緊接linux基礎(chǔ)（一）的內(nèi)容
11、安裝 miniconda,建立虛擬環(huán)境，在虛擬環(huán)境中安裝生信必備軟件
# miniconda官網(wǎng)下載linux版安裝包：Miniconda2-4.7.10-Linux-x86_64.sh*
>./Miniconda2-4.7.10-Linux-x86_64.sh* #進入安裝包所在目錄，運行安裝包
>conda config --add channels r
>conda config --add channels conda-forge
>conda config --add channels bioconda
>conda create -n rna python=2 #建立python2環(huán)境的名叫rna的虛擬conda環(huán)境
>souce activate rna #激活進入rna環(huán)境
>conda install -y bwa gatk4 sra-tools fastqc trim-galore star hisat2 bowtie2 subread htseq multiqc samtools salmon #安裝軟件
>source deactivate #退出環(huán)境
12、產(chǎn)生bam文件的命令，示例：
>bowtie2-align-s --wrapper basic-0 -p 20 -x /yanke/yanke21/database/index/bowtie2/hg38 -S WT_rep2_Input.sam -U /tmp/41440.unp
>samtools view tmp.sorted.bam | head -n 2 #運行虛擬環(huán)境中的軟件要先進入conda環(huán)境
SRR1042600.42157053 0 chr1 629895 42 51M * 0 0 ATAACCAATACTACCAATCANTACTCATCATTAATAATCATAATGGCTATA CCCFFFFFHHHHHJJJJJJJ#4AGHJJIIJJIIIIIJJJJIJIIIIJJIJI AS:i:-6 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:11C8A30 YT:Z:UU
SRR1042600.42212881 0 chr1 629895 42 51M * 0 0 ATAACCAATACTACCAATCANTACTCATCATTAATAATCATAATGGCTATA @@<FDFFBFDHHFJEIIGJI#3AFHGEHEIJIIGIIGGIJIIJIGIIGIIJ AS:i:-6 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:11C8A30 YT:Z:UU
SRR1042600.12010763 16 chr1 629895 24 51M * 0 0 ATAACCAATACTTCTAATCAAAACTCATCATTAATAATCATAATGGCTATA ?4B?1*4DD?11*1*?+22+<3F:3@EC:CC4EA,DEDDDDD?D3B:==+; AS:i:-10 XN:i:0 XM:i:4 XO:i:0 XG:i:0 NM:i:4 MD:Z:11C0A1C6T29YT:Z:UU
SRR1042600.29629551 16 chr1 629895 40 51M * 0 0 ATAACCAATACTACCAATCACTACTCATCATTAATAATCATAATGGCTATA HGF?JJHHFDHHGJJIHDFA+E?JIJJIIHGJJJJJJJHHHHHFFFFFCC@ AS:i:-8 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:11C8A30 YT:Z:UU
SRR1042600.41910745 0 chr1 629896 42 51M * 0 0 TAACCAATACTACCAATCAANACTCATCATTAATAATCATAATGGCTATAG CC@FFFFFHHHHGIIHIJJJ#3<CFHCGGIIIJJJJJJJJIGGFHIIJFII AS:i:-6 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:10C9T30 YT:Z:UU
SRR1042600.14329856 16 chr1 629896 8 18M1I32M * 0 0 AAACCAAATCCTCCAATCAAATCCTCATCATTAATAATCATAATGGCTATA #############################@IHHGCE9GHFHHHDDDDD<@@ AS:i:-18 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:0T6T0A2A9A28 YT:Z:UU
13、查看使用的參考基因組 gencode.v29.annotation.gtf.gz 具體有多少條染色體
>zcat gencode.v29.annotation.gtf.gz | grep -v "#" | cut -f 1| sort | uniq -c
250524 chr1
99843 chr10
167540 chr11
159530 chr12
42767 chr13
96124 chr14
100426 chr15
130878 chr16
171137 chr17
48848 chr18
168227 chr19
204338 chr2
61485 chr20
29652 chr21
59764 chr22
172809 chr3
108328 chr4
120515 chr5
124117 chr6
130671 chr7
98583 chr8
97204 chr9
143 chrM
89500 chrX
9064 chrY
14、上面的后綴為BAM 的文件的第二列，只有 0 和 16 兩個數(shù)字，用 cut/sort/uniq等命令統(tǒng)計它們的個數(shù)。
>samtools view tmp.sorted.bam | cut -f 2 | sort | uniq -c
29 0
24 16
15、重新打開 rmDuplicate/samtools/paired 文件夾下面的后綴為BAM 的文件，再次查看第二列，并且統(tǒng)計
>cd ~/rmDuplicate/samtools/paired
>ll
total 52
drwxrwxr-x 2 yanke21 yanke21 4096 Nov 12 2016 ./
drwxrwxr-x 4 yanke21 yanke21 4096 Nov 12 2016 ../
-rw-rw-r-- 1 yanke21 yanke21 354 Nov 12 2016 readme.txt
-rw-rw-r-- 1 yanke21 yanke21 3111 Nov 12 2016 tmp.header
-rw-rw-r-- 1 yanke21 yanke21 4379 Nov 12 2016 tmp.rmdup.bam
-rw-rw-r-- 1 yanke21 yanke21 1886 Nov 12 2016 tmp.rmdup.vcf.gz
-rw-rw-r-- 1 yanke21 yanke21 11649 Nov 12 2016 tmp.sam
-rw-rw-r-- 1 yanke21 yanke21 4543 Nov 12 2016 tmp.sorted.bam
-rw-rw-r-- 1 yanke21 yanke21 1888 Nov 12 2016 tmp.sorted.vcf.gz
>samtools view tmp.sorted.bam | cut -f 2 | sort | uniq -c
8 147
3 163
1 323
1 353
1 371
1 387
1 433
3 83
2 97
9 99
16、下載 http://www.biotrainee.com/jmzeng/sickle/sickle-results.zip 文件，并且解壓，查看里面的文件夾結(jié)構(gòu)， 這個文件有2.3M，注意留心下載時間及下載速度。
>wget -c http://www.biotrainee.com/jmzeng/sickle/sickle-results.zip
>unzip sickle-results.zip
>tree sickle-results
sickle-results
├── command.txt
├── single_tmp_fastqc.html
├── single_tmp_fastqc.zip
├── test1_fastqc.html
├── test1_fastqc.zip
├── test2_fastqc.html
├── test2_fastqc.zip
├── trimmed_output_file1_fastqc.html
├── trimmed_output_file1_fastqc.zip
├── trimmed_output_file2_fastqc.html
└── trimmed_output_file2_fastqc.zip
17、解壓 sickle-results/single_tmp_fastqc.zip 文件，并且進入解壓后的文件夾，找到 fastqc_data.txt 文件，并且搜索該文本文件以 >>開頭的有多少行？
>unzip sickle-results/single_tmp_fastqc.zip
>cd single_tmp_fastqc/ && ll
total 296
drwxrwxr-x 4 yanke21 yanke21 4096 Oct 6 2016 ./
drwxr-xr-x 13 yanke21 yanke 4096 Aug 24 11:29 ../
-rw-rw-r-- 1 yanke21 yanke21 12048 Oct 6 2016 fastqc_data.txt
-rw-rw-r-- 1 yanke21 yanke21 5186 Oct 6 2016 fastqc.fo
-rw-rw-r-- 1 yanke21 yanke21 259290 Oct 6 2016 fastqc_report.html
drwxrwxr-x 2 yanke21 yanke21 4096 Oct 6 2016 Icons/
drwxrwxr-x 2 yanke21 yanke21 4096 Oct 6 2016 Images/
-rw-rw-r-- 1 yanke21 yanke21 542 Oct 6 2016 summary.txt
>grep -E '^>>' fastqc_data.txt | wc -l
24
18、下載 http://www.biotrainee.com/jmzeng/tmp/hg38.tss文件，去NCBI找到TP53/BRCA1等自己感興趣的基因?qū)?yīng)的 refseq數(shù)據(jù)庫ID，然后找到它們的hg38.tss文件的哪一行。
https://www.ncbi.nlm.nih.gov/gene/7157
>wget -c http://www.biotrainee.com/jmzeng/tmp/hg38.tss
>less hg38.tss
NM_012279 chr5 177020680 177024680 0
NR_131773 chr5 177020680 177024680 0
NR_131774 chr5 177020680 177024680 0
NM_001308213 chr5 177020680 177024680 0
NM_001308219 chr5 177020680 177024680 0
NM_001318082 chr7 99502644 99506644 0
NM_001291980 chr5 177084872 177088872 0
NM_001283039 chr11 3795819 3799819 0
NM_001283040 chr11 3795819 3799819 0
NM_138964 chr2 68641588 68645588 0
NM_139057 chr15 100340005 100344005 1
>grep 'NM_001143990' hg38.tss
NM_001143990 chr17 7684071 7688071 0
>grep 'NM_001291588' hg38.tss
NM_001291588 chr17 43209827 43213827 0
19、解析hg38.tss 文件，統(tǒng)計每條染色體的基因個數(shù)。
cut -f 2 hg38.tss | sort | uniq -c
20、解析hg38.tss 文件，統(tǒng)計NM和NR開頭的熟練，了解NM和NR開頭的含義
grep -E '^NM' hg38.tss | wc -l
51064
grep -E '^NR' hg38.tss | wc -l
15954
NM開頭表示轉(zhuǎn)錄產(chǎn)物序列，成熟的mRNA序列
NR開頭表示非編碼的轉(zhuǎn)錄子序列，包括RNAs,假基因轉(zhuǎn)子等
