{"id":623,"date":"2013-03-09T08:42:40","date_gmt":"2013-03-09T00:42:40","guid":{"rendered":"http:\/\/www.hzaumycology.com\/chenlianfu_blog\/?p=623"},"modified":"2013-06-16T16:50:48","modified_gmt":"2013-06-16T08:50:48","slug":"cufllinks%e7%9a%84%e5%ae%89%e8%a3%85%e4%b8%8e%e4%bd%bf%e7%94%a8","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=623","title":{"rendered":"Cufllinks\u7684\u5b89\u88c5\u4e0e\u4f7f\u7528"},"content":{"rendered":"

\u4e00. \u7b80\u4ecb<\/h1>\n

Cufflinks<\/a>\u4e0b\u4e3b\u8981\u5305\u542bcufflinks,cuffmerge,cuffcompare\u548ccuffdiff\u7b49\u51e0\u652f\u4e3b\u8981\u7684\u7a0b\u5e8f\u3002\u4e3b\u8981\u7528\u4e8e\u57fa\u56e0\u8868\u8fbe\u91cf\u7684\u8ba1\u7b97\u548c\u5dee\u5f02\u8868\u8fbe\u57fa\u56e0\u7684\u5bfb\u627e\u3002<\/p>\n

\u4e8c. \u5b89\u88c5<\/h1>\n

Cufflinks\u4e0b\u8f7d\u7f51\u9875<\/a>\u3002
\n1. \u4e3a\u4e86\u5b89\u88c5Cufflinks\uff0c\u5fc5\u987b\u6709Boost C++ libraries<\/a>\u3002\u4e0b\u8f7dBoost<\/a>\u5e76\u5b89\u88c5\u3002\u9ed8\u8ba4\u5b89\u88c5\u5728\/usr\/local\u3002<\/p>\n

$ tar jxvf boost_1_53_0.tar.bz2\r\n$ cd boost_1_53_0\r\n$ .\/bootstrap.sh\r\n$ sudo .\/b2 install<\/pre>\n
2.\u5b89\u88c5SAM tools\u3002<\/p>\n
\u4e0b\u8f7dSAM tools<\/a>\u3002\r\n$ tar jxvf samtools-0.1.18.tar.bz2\r\n$ cd samtools-0.1.18\r\n$ make\r\n$ sudo su \r\n# mkdir \/usr\/local\/include\/bam\r\n# cp libbam.a \/usr\/local\/lib\r\n# cp *.h \/usr\/local\/include\/bam\/\r\n# cp samtools \/usr\/bin\/<\/pre>\n
3. \u5b89\u88c5 Eigen libraries\u3002<\/p>\n
\u4e0b\u8f7dEigen<\/a>\r\n$ tar jxvf 3.1.2.tar.bz2\r\n$ cd eigen-eigen-5097c01bcdc4\r\n$ sudo cp -r Eigen\/ \/usr\/local\/include\/<\/pre>\n
4. \u5b89\u88c5Cufflinks\u3002<\/p>\n
$ tar zxvf cufflinks-2.0.2.tar.gz\r\n$ cd cufflinks-2.0.2\r\n$ .\/configure --prefix=\/path\/to\/cufflinks\/install --with-boost=\/usr\/local\/ --with-eigen=\/usr\/local\/include\/\/Eigen\/\r\n$ make\r\n$ make install<\/pre>\n
5. \u53ef\u4ee5\u76f4\u63a5\u4e0b\u8f7dLinux x86_64 binary\u3002\u4e0d\u9700\u8981\u4e0a\u8ff0\u7e41\u7410\u6b65\u9aa4\uff0c\u89e3\u538b\u540e\u7684\u7a0b\u5e8f\u76f4\u63a5\u53ef\u7528\u3002<\/p>\n
\u4e09. Cufflinks<\/a>\u7684\u4f7f\u7528<\/h1>\n
1. Cufflinks\u7b80\u4ecb<\/h2>\n
Cufflinks\u7a0b\u5e8f\u4e3b\u8981\u6839\u636eTophat\u7684\u6bd4\u5bf9\u7ed3\u679c\uff0c\u4f9d\u6258\u6216\u4e0d\u4f9d\u6258\u4e8e\u53c2\u8003\u57fa\u56e0\u7ec4\u7684GTF\u6ce8\u91ca\u6587\u4ef6\uff0c\u8ba1\u7b97\u51fa(\u5404\u4e2agene\u7684)isoform\u7684FPKM\u503c\uff0c\u5e76\u7ed9\u51fatrascripts.gtf\u6ce8\u91ca\u7ed3\u679c(\u7ec4\u88c5\u51fa\u8f6c\u5f55\u7ec4)\u3002<\/p>\n
2. \u4f7f\u7528\u65b9\u6cd5<\/h2>\n
$ cufflinks [options]* <aligned_reads.(sam\/bam)>\r\n\r\n\u4e00\u4e2a\u5e38\u7528\u7684\u4f8b\u5b50\uff1a\r\n$ cufflinks -p 8 -G transcript.gtf --library-type fr-unstranded -o cufflinks_output tophat_out\/accepted_hits.bam<\/pre>\n
3. \u666e\u901a\u53c2\u6570<\/h2>\n
-h | --help\r\n-o | --output-dir <sting>  default: .\/<\/span>\r\n    \u8bbe\u7f6e\u8f93\u51fa\u7684\u6587\u4ef6\u5939\u540d\u79f0\r\n\r\n-p | --num-threads  default: 1<\/span>\r\n    \u7528\u4e8e\u6bd4\u5bf9reads\u7684CPU\u7ebf\u7a0b\u6570\r\n\r\n-G | --GTF <reference_annotation.(gtf\/gff)><\/span>\r\n    \u63d0\u4f9b\u4e00\u4e2aGFF\u6587\u4ef6\uff0c\u4ee5\u6b64\u6765\u8ba1\u7b97isoform\u7684\u8868\u8fbe\u3002\u6b64\u65f6\uff0c\u5c06\u4e0d\u4f1a\u7ec4\u88c5\u65b0\u7684transcripts\uff0c\r\n\u7a0b\u5e8f\u4f1a\u5ffd\u7565\u548creference transcript\u4e0d\u517c\u5bb9\u7684\u6bd4\u5bf9\u7ed3\u679c\r\n\r\n-g | --GTF-guide <reference_annotation.(gtf\/gff)><\/span>\r\n    \u63d0\u4f9bGFF\u6587\u4ef6\uff0c\u4ee5\u6b64\u6765\u6307\u5bfc\u8f6c\u5f55\u5b50\u7ec4\u88c5(RABT assembly)\u3002\u6b64\u65f6\uff0c\u8f93\u51fa\u7ed3\u679c\u4f1a\u5305\u542bref\r\nerence transcripts\u548cnovel genes and isforms\u3002\r\n\r\n-M | --mask-file <mask.(gtf\/gff)><\/span>\r\n    \u63d0\u4f9bGFF\u6587\u4ef6\u3002Cufflinks\u5c06\u5ffd\u7565\u6bd4\u5bf9\u5230\u8be5GTF\u6587\u4ef6\u7684transcripts\u4e2d\u7684reads\u3002\u8be5\r\n\u6587\u4ef6\u4e2d\u5e38\u5e38\u662frRNA\u7684\u6ce8\u91ca\uff0c\u4e5f\u53ef\u4ee5\u5305\u542b\u7ebf\u7acb\u4f53\u548c\u5176\u5b83\u5e0c\u671b\u5ffd\u7565\u7684transcripts\u7684\u6ce8\u91ca\u3002\u5c06\u8fd9\r\n\u4e9b\u4e0d\u9700\u8981\u7684RNA\u53bb\u9664\u540e\uff0c\u5bf9\u8ba1\u7b97mRNA\u7684\u8868\u8fbe\u91cf\u662f\u6709\u5229\u7684\u3002\r\n\r\n-b | --frag-bias-correct <genome.fa><\/span>\r\n    \u63d0\u4f9b\u4e00\u4e2afasta\u6587\u4ef6\u6765\u6307\u5bfcCufflinks\u8fd0\u884c\u65b0\u7684bias detection and correct\r\nion algorithm\u3002\u8fd9\u6837\u80fd\u660e\u663e\u63d0\u9ad8\u8f6c\u5f55\u5b50\u4e30\u5ea6\u8ba1\u7b97\u7684\u7cbe\u786e\u6027\u3002\r\n\r\n-u | --multi-read-correct<\/span>\r\n    \u8ba9Cufflinks\u6765\u505ainitial estimation\u6b65\u9aa4\uff0c\u4ece\u800c\u66f4\u7cbe\u786e\u8861\u91cf\u6bd4\u5bf9\u5230genome\u591a\u4e2a\r\n\u4f4d\u70b9\u7684reads\u3002\r\n\r\n--library-type  default:fr-unstranded<\/span>\r\n    \u5904\u7406\u7684reads\u5177\u6709\u94fe\u7279\u5f02\u6027\u3002\u6bd4\u5bf9\u7ed3\u679c\u4e2d\u5c06\u4f1a\u6709\u4e2aXS\u6807\u7b7e\u3002\u4e00\u822cIllumina\u6570\u636e\u7684lib\r\nrary-type\u4e3a fr-unstranded\u3002<\/pre>\n
4. \u4e30\u5ea6\u8bc4\u4f30\u53c2\u6570<\/h2>\n
-m | --frag-len-mean default: 200<\/span>\r\n\u63d2\u5165\u7247\u6bb5\u7684\u5e73\u5747\u957f\u5ea6\u3002\u4e0d\u8fc7\u73b0\u5728Cufflinks\u80fdlearns\u63d2\u5165\u7247\u6bb5\u7684\u5e73\u5747\u957f\u5ea6\uff0c\u56e0\u6b64\u4e0d\u63a8\u8350\u81ea\u4e3b\r\n\u8bbe\u7f6e\u6b64\u503c\u3002\r\n\r\n-s | --frag-len-std-dev default: 80<\/span>\r\n\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\u7684\u6807\u51c6\u5dee\u3002\u4e0d\u8fc7\u73b0\u5728Cufflinks\u80fdlearns\u63d2\u5165\u7247\u6bb5\u7684\u5e73\u5747\u957f\u5ea6\uff0c\u56e0\u6b64\u4e0d\u63a8\u8350\u81ea\r\n\u4e3b\u8bbe\u7f6e\u6b64\u503c\u3002\r\n\r\n-N | --upper-quartile-form<\/span>\r\n\u4f7f\u752875%\u5206\u4e3a\u6570\u7684\u503c\u6765\u4ee3\u66ff\u603b\u7684\u503c(\u6bd4\u5bf9\u5230\u5355\u4e00\u4f4d\u70b9\u7684fragments\u7684\u6570\u503c)\uff0c\u4f5cnormal\r\nize\u3002\u8fd9\u6837\u6709\u5229\u4e8e\u5728\u4f4e\u4e30\u5ea6\u57fa\u56e0\u548c\u8f6c\u5f55\u5b50\u4e2d\u5bfb\u627e\u5dee\u5f02\u57fa\u56e0\u3002\r\n\r\n--total-hits-norm default: TRUE<\/span>\r\nCufflinks\u5728\u8ba1\u7b97FPKM\u65f6,\u7b97\u5165\u6240\u6709\u7684fragments\u548c\u6bd4\u5bf9\u4e0a\u7684reads\u3002\u548c\u4e0b\u4e00\u4e2a\u53c2\u6570\r\n\u5bf9\u7acb\u3002\u9ed8\u8ba4\u6fc0\u6d3b\u8be5\u53c2\u6570\u3002\r\n\r\n--compatible-hits-norm <\/span>\r\nCufflinks\u5728\u8ba1\u7b97FPKM\u65f6\uff0c\u53ea\u9488\u5bf9\u548creference transcripts\u517c\u5bb9\u7684fragmen\r\nts\u4ee5\u53ca\u6bd4\u5bf9\u4e0a\u7684reads\u3002\u8be5\u53c2\u6570\u9ed8\u8ba4\u4e0d\u6fc0\u6d3b\uff0c\u53ea\u80fd\u5728\u6709 --GTF \u53c2\u6570\u4e0b\u6709\u6548\uff0c\u5e76\u4e14\u4f5c RABT\r\n\u6216 ab initio \u7684\u65f6\u5019\u65e0\u6548\u3002<\/pre>\n
5. \u7ec4\u88c5\u5e38\u7528\u53c2\u6570<\/h2>\n
-L | --label  default: CUFF<\/span>\r\n    Cufflink\u4ee5GTF\u683c\u5f0f\u6765\u62a5\u544a\u8f6c\u5f55\u5b50\u7247\u6bb5(transfrags),\u8be5\u53c2\u6570\u662fGTF\u6587\u4ef6\u7684\u524d\u7f00\r\n\r\n--min-frags-per-transfrag <int>  default: 10<\/span>\r\n    \u7ec4\u88c5\u51fa\u7684transfrags\u88ab\u652f\u6301\u7684RNA-seq\u7684fragments\u6570\u5c11\u4e8e\u8be5\u503c\u5219\u4e0d\u88ab\u62a5\u9053\u3002\r\n\r\n--min-intron-length <int>  default: 50<\/span>\r\n    \u6700\u5c0f\u7684intron\u5927\u5c0f\u3002\r\n\r\n--overlap-radius <int>  default: 50<\/span>\r\n    Transfrags\u4e4b\u95f4\u7684\u8ddd\u79bb\u5c11\u4e8e\u8be5\u503c\uff0c\u5219\u5c06\u5176\u8fde\u5230\u4e00\u8d77\u3002<\/pre>\n
6. Cufflinks\u8f93\u51fa\u7ed3\u679c<\/h2>\n
1. transcripts.gtf
\n\u8be5\u6587\u4ef6\u5305\u542bCufflinks\u7684\u7ec4\u88c5\u7ed3\u679cisoforms\u3002\u524d7\u5217\u4e3a\u6807\u51c6\u7684GTF\u683c\u5f0f\uff0c\u6700\u540e\u4e00\u5217\u4e3aattributes\u3002\u5176\u6bcf\u4e00\u5217\u7684\u610f\u4e49\uff1a<\/p>\n
\u5217\u6570   \u5217\u7684\u540d\u79f0  \u4f8b\u5b50         \u63cf\u8ff0\r\n1     \u5e8f\u5217\u540d    chrX        \u67d3\u8272\u4f53\u6216contig\u540d\r\n2     \u6765\u6e90      Cufflinks   \u4ea7\u751f\u8be5\u6587\u4ef6\u7684\u7a0b\u5e8f\u540d\r\n3     \u7c7b\u578b      exon        \u8bb0\u5f55\u7684\u7c7b\u578b\uff0c\u4e00\u822c\u662ftranscript\u6216exon\r\n4     \u8d77\u59cb      1           1-base\u7684\u503c\r\n5     \u7ed3\u675f      1000        \u7ed3\u675f\u4f4d\u7f6e\r\n6     \u5f97\u5206      1000        \r\n7     \u94fe        +          Cufflinks\u731c\u6d4bisoform\u6765\u81ea\u53c2\u8003\u5e8f\u5217\u7684\u90a3\u4e00\u6761\u94fe\uff0c\r\n\u4e00\u822c\u662f'+','-'\u6216'.'\r\n8     frame    .           Cufflinks\u4e0d\u53bb\u9884\u6d4b\u8d77\u59cb\u6216\u7ec8\u6b62\u5bc6\u7801\u5b50\u6846\u7684\u4f4d\u7f6e\r\n9     attributes  ...      \u8be6\u89c1\u4e0b<\/pre>\n
\u6bcf\u4e00\u4e2aGTF\u8bb0\u5f55\u5305\u542b\u5982\u4e0battributes\uff1a<\/p>\n
Attribute      \u4f8b\u5b50       \u63cf\u8ff0\r\ngene_id        CUFF.1    Cufflinks\u7684gene id\r\ntranscript_id  CUFF.1.1  Cufflinks\u7684\u8f6c\u5f55\u5b50 id\r\nFPKM           101.267   isoform\u6c34\u5e73\u4e0a\u7684\u4e30\u5ea6, F<\/strong>ragments P<\/strong>er K<\/strong>ilobase\r\n of exon model per M<\/strong>illion mapped fragments\r\nfrac           0.7647    \u4fdd\u7559\u7740\u7684\u4e00\u9879\uff0c\u5ffd\u7565\u5373\u53ef\uff0c\u4ee5\u540e\u53ef\u80fd\u4f1a\u53d6\u6d88\u8fd9\u4e2a\r\nconf_lo        0.07      isoform\u4e30\u5ea6\u768495%\u7f6e\u4fe1\u533a\u95f4\u7684\u4e0b\u8fb9\u754c\uff0c\u5373 \u4e0b\u8fb9\u754c\u503c =\r\n FPKM * ( 1.0 - conf_lo )\r\nconf_hi        0.1102    isoform\u4e30\u5ea6\u768495%\u7f6e\u4fe1\u533a\u95f4\u7684\u4e0a\u8fb9\u754c\uff0c\u5373 \u4e0a\u8fb9\u754c\u503c =\r\n FPKM * ( 1.0 + conf_hi )\r\ncov            100.765   \u8ba1\u7b97\u6574\u4e2atranscript\u4e0aread\u7684\u8986\u76d6\u5ea6\r\nfull_read_support   yes  \u5f53\u4f7f\u7528 RABT assembly \u65f6\uff0c\u8be5\u9009\u9879\u62a5\u544a\u6240\u6709\u7684intr\r\nons\u548cexons\u662f\u5426\u5b8c\u5168\u88abreads\u6240\u8986\u76d6<\/pre>\n
2. ispforms.fpkm_tracking
\nisoforms(\u53ef\u4ee5\u7406\u89e3\u4e3agene\u7684\u5404\u4e2a\u5916\u663e\u5b50)\u7684fpkm\u8ba1\u7b97\u7ed3\u679c
\n3. genes.fpkm_tracking
\ngene\u7684fpkm\u8ba1\u7b97\u7ed3\u679c<\/p>\n
\u56db. Cuffmerge<\/a>\u7684\u4f7f\u7528<\/h1>\n
1. Cuffmerge\u7b80\u4ecb<\/h2>\n
Cuffmerge\u5c06\u5404\u4e2aCufflinks\u751f\u6210\u7684transcripts.gtf\u6587\u4ef6\u878d\u5408\u79f0\u4e3a\u4e00\u4e2a\u66f4\u52a0\u5168\u9762\u7684transcripts\u6ce8\u91ca\u7ed3\u679c\u6587\u4ef6merged.gtf\u3002\u4ee5\u5229\u4e8e\u7528Cuffdiff\u6765\u5206\u6790\u57fa\u56e0\u5dee\u5f02\u8868\u8fbe\u3002<\/p>\n
2. \u4f7f\u7528\u65b9\u6cd5<\/h2>\n
$ cuffmerge [options]* <assembly_GTF_list.txt>\r\n\u8f93\u5165\u6587\u4ef6\u4e3a\u4e00\u4e2a\u6587\u672c\u6587\u4ef6\uff0c\u662f\u5305\u542b\u7740GTF\u6587\u4ef6\u8def\u5f84\u7684list\u3002\u5e38\u7528\u4f8b\u5b50\uff1a\r\n$ cuffmerge -o .\/merged_asm -p 8 assembly_list.txt<\/pre>\n
3. \u4f7f\u7528\u53c2\u6570<\/h2>\n
-h | --help\r\n-o <output_dir> default: .\/merged_asm<\/span>\r\n\u5c06\u7ed3\u679c\u8f93\u51fa\u81f3\u8be5\u6587\u4ef6\u5939\u3002\r\n\r\n-g | --ref-gtf<\/span>\r\n\u5c06\u8be5reference GTF\u4e00\u8d77\u878d\u5408\u5230\u6700\u7ec8\u7ed3\u679c\u4e2d\u3002\r\n\r\n-p | --num-threads <int> defautl: 1<\/span>\r\n\u4f7f\u7528\u7684CPU\u7ebf\u7a0b\u6570\r\n\r\n-s | --ref-sequence <seq_dir>\/<seq_fastq><\/span>\r\n\u8be5\u53c2\u6570\u6307\u5411\u57fa\u56e0\u7ec4DNA\u5e8f\u5217\u3002\u5982\u679c\u662f\u4e00\u4e2a\u6587\u4ef6\u5939\uff0c\u5219\u6bcf\u4e2acontig\u5219\u662f\u4e00\u4e2afasta\u6587\u4ef6\uff1b\u5982\u679c\u662f\r\n\u4e00\u4e2afasta\u6587\u4ef6\uff0c\u5219\u6240\u6709\u7684contigs\u90fd\u9700\u8981\u5728\u91cc\u9762\u3002Cuffmerge\u5c06\u4f7f\u7528\u8be5ref-sequence\u6765\r\n\u5e2e\u52a9\u5bf9transfrags\u5206\u7c7b\uff0c\u5e76\u6392\u9664repeats\u3002\u6bd4\u5982transcripts\u5305\u542b\u4e00\u4e9b\u5c0f\u5199\u78b1\u57fa\u7684\u5c06\u5f52\u7c7b\r\n\u5230repeats.<\/pre>\n
4. Cuffmerge\u8f93\u51fa\u7ed3\u679c<\/h2>\n
\u8f93\u51fa\u7684\u7ed3\u679c\u6587\u4ef6\u9ed8\u8ba4\u4e3a <output_dir>\/merged.gtf<\/p>\n
\u4e94. Cuffcompare<\/a>\u7684\u4f7f\u7528<\/h1>\n
1. Cuffcompare\u7b80\u4ecb<\/h2>\n
Cuffcompare\u4f7f\u7528Cufflinks\u7684GTF\u7ed3\u679c\uff0c\u5bf9GTF\u7ed3\u679c\u8fdb\u884c\u6bd4\u8f83\u3002\u548creference gtf\u6bd4\u8f83\u5bfb\u627enovel\u8f6c\u5f55\u5b50\u7b49\u3002<\/p>\n
2. Cuffcompare\u7684\u4f7f\u7528\u65b9\u6cd5<\/h2>\n
$ cuffcompare [options]* <cuff1.gtf> [cuff2.gtf] ... [cuffN.gtf]\r\n\r\n\u4f7f\u7528\u4f8b\u5b50\uff1a\r\n$ cuffcompare -o cuffcmp cuff1.gtf cuff2.gtf<\/pre>\n
3. \u4f7f\u7528\u53c2\u6570<\/h2>\n
-h\r\n-V\r\n-o <outprefix> default: cuffcmp<\/span>\r\n\u8f93\u51fa\u6587\u4ef6\u7684\u524d\u7f00\r\n\r\n-r <reference_mrna.gtf><\/span>\r\n\u53c2\u8003\u7684GFF\u6587\u4ef6\u3002\u7528\u6765\u8bc4\u4f30\u8f93\u5165\u7684gtf\u6587\u4ef6\u4e2dgene models\u7684\u7cbe\u786e\u6027\u3002\u6bcf\u4e00\u4e2a\u8f93\u5165\u7684gtf\u7684is\r\noforms\u5c06\u548c\u8be5\u53c2\u8003\u6587\u4ef6\u8fdb\u884c\u6bd4\u8f83\uff0c\u5e76\u88ab\u6807\u6ce8\u4e3a overlapping, matching \u6216 novel\u3002\r\n\r\n-R<\/span>\r\n\u5f53\u6709\u4e86 -r \u53c2\u6570\u65f6\uff0c\u6307\u5b9a\u8be5\u53c2\u6570\u65f6\uff0c\u5c06\u5ffd\u7565\u53c2\u8003GFF\u6587\u4ef6\u4e2d\u7684\u4e00\u4e9btranscripts\u3002\u8fd9\u4e9btran\r\nscripts\u4e0d\u548c\u4efb\u4f55\u8f93\u5165\u7684GTF\u6587\u4ef6overlapped\u3002\r\n\r\n-s <seq_dir>\/<seq_fastq><\/span>\r\n\u8be5\u53c2\u6570\u6307\u5411\u57fa\u56e0\u7ec4DNA\u5e8f\u5217\u3002\u5982\u679c\u662f\u4e00\u4e2a\u6587\u4ef6\u5939\uff0c\u5219\u6bcf\u4e2acontig\u5219\u662f\u4e00\u4e2afasta\u6587\u4ef6\uff1b\u5982\u679c\u662f\r\n\u4e00\u4e2afasta\u6587\u4ef6\uff0c\u5219\u6240\u6709\u7684contigs\u90fd\u9700\u8981\u5728\u91cc\u9762\u3002\u5c0f\u5199\u5b57\u6bcd\u7684\u78b1\u57fa\u7528\u6765\u5c06\u76f8\u5e94\u7684transcri\r\npts\u4f5c\u4e3arepeats\u5904\u7406\u3002<\/pre>\n
4. \u8f93\u51fa\u7ed3\u679c<\/a><\/h2>\n
\u5728\u5f53\u524d\u76ee\u5f55\u4e0b\u8f93\u51fa3\u4e2a\u6587\u4ef6\uff1a<coutprefix>.stats\uff0c<coutprefix>.combined.gtf \u548c <coutprefix>.tracking; \u5728\u8f93\u5165\u7684GTF\u7684\u540c\u76ee\u5f55\u4e0b\u8f93\u51fa<cuff_in>.refmap \u548c <cuff_in>.tmap \u6587\u4ef6\u3002<\/p>\n
\u516d. Cuffdiff<\/a>\u7684\u4f7f\u7528<\/h1>\n
1. Cuffdiff\u7b80\u4ecb<\/h2>\n
\u7528\u4e8e\u5bfb\u627e\u8f6c\u5f55\u5b50\u8868\u8fbe\u7684\u663e\u8457\u6027\u5dee\u5f02\u3002<\/p>\n
2. Cuffdiff\u4f7f\u7528\u65b9\u6cd5<\/h2>\n
$ cuffdiff [options]* <transcripts.gtf> <sample1_1.sam[,...,sample1_M.sam]> <sample2_1.sam[,...,sample2_M.sam]>...[sampleN_1.sam[,...,sampleN_M.sam]]\r\n\u5176\u4e2dtranscripts.gtf\u662f\u7531cufflinks\uff0ccuffcompare\uff0ccuffmerge\u6240\u751f\u6210\u7684\u6587\u4ef6\uff0c\u6216\u662f\u7531\u5176\u5b83\u7a0b\u5e8f\u751f\u6210\u7684\u3002\r\n\r\n\u4e00\u4e2a\u5e38\u7528\u4f8b\u5b50\uff1a\r\n$ cuffdiff --lables lable1,lable2 -p 8 --time-series --multi-read-correct --library-type fr-unstranded --poisson-dispersion transcripts.gtf sample1.sam sample2.sam<\/pre>\n
3. \u4f7f\u7528\u53c2\u6570<\/h2>\n
-h | --help\r\n-o | --output-dir <sting> default: .\/<\/span>\r\n\u8f93\u51fa\u7684\u6587\u4ef6\u5939\u76ee\u5f55\u3002\r\n-L | --lables <lable1,lable2,...,lableN>  default: q1,q2,...qN<\/span>\r\n\u7ed9\u6bcf\u4e2asample\u4e00\u4e2a\u6837\u54c1\u540d\r\n\r\n-p | --num-threads <int> default: 1<\/span>\r\n\u4f7f\u7528\u7684CPU\u7ebf\u7a0b\u6570\r\n\r\n-T | --time-series<\/span>\r\n\u8ba9Cuffdiff\u6765\u6309\u6837\u54c1\u987a\u5e8f\u6765\u6bd4\u5bf9\u6837\u54c1\uff0c\u800c\u4e0d\u662f\u5bf9\u6240\u6709\u7684samples\u90fd\u8fdb\u884c\u4e24\u4e24\u6bd4\u5bf9\u3002\u5373\u7b2c\u4e8c\u4e2a\r\nSAM\u548c\u7b2c\u4e00\u4e2aSAM\u6bd4\uff1b\u7b2c\u4e09\u4e2aSAM\u548c\u7b2c\u4e8c\u4e2aSAM\u6bd4\uff1b\u7b2c\u56db\u4e2aSAM\u548c\u7b2c\u4e09\u4e2aSAM\u6bd4...\r\n\r\n-N | --upper-quartile-form<\/span>\r\n\u4f7f\u752875%\u5206\u4e3a\u6570\u7684\u503c\u6765\u4ee3\u66ff\u603b\u7684\u503c(\u6bd4\u5bf9\u5230\u5355\u4e00\u4f4d\u70b9\u7684fragments\u7684\u6570\u503c)\uff0c\u4f5cnormalize\u3002\r\n\u8fd9\u6837\u6709\u5229\u4e8e\u5728\u4f4e\u4e30\u5ea6\u57fa\u56e0\u548c\u8f6c\u5f55\u5b50\u4e2d\u5bfb\u627e\u5dee\u5f02\u57fa\u56e0\u3002\r\n\r\n--total-hits-norm default: TRUE<\/span>\r\nCufflinks\u5728\u8ba1\u7b97FPKM\u65f6,\u7b97\u5165\u6240\u6709\u7684fragments\u548c\u6bd4\u5bf9\u4e0a\u7684reads\u3002\u548c\u4e0b\u4e00\u4e2a\u53c2\u6570\u5bf9\u7acb\u3002\r\n\u9ed8\u8ba4\u6fc0\u6d3b\u8be5\u53c2\u6570\u3002\r\n\r\n--compatible-hits-norm<\/span>\r\nCufflinks\u5728\u8ba1\u7b97FPKM\u65f6\uff0c\u53ea\u9488\u5bf9\u548creference transcripts\u517c\u5bb9\u7684fragments\u4ee5\u53ca\r\n\u6bd4\u5bf9\u4e0a\u7684reads\u3002\u8be5\u53c2\u6570\u9ed8\u8ba4\u4e0d\u6fc0\u6d3b\uff0c\u53ea\u80fd\u5728\u6709 --GTF \u53c2\u6570\u4e0b\u6709\u6548\uff0c\u5e76\u4e14\u4f5c RABT \u6216 ab\r\n initio \u7684\u65f6\u5019\u65e0\u6548\u3002\r\n\r\n-b | --frag-bias-correct<\/span>\r\n\u63d0\u4f9b\u4e00\u4e2afasta\u6587\u4ef6\u6765\u6307\u5bfcCufflinks\u8fd0\u884c\u65b0\u7684bias detection and correction \r\nalgorithm\u3002\u8fd9\u6837\u80fd\u660e\u663e\u63d0\u9ad8\u8f6c\u5f55\u5b50\u4e30\u5ea6\u8ba1\u7b97\u7684\u7cbe\u786e\u6027\u3002\r\n\r\n-u | --multi-read-correct<\/span>\r\n\u8ba9Cufflinks\u6765\u505ainitial estimation\u6b65\u9aa4\uff0c\u4ece\u800c\u66f4\u7cbe\u786e\u8861\u91cf\u6bd4\u5bf9\u5230genome\u591a\u4e2a\u4f4d\u70b9\r\n\u7684reads\u3002\r\n\r\n-c | --min-alignment-count <int>  default: 10<\/span>\r\n\u5982\u679c\u6bd4\u5bf9\u5230\u67d0\u4e00\u4e2a\u4f4d\u70b9\u7684fragments\u6570\u76ee\u5c11\u4e8e\u8be5\u503c\uff0c\u5219\u4e0d\u505a\u8be5\u4f4d\u70b9\u7684\u663e\u8457\u6027\u5206\u6790\u3002\u8ba4\u4e3a\u8be5\u4f4d\u70b9\r\n\u7684\u8868\u8fbe\u91cf\u6ca1\u6709\u663e\u8457\u6027\u5dee\u5f02\u3002\r\n\r\n-M | --mask-file <mask.(gtf\/gff)><\/span>\r\n\u63d0\u4f9bGFF\u6587\u4ef6\u3002Cufflinks\u5c06\u5ffd\u7565\u6bd4\u5bf9\u5230\u8be5GTF\u6587\u4ef6\u7684transcripts\u4e2d\u7684reads\u3002\u8be5\u6587\u4ef6\u4e2d\r\n\u5e38\u5e38\u662frRNA\u7684\u6ce8\u91ca\uff0c\u4e5f\u53ef\u4ee5\u5305\u542b\u7ebf\u7acb\u4f53\u548c\u5176\u5b83\u5e0c\u671b\u5ffd\u7565\u7684transcripts\u7684\u6ce8\u91ca\u3002\u5c06\u8fd9\u4e9b\u4e0d\u9700\r\n\u8981\u7684RNA\u53bb\u9664\u540e\uff0c\u5bf9\u8ba1\u7b97mRNA\u7684\u8868\u8fbe\u91cf\u662f\u6709\u5229\u7684\u3002\r\n\r\n-FDR <float> default: 0.05<\/span>\r\n\u5141\u8bb8\u7684false discovery rate.\r\n\r\n--library-type default:fr-unstranded<\/span>\r\n\u5904\u7406\u7684reads\u5177\u6709\u94fe\u7279\u5f02\u6027\u3002\u6bd4\u5bf9\u7ed3\u679c\u4e2d\u5c06\u4f1a\u6709\u4e2aXS\u6807\u7b7e\u3002\u4e00\u822cIllumina\u6570\u636e\u7684library-\r\ntype\u4e3a fr-unstranded\u3002\r\n\r\n-m | --frag-len-mean default: 200<\/span>\r\n\u63d2\u5165\u7247\u6bb5\u7684\u5e73\u5747\u957f\u5ea6\u3002\u4e0d\u8fc7\u73b0\u5728Cufflinks\u80fdlearns\u63d2\u5165\u7247\u6bb5\u7684\u5e73\u5747\u957f\u5ea6\uff0c\u56e0\u6b64\u4e0d\u63a8\u8350\u81ea\u4e3b\r\n\u8bbe\u7f6e\u6b64\u503c\u3002\r\n\r\n-s | --frag-len-std-dev default: 80<\/span>\r\n\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\u7684\u6807\u51c6\u5dee\u3002\u4e0d\u8fc7\u73b0\u5728Cufflinks\u80fdlearns\u63d2\u5165\u7247\u6bb5\u7684\u5e73\u5747\u957f\u5ea6\uff0c\u56e0\u6b64\u4e0d\u63a8\u8350\u81ea\r\n\u4e3b\u8bbe\u7f6e\u6b64\u503c\u3002\r\n\r\n--poisson-dispersion<\/span>\r\nUse the Poisson fragment dispersion model instead of learning one \r\nin each condition.<\/pre>\n
4. Cuffdiff\u8f93\u51fa<\/a><\/h2>\n
1. FPKM tracking files
\n2. Count tracking files
\n3. Read group tracking files
\n4. Differential expression test
\n5. Differential splicing tests – splicing.diff
\n6. Differential coding output – cds.diff
\n7. Differential promoter use – promoters.diff
\n8. Read group info – read_groups.info
\n9. Run info – run.info<\/p>\n
\u4e03. cufflinks\u4f7f\u7528\u4e2d\u9047\u5230\u7684\u95ee\u9898<\/h1>\n
1. \u4f7f\u7528cuffdiff\u65f6\u5019\uff0c\u5728\u6700\u65b0\u7248\u672c\u4e0b\uff0c\u65e0\u91cd\u590d\u7684RNA-seq\u6837\u4f5c\u6bd4\u8f83\uff0c\u7ed3\u679c\u4e2d\u6ca1\u6709\u5dee\u5f02\u8868\u8fbe\u57fa\u56e0\uff1f<\/strong>
\n\u5728v2.0.1\u53ca\u4e4b\u540e\u7684\u7248\u672c\u4e2dcuffdiff\u8c8c\u4f3c\u4e0d\u652f\u6301\u65e0\u91cd\u590d\u7684RNA-seq\u6570\u636e\u4e86\u3002\u4f7f\u7528\u4e4b\u524d\u7684\u7248\u672c\u5373\u53ef\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"
\u4e00. \u7b80\u4ecb Cufflinks\u4e0b\u4e3b\u8981\u5305\u542bcufflinks,cuffmerge, … \u7ee7\u7eed\u9605\u8bfb →<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[],"tags":[],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/623"}],"collection":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=623"}],"version-history":[{"count":51,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/623\/revisions"}],"predecessor-version":[{"id":1609,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/623\/revisions\/1609"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=623"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=623"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=623"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}