$ mkdir ref #新建reference文件夹
xingkx 17:41:23 ~
$ cd ref
xingkx 17:41:31 ~/ref
$ mkdir mm9
xingkx 17:41:48 ~/ref
$ cd mm9
xingkx 17:41:54 ~/ref/mm9
$ wget --timestamping \
'ftp://hgdownload.cse.ucsc.edu/goldenPath/mm9/chromosomes/*'
--2023-04-03 17:41:57-- ftp://hgdownload.cse.ucsc.edu/goldenPath/mm9/chromosomes/*
=> ‘.listing’
Resolving hgdownload.cse.ucsc.edu (hgdownload.cse.ucsc.edu)... 128.114.119.163
Connecting to hgdownload.cse.ucsc.edu (hgdownload.cse.ucsc.edu)|128.114.119.163|:21... ^C
xingkx 17:42:26 ~/ref/mm9
试图通过网页链接下载小鼠mm9参考基因组失败，于是手动下载。
image.png
image.png
image.png
image.png
不要random文件，要下载md5文件
$ cd ..
xingkx 17:46:30 ~/ref
$ vim md5.txt
xingkx 17:49:04 ~/ref
$ cat md5.txt #查看md5文件，包含了要检测的文件
79d6d3a0198d6dccaa6c2af23ec9be00 chr1.fa.gz
24893d2118e606d341f3a91836f6267d chr10.fa.gz
f01fd408af17f393720044a44b3c4ee0 chr11.fa.gz
d882d9ba5696b6e6fdd80179670bc463 chr12.fa.gz
d400282edeb4c0529d5196019372d3c1 chr13.fa.gz
e4735f6e161a9498c2487958a6153891 chr13_random.fa.gz
1797fcbad80d0794b598d6fb6053e299 chr14.fa.gz
f182f5e0e3e5f90b3775b6e32274319b chr15.fa.gz
ef9806225139e113257e0e20717e2285 chr16.fa.gz
46cc30a95e412eb0320255335a42863f chr16_random.fa.gz
57e5044feac506810ed03d5bab6bb231 chr17.fa.gz
e16f391324164d5499fc48c05ef529e8 chr17_random.fa.gz
d409da85d239f0d8b1bfaa3a494be121 chr18.fa.gz
94ad4a7ceb60ad29feadb3d8aa2a925a chr19.fa.gz
c06a6759bd5f7a14c7fe4aa0a6423e7e chr1_random.fa.gz
14eaca978b51529479b5497634bf90ce chr2.fa.gz
180b4b2179fd550a39d1cd0b841e4834 chr3.fa.gz
ddd635cdcebd2c6666b4dadbf1c52fd2 chr3_random.fa.gz
c86d544389b3c77b54070d8d041fbc19 chr4.fa.gz
de2a1bb1505fd53cf2b9849d3a16358d chr4_random.fa.gz
d0d1e3c1a3914c1d2ec807330ee4d474 chr5.fa.gz
2e6a7618b193c877b9534101136bde47 chr5_random.fa.gz
4276c21b867322940abfccfd98e9c864 chr6.fa.gz
6e609fe100868e5b3051b9801b9efcf8 chr7.fa.gz
1f74d499fd66fbc3d2b239b4076e2177 chr7_random.fa.gz
e9b1b3ce3a1ce0a540bfb7eb2af44f59 chr8.fa.gz
0fad939822aab8a83fd9ef155bd0b52a chr8_random.fa.gz
0b2c504c618a5dd04619bd82171cc6dd chr9.fa.gz
4e30bb88d367d149a0aa050cc4e423cd chr9_random.fa.gz
ea02ff9a4831504423d63f26a329b74e chrM.fa.gz
e56577087f9765e2ecdf9d605ad2f3b8 chrUn_random.fa.gz
28b4c65ff0bf555bdf305d9b629794b4 chrX.fa.gz
a3c21a22e22d8fb4af962195e8b81e22 chrX_random.fa.gz
39079a4b2762f2237eca347d52c11215 chrY.fa.gz
5e013cab47168d75e96bdfc123771d54 chrY_random.fa.gz
xingkx 17:50:58 ~/ref
$ mv md5.txt mm9
xingkx 17:57:28 ~/ref
$ ls
mm9
xingkx 17:57:35 ~/ref
$ cd mm9
xingkx 17:58:03 ~/ref/mm9
$ ls
chr10.fa.gz chr14.fa.gz chr18.fa.gz chr3.fa.gz chr7.fa.gz chrX.fa.gz
chr11.fa.gz chr15.fa.gz chr19.fa.gz chr4.fa.gz chr8.fa.gz chrY.fa.gz
chr12.fa.gz chr16.fa.gz chr1.fa.gz chr5.fa.gz chr9.fa.gz md5.txt
chr13.fa.gz chr17.fa.gz chr2.fa.gz chr6.fa.gz chrM.fa.gz
xingkx 17:59:56 ~/ref/mm9
$ md5sum -c md5.txt #检测下载数据的md5值
chr1.fa.gz: OK
chr10.fa.gz: OK
chr11.fa.gz: OK
chr12.fa.gz: OK
chr13.fa.gz: OK
md5sum: chr13_random.fa.gz: No such file or directory
chr13_random.fa.gz: FAILED open or read
chr14.fa.gz: OK
chr15.fa.gz: OK
chr16.fa.gz: OK
md5sum: chr16_random.fa.gz: No such file or directory
chr16_random.fa.gz: FAILED open or read
chr17.fa.gz: OK
md5sum: chr17_random.fa.gz: No such file or directory
chr17_random.fa.gz: FAILED open or read
chr18.fa.gz: OK
chr19.fa.gz: OK
md5sum: chr1_random.fa.gz: No such file or directory
chr1_random.fa.gz: FAILED open or read
chr2.fa.gz: OK
chr3.fa.gz: OK
md5sum: chr3_random.fa.gz: No such file or directory
chr3_random.fa.gz: FAILED open or read
chr4.fa.gz: OK
md5sum: chr4_random.fa.gz: No such file or directory
chr4_random.fa.gz: FAILED open or read
chr5.fa.gz: OK
md5sum: chr5_random.fa.gz: No such file or directory
chr5_random.fa.gz: FAILED open or read
chr6.fa.gz: OK
chr7.fa.gz: OK
md5sum: chr7_random.fa.gz: No such file or directory
chr7_random.fa.gz: FAILED open or read
chr8.fa.gz: OK
md5sum: chr8_random.fa.gz: No such file or directory
chr8_random.fa.gz: FAILED open or read
chr9.fa.gz: OK
md5sum: chr9_random.fa.gz: No such file or directory
chr9_random.fa.gz: FAILED open or read
chrM.fa.gz: OK
md5sum: chrUn_random.fa.gz: No such file or directory
chrUn_random.fa.gz: FAILED open or read
chrX.fa.gz: OK
md5sum: chrX_random.fa.gz: No such file or directory
chrX_random.fa.gz: FAILED open or read
chrY.fa.gz: OK
md5sum: chrY_random.fa.gz: No such file or directory
chrY_random.fa.gz: FAILED open or read
md5sum: WARNING: 13 listed files could not be read
xingkx 18:00:31 ~/ref/mm9
$ gunzip *gz #解压当前目录下的所有gz文件
xingkx 18:01:22 ~/ref/mm9
$ ls #解压成功，全部以fa结尾
chr10.fa chr13.fa chr16.fa chr19.fa chr3.fa chr6.fa chr9.fa chrY.fa
chr11.fa chr14.fa chr17.fa chr1.fa chr4.fa chr7.fa chrM.fa md5.txt
chr12.fa chr15.fa chr18.fa chr2.fa chr5.fa chr8.fa chrX.fa
xingkx 18:01:58 ~/ref/mm9
$ cat *fa > mm9.fa #将所有fa文件重定向到mm9.fa,拼接起来
xingkx 18:02:39 ~/ref/mm9
$ ls #重定向后，原来的文件依然存在
chr10.fa chr14.fa chr18.fa chr3.fa chr7.fa chrX.fa mm9.fa.fai
chr11.fa chr15.fa chr19.fa chr4.fa chr8.fa chrY.fa nohup.out
chr12.fa chr16.fa chr1.fa chr5.fa chr9.fa md5.txt
chr13.fa chr17.fa chr2.fa chr6.fa chrM.fa mm9.fa
(chipseq) xingkx 19:21:38 ~/ref/mm9
激活chipseq环境，使用samtools工具
conda activate chipseq
samtools
samtools faidx mm9.fa
ls
$ cat mm9.fa.fai
chr10 129993255 7 50 51
chr11 121843856 132593135 50 51
chr12 121257530 256873876 50 51
chr13 120284312 380556564 50 51
chr14 125194864 503246570 50 51
chr15 103494974 630945339 50 51
chr16 98319150 736510220 50 51
chr17 95272651 836795760 50 51
chr18 90772031 933973872 50 51
chr19 61342430 1026561351 50 51
chr1 197195432 1089130636 50 51
chr2 181748087 1290269983 50 51
chr3 159599783 1475653038 50 51
chr4 155630120 1638444823 50 51
chr5 152537259 1797187552 50 51
chr6 149517037 1952775563 50 51
chr7 152524553 2105282947 50 51
chr8 131738871 2260857998 50 51
chr9 124076172 2395231653 50 51
chrM 16299 2521789355 50 51
chrX 166650296 2521805986 50 51
chrY 15902555 2691789294 50 51
(chipseq) xingkx 19:28:35 ~/ref/mm9
使用bowtie2工具比对工具,bowtie2-build构建索引
345 bowtie
346 bowtie2
347 bowtie2-build -h
348 nohup bowtie2-build --threads 4 mm9.fa mm9 & #线程数是4，前缀是mm9
image.png
表示任务跑完了 1.bg 2.top
bg命令来自于英文单词background的缩写，中文译为“背景、后台”，其功能是用于将作业放到后台运行。在Linux系统终端中执行命令时，如遇到备份、打包、下载等长时间的任务，就会很长一段时间占用宝贵的终端执行界面，无法执行其他任务。而bg命令则可以将指定的命令任务放到系统后台去执行，使得终端界面可以继续其他工作，效果等同于“命令 &”的执行效果。
$ ls
chr10.fa chr15.fa chr1.fa chr6.fa chrX.fa mm9.3.bt2 mm9.rev.2.bt2
chr11.fa chr16.fa chr2.fa chr7.fa chrY.fa mm9.4.bt2 nohup.out
chr12.fa chr17.fa chr3.fa chr8.fa md5.txt mm9.fa
chr13.fa chr18.fa chr4.fa chr9.fa mm9.1.bt2 mm9.fa.fai
chr14.fa chr19.fa chr5.fa chrM.fa mm9.2.bt2 mm9.rev.1.bt2
xingkx 09:24:49 ~/ref/mm9
bt2结尾的文件，索引构建成功
$ cd clean #使用剪切过后的文件
(chipseq) xingkx 22:58:19 ~/chipseq/clean
$ ls
fastqc SRR391033.fastq.gz_trimming_report.txt SRR391033_trimmed.fq.gz
(chipseq) xingkx 22:58:21 ~/chipseq/clean
$ bowtie2 -p 20 -x ../../ref/mm9/mm9 -U SRR391033_trimmed.fq.gz -b ../align/sRR391033.bam
# 最后一个mm9指前缀
Warning: Output file '../align/sRR391033.bam' was specified without -S. This will not work in future Bowtie 2 versions. Please use -S instead.
0 reads
0.00% overall alignment rate
(chipseq) xingkx 23:03:40 ~/chipseq/clean
$ bowtie2 -p 20 -x ../../ref/mm9/mm9 -U SRR391033_trimmed.fq.gz -S ../align/SRR391033.sam
#bam文件不行，使用sam文件
Error while flushing and closing output
terminate called after throwing an instance of 'int'
Aborted (core dumped)
(ERR): bowtie2-align exited with value 134
(chipseq) xingkx 23:05:21 ~/chipseq/clean
假设你发现前台运行的一个程序需要很长的时间,但是需要干其他的事情,你就可以用 Ctrl-Z ,终止这个程序,然后可以看到系统提示:
[1]+ Stopped /root/bin/rsync.sh
然后我们可以把程序调度到后台执行:(bg 后面的数字为作业号)
#bg 1
[1]+ /root/bin/rsync.sh &
用 jobs 命令查看正在运行的任务:
#jobs
[1]+ Running /root/bin/rsync.sh &
如果想把它调回到前台运行,可以用
#fg 1
/root/bin/rsync.sh
这样,你在控制台上就只能等待这个任务完成了.
& 将指令丢到后台中去执行
[ctrl]+z 将前台任务丢到后台中暂停
jobs 查看后台的工作状态
fg %jobnumber 将后台的任务拿到前台来处理
bg %jobnumber 将任务放到后台中去处理
kill 管理后台的任务
命令运行时使用CTRL+Z，强制当前进程转为后台，并使之停止。
ctrl+z 加 bg 效果等同于 nohup &
image.png
存储空间问题，home下你个人就6G的空间，mnt下你应该会有几T的空间，限制不一样的，把chipseq 文件夹移动到/mnt,然后重新跑任务
cp /home/xingkx/chipseq /mnt/disk4/xingkx
$ conda create -n chipseq2 python=3.9
Retrieving notices: ...working... done
Collecting package metadata (current_repodata.json): failed
CondaHTTPError: HTTP 000 CONNECTION FAILED for url <https://mirrors.tuna.tsinghua.edu.cn/anaconda/cloud/bioconda/linux-64/current_repodata.json>
Elapsed: -
An HTTP error occurred when trying to retrieve this URL.
HTTP errors are often intermittent, and a simple retry will get you on your way.
'https//mirrors.tuna.tsinghua.edu.cn/anaconda/cloud/bioconda/linux-64'
(chipseq) xingkx 22:44:00 /mnt/disk4/xingkx/chipseq/align
bowtie2 -p 1 -x /home/xingkx/ref/mm9/mm9 -U SRR391033_trimmed.fq.gz -S /home/xingkx/chipseq/align/test.sam
image.png
线程数改小，移动到home目录下跑
查看sam文件的两种方式：
less SRR391033.sam
samtools view SRR391033.sam |less
sam文件转bam文件，使用samtools工具,bam 文件是排过序的
samtools sort -o SRR391033.sort.bam -O BA M -@ 4 SRR391033.sam
查看bam 文件：
less SRR391033.sort.bam
samtools view SRR391033.sort.bam |less
bam文件各列的含义：
https://www.cnblogs.com/xudongliang/p/5437850.html
使用macs2 call peak
macs2 callpeak -t SRR391033.sort.bam -g mm --outdir ../peaks/ -n SRR391033 -q 0.05
这里有p value,q value的概念，多重校正，q是校正过后的p值
报错
python 版本是2.7,改成3
python -V
pip install numpy -U #Python使用pip安装Numpy模块
conda create chipseq2 python=3.9
image.png
一直因为网络问题报错 服务器没联网
