情况一：tophat2必须在python2.7下安装，选择在conda内创个小环境即可：

安装tophat2

##下载
wget http://ccb.jhu.edu/software/tophat/downloads/tophat-2.1.0.Linux_x86_64.tar.gz

##解压
tar -zxvf tophat-2.1.0.Linux_x86_64.tar.gz

##加入环境变量path
export PATH=/home/u20111230014/workspace/software/tophat-2.1.0.Linux_x86_64/:$PATH

激活conda，为tophat创建适合它的python2.7

conda create --name python27 python=2.7 -y
conda activate python27

在此时python2.7的环境下，进入tophat-2.1.0.Linux_x86_64目录下

cd tophat-2.1.0.Linux_x86_64
找到tophat，vim tophat进行修改

找到你的python2 ：whereis python2

(python27) [u20111230014@cpu10 tophat-2.1.0.Linux_x86_64]$ whereis python2
python2: /usr/bin/python2 /usr/bin/python2.7 /usr/lib/python2.7 /usr/lib64/python2.7 /usr/include/python2.7 /home/u20111230014/miniconda3/envs/python27/bin/python2 /home/u20111230014/miniconda3/envs/python27/bin/python2.7-config /home/u20111230014/miniconda3/envs/python27/bin/python2.7 /home/public/software/miniconda2/bin/python2 /home/public/software/miniconda2/bin/python2.7-config /home/public/software/miniconda2/bin/python2.7

选择conda下安装的python2.7环境路径 /home/u20111230014/miniconda3/envs/python27/bin/python2

将tophat文件的第一句#!/usr/bin/env python

改为 /home/u20111230014/miniconda3/envs/python27/bin/python2 后保存
PS: 路径必须要带上/bin/python2
tophat2 -h测试即可！

另一种情况：

报错：Error: Could not find Bowtie 2 index files (genome.*.bt2) 或 Warning: Could not find FASTA file bowtie2_index/GRCm39_index.fa但不提示error，照常preparing reads，但生成不出有效文件，报错如下：
[2022-02-21 00:23:56] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2022-02-21 00:23:56] Checking for Bowtie
                  Bowtie version:        2.2.5.0
[2022-02-21 00:23:58] Checking for Bowtie index files (genome)..
[2022-02-21 00:23:58] Checking for reference FASTA file
        Warning: Could not find FASTA file bowtie2_index/GRCm39_index.fa
[2022-02-21 00:23:58] Reconstituting reference FASTA file from Bowtie index
  Executing: /home/public/software/miniconda2/bin/bowtie2-inspect bowtie2_index/GRCm39_index > Top
hat_Cufflinks_DPP-0_clean.fq/tmp/GRCm39_index.fa
[2022-02-21 00:25:32] Generating SAM header for bowtie2_index/GRCm39_index
[2022-02-21 00:25:36] Preparing reads
         left reads: min. length=50, max. length=345, 29935913 kept reads (1 discarded)
[2022-02-21 00:31:57] Mapping left_kept_reads to genome GRCm39_index with Bowtie2 
[2022-02-21 00:50:15] Mapping left_kept_reads_seg1 to genome GRCm39_index with Bowtie2 (1/14)

解决方法：

在含有具体fasta文件的bowtie2_index文件夹内，重新对GRCm39.fa作一次软连接，命名为GRCm39_index.fa
[u20111230014@cpu10 bowtie2_index]$ ln -s GRCm39.fa GRCm39_index.fa

再配上slurm系统下的脚本，即可，如下：

PS:   go_FPKM_SE.sh,  序列DPP-0_clean.fq，注释文件GRCm39.gtf，bowtie2_index文件夹必须在同一个文件夹内
[u20111230014@cpu10 GRCm39]$ more tophat.slurm 
#!/bin/bash
#SBATCH -J Job.SRR8777   
#SBATCH -p dna             
#SBATCH -N 1  
#SBATCH --mem=50G               
#SBATCH --cpus-per-task=25    
#SBATCH -t 1-10:00:00        
#SBATCH -o Out.SRR8777   
#SBATCH --mail-user=394710725@qq.com

go_FPKM_SE.sh 24 DPP-0_clean.fq GRCm39.gtf bowtie2_index/GRCm39_index

成功！

[u20111230014@cpu10 GRCm39]$ go_FPKM_SE.sh 24 DPP-0_clean.fq GRCm39.gtf bowtie2_index/GRCm39_index
Usage:
   go_FPKM_SE.sh Threads Read1.fq transcriptome-index bowtie2-index 

Running info
Read:       DPP-0_clean.fq
Annotation: GRCm39.gtf
Genome:     bowtie2_index/GRCm39_index
 

[2022-02-21 14:29:28] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2022-02-21 14:29:28] Checking for Bowtie
                  Bowtie version:        2.2.5.0
[2022-02-21 14:29:28] Checking for Bowtie index files (genome)..
[2022-02-21 14:29:28] Checking for reference FASTA file
[2022-02-21 14:29:28] Generating SAM header for bowtie2_index/GRCm39_index
[2022-02-21 14:29:32] Preparing reads