最近试了很多Smart-seq2上游处理的方法，参照了Jimmy老师的教程，以及阿则的生信小站公众号文章。对代码进行了整理。但是过程中仍然有一些问题尚待解决。

1. 数据、软件准备

• SRR文件下载到SRRfile文件下

mkdir SRRfile
prefetch --option-file SRR_Acc_List.txt

• hisat2下载

conda install hisat2

• hisat2比对索引下载

axel https://genome-idx.s3.amazonaws.com/hisat/grch38_tran.tar.gz
# wget https://genome-idx.s3.amazonaws.com/hisat/grch38_tran.tar.gz

• conda安装其他软件

conda install fastqc multiqc samtools sambamba featureCounts

2. 数据处理

• 解压缩

gunzip *.gz

• SRA文件转fastq

mkdir fastq
ls SRRfile/SRR* | while read id;do \
    (fastq-dump --gzip --split-3 -A `basename $id` -O fastq/$id &);done

• 质控

mkdir fastqc
fastqc -o ./fastqc -t 20 ./fastq/*.fastq &&multiqc ./fastqc -o ./fastqc

• 去接头

# trim_galore
mkdir clean
cat SRR_Acc_List.txt | while read id;do\
  trim_galore --quality 20 --phred33 \
  --length 20 -j 20 \
  -o ../clean \
  --paried ./fastq/${id}_1.fastq ./fastq/${id}_2.fastq
done
gunzip ./clean/*.gz

3. 序列比对

• hisat2

mkdir aligned
cat SRR_Acc_List.txt | while read id;do
  hisat2 -p 20 -x ~/ref/genome_tran/genome_tran \
  -S ./aligned/${id}.sam \
 -1./clean/${id}_1_val_1.fq -2 ./clean/${id}_2_val_2.fq
done

• sam转bam

mkdir bam
cat SRR_Acc_List.txt | while read id;do
  samtools sort -n -@ 20 \
  -o ./bam/${id}.bam ./aligned/${id}.sam
done

• bam文件排序

cat SRR_Acc_List.txt | while read id;do
  sambamba sort -t 20 -o ./bam/${id}.bam ./bam/${id}.bam
done

• 输出为count

mkdir count
cat SRR_Acc_List.txt | while read id;do
  featureCounts -T 20 -p -t exon -g gene_name \
  -a ~/ref/gencode.v40.annotation.gtf \
  -o count/${id}.all_feature.txt \
  bam/${id}.sorted.bam
done

4.批处理文件

根据以上，写成批处理文件，批量处理SRR格式文件。

### srr2fastq
mkdir fastq
ls SRRfile/SRR* | while read id;do \
    (fastq-dump --gzip --split-3 -A `basename $id` -O fastq/$id &);done

### fastqc
mkdir fastqc
fastqc -o ./fastqc -t 20 ./fastq/*.fastq &&multiqc ./fastqc -o ./fastqc

### trim_galore
mkdir clean
cat SRR_Acc_List.txt | while read id;do\
  trim_galore --quality 20 --phred33 \
  --length 20 -j 20 \
  -o ../clean \
  --paried ./fastq/${id}_1.fastq ./fastq/${id}_2.fastq
done

### hisat2
mkdir aligned
cat SRR_Acc_List.txt | while read id;do
  hisat2 -p 20 -x ~/ref/genome_tran/genome_tran \
  -S ./aligned/${id}.sam \
 -1./clean/${id}_1_val_1.fq -2 ./clean/${id}_2_val_2.fq
done

### sam转bam
mkdir bam
cat SRR_Acc_List.txt | while read id;do
  samtools sort -n -@ 20 \
  -o ./bam/${id}.bam ./aligned/${id}.sam
done

### bam文件排序
cat SRR_Acc_List.txt | while read id;do
  sambamba sort -t 20 -o ./bam/${id}.bam ./bam/${id}.bam
done

### featurecounts 
mkdir count
cat SRR_Acc_List.txt | while read id;do
  featureCounts -T 20 -p -t exon -g gene_name \
  -a ~/ref/gencode.v40.annotation.gtf \
  -o count/${id}.all_feature.txt \
  bam/${id}.sorted.bam
done


### 
echo "------- end -------"

参考资料

1. Jimmy老师的教程
2. 阿则的生信小站公众号文章