.island_epk.tsv \
./DRE/SRR1200852.island_epk.tsv,./DRE/SRR1200853.island_epk.tsv,./DRE/SRR1200856.island_epk.tsv,./DRE/SRR1200857.island_epk.tsv,./DRE/SRR1200860.island_epk.tsv,./DRE/SRR1200861.island_epk.tsv,./DRE/SRR1200864.island_epk.tsv,./DRE/SRR1200865.island_epk.tsv,./DRE/SRR1200868.island_epk.tsv,./DRE/SRR1200869.island_epk.tsv,./DRE/SRR1200870.island_epk.tsv,./DRE/SRR1200871.island_epk.tsv,./DRE/SRR1200876.island_epk.tsv,./DRE/SRR1200877.island_epk.tsv,./DRE/SRR1200880.island_epk.tsv,./DRE/SRR1200881.island_epk.tsv,./DRE/SRR1200884.island_epk.tsv,./DRE/SRR1200885.island_epk.tsv  \
> ./DRE/diff_islands.tsv

DREtools结果展示

image.png

MET-DB交集