目前hisat2是最流行的轉(zhuǎn)錄組比對軟件，其比對速度快，且準(zhǔn)確度較高。具體使用方法如下：
用到的軟件版本為hisat2-2.2.1，samtools-1.6

1. 構(gòu)建hisat2參考基因組比對庫

hisat2-build -p 10 ref.fa ref

2. 用hisat2進(jìn)行比對

#對于雙端序列
hisat2 -p 20 --dta -x ref -1 ${sample}_1.fastq.gz -2 ${sample}_2.fastq.gz -S ${sample}.sam
#對于單端序列
hisat2 -p 20 -x ref -U ${sample}.fastq.gz -S ${sample}.sam

3. 用samtools將sam轉(zhuǎn)換成bam

samtools view -Sb ${sample}.sam -@ 20 > ${sample}.bam

4. 用samtools將bam排序

samtools sort ${sample}.bam -@ 20 -o ${sample}.sort

5. 用featureCounts進(jìn)行計數(shù)

featureCounts -T 10 -a ref.gtf -o ${sample}.txt -f -t gene ${sample}.sort.bam

6. 用自己寫的腳本算TPM值

import sys
if len(sys.argv) !=3 or "--help" in sys.argv:
        print("Usage: python counts2_TPM.py featurecounts.results Tpm.results")
        exit()
file=open(sys.argv[1],"r")
line =file.readlines()
out=open(sys.argv[2],"w")
list=[]
for i in line[2:]:
        l=i.strip().split("\t")
        num=l[-1]
        len=l[-2]
        N=int(num)/int(len)
        list.append(N)
for i in line[2:]:
        l=i.strip().split("\t")
        id=l[0]
        num=l[-1]
        len=l[-2]
        tpm=((int(num)/int(len))*(10**6))/sum(list)
        out.write(id+"\t"+str(tpm)+"\n")
file.close()
out.close()