{"id":910,"date":"2013-04-15T13:33:07","date_gmt":"2013-04-15T05:33:07","guid":{"rendered":"http:\/\/www.hzaumycology.com\/chenlianfu_blog\/?p=910"},"modified":"2013-06-11T10:49:05","modified_gmt":"2013-06-11T02:49:05","slug":"tophat%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=910","title":{"rendered":"Tophat\u7684\u5b89\u88c5\u4e0e\u4f7f\u7528"},"content":{"rendered":"

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

Tophat<\/a>\u4f7f\u7528RNA-seq\u7684reads\u6570\u636e\u6765\u5bfb\u627e\u57fa\u56e0\u7684\u526a\u5207\u70b9(splice junction\uff09\u3002\u8be5\u8f6f\u4ef6\u8c03\u7528Bowtie,\u6216Bowtie2\u6765\u5c06reads\u6bd4\u5bf9\u5230\u53c2\u8003\u57fa\u56e0\u7ec4\u4e0a\uff0c\u5206\u6790\u6bd4\u5bf9\u7ed3\u679c\uff0c\u4ece\u800c\u5bfb\u627e\u51fa\u5916\u663e\u5b50\u4e4b\u95f4\u7684\u7ed3\u5408\u4f4d\u70b9\u3002<\/p>\n

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

\u76f4\u63a5\u4e0b\u8f7d\u9002\u5408\u4e8eLinux x86_64\u7684\u4e8c\u8fdb\u5236\u6587\u4ef6\uff0c\u89e3\u538b\u7f29\u5373\u53ef\u4f7f\u7528\u3002<\/p>\n

$ wget http:\/\/tophat.cbcb.umd.edu\/downloads\/tophat-2.0.8b.Linux_x86_64.tar.gz\r\n$ tar zxf tophat-2.0.8b.Linux_x86_64.tar.gz<\/pre>\n
\u524d\u63d0\u6761\u4ef6\u5f53\u7136\u8981\u5b89\u88c5Bowtie, Bowtie2, SAM tools, Boost C++ libraries\u7b49\u3002<\/p>\n
\u4e09. Tophat\u7684\u4f7f\u7528\u53c2\u6570<\/h1>\n
\u4f7f\u7528Tohat\u65f6\uff0cbowtie2\uff08\u6216bowtie\uff0c\u4e0b\u540c\uff09, bowtie2-align, bowtie2-inspect, bowtie2-build \u548c samtools \u5fc5\u987b\u8981\u5728\u7cfb\u7edf\u8def\u5f84\u4e2d\u3002<\/p>\n
1. \u7528\u6cd5<\/h2>\n
$ tophat [options]* <index_base> <reads1_1[,...,readsN_1]> <reads1_2[,...,readsN_2]>\r\n\u53ef\u4ee5\u770b\u51fa\uff0ctophat\u5fc5\u987b\u8981\u7684\u6761\u4ef6\u662f\u6bd4\u5bf9\u7684index\u6570\u636e\u5e93\uff0c\u4ee5\u53ca\u8981\u6bd4\u5bf9\u7684reads\u3002\u53ef\u4ee5\u4e3a\u591a\u4e2a\r\npaired-end reads\u6570\u636e\u4ee5\u9017\u53f7\u5206\u5f00\u3002<\/pre>\n
\u503c\u5f97\u6ce8\u610f\u7684\uff1aTophat\u80fd\u6bd4\u5bf9\u7684\u6700\u5927reads\u4e3a1024bp\uff1b \u80fd\u6bd4\u5bf9paired-end reads\uff1b \u4e0d\u80fd\u5c06\u591a\u79cd\u4e0d\u540c\u7c7b\u578b\u7684reads\u6df7\u5408\u8d77\u6765\u8fdb\u884c\u6bd4\u5bf9\uff0c\u8fd9\u6837\u4f1a\u7ed9\u51fa\u4e0d\u597d\u7684\u7ed3\u679c\u3002<\/p>\n
\u5982\u679c\u6709\u591a\u79cd\u4e0d\u540c\u7c7b\u578b\u7684reads\u8fdb\u884c\u6bd4\u5bf9\uff0c\u5219\u53ef\u4ee5\uff1a<\/p>\n
\u9996\u5148\uff0c\u5bf9\u4e00\u79cd\u7c7b\u578b\u7684reads\u4f7f\u7528\u5408\u9002\u7684\u53c2\u6570\u8fd0\u884ctophat\uff1b\r\n\u63a5\u7740\uff0c\u4f7f\u7528bed_to_juncs\u5c06\u524d\u4e00\u6b21\u7684\u8fd0\u884c\u7ed3\u679cjunctions.bed\u8f6c\u6362\u6210\u4e0b\u4e00\u6b21\u8fd0\u884ctophat\r\n\u6240\u7684-j\u53c2\u6570\u6240\u9700\u7684junction\u6587\u4ef6\uff1b\r\n\u6700\u540e\uff0c\u518d\u4e00\u6b21\u4f7f\u7528-j\u53c2\u6570\u8fd0\u884ctophat\u3002<\/pre>\n
2. \u5e38\u7528\u4e00\u822c\u53c2\u6570<\/h2>\n
-h | --help\r\n-v | --version\r\n\r\n-N | --read-mismatches  default: 2<\/span>\r\n    \u4e22\u5f03\u4e0d\u5339\u914d\u78b1\u57fa\u6570\u8d85\u8fc7\u8be5\u6570\u76ee\u7684\u6bd4\u5bf9\u7ed3\u679c\r\n\r\n--read-gap-length  default: 2<\/span>\r\n    \u4e22\u5f03gap\u603b\u957f\u5ea6\u8d85\u8fc7\u8be5\u6570\u76ee\u7684\u6bd4\u5bf9\u7ed3\u679c\r\n\r\n--read-edit-dist  default: 2<\/span>\r\n    \u4e22\u5f03read\u7684edit distance\u5927\u4e8e\u8be5\u503c\u7684\u6bd4\u5bf9\u7ed3\u679c\r\n\r\n--read-realign-edit-dist <int> default: \"read-edit-dist\" + 1<\/span>\r\n    \u4e00\u4e9b\u8de8\u8d8a\u591a\u4e2aexons\u7684reads\u53ef\u80fd\u4f1a\u88ab\u9519\u8bef\u5730\u6bd4\u5bf9\u5230geneome\u4e0a\u3002Tophat\u6709\u591a\u4e2a\u6bd4\u5bf9\r\n\u6b65\u9aa4\uff0c\u6bcf\u4e2a\u6bd4\u5bf9\u6b65\u9aa4\u8fc7\u540e\uff0c\u6bd4\u5bf9\u7ed3\u679c\u4e2d\u5305\u542b\u4e86edit distance\u7684\u503c\u3002\u8be5\u53c2\u6570\u80fd\u8ba9Tophat\u5bf9\r\n\u90a3\u4e9bedit distance\u7684\u503c >= \u8be5\u53c2\u6570\u7684reads\u91cd\u65b0\u8fdb\u884c\u6bd4\u5bf9\u3002\u82e5\u8bbe\u7f6e\u8be5\u53c2\u6570\u503c\u4e3a0\uff0c\u5219\u6bcf\u4e2a\r\nread\u5728\u591a\u4e2a\u6bd4\u5bf9\u6b65\u9aa4\u4e2d\u6bcf\u6b21\u90fd\u8981\u8fdb\u884c\u6bd4\u5bf9\u3002\u8fd9\u6837\u4f1a\u52a0\u5927\u5730\u589e\u52a0\u6bd4\u5bf9\u7cbe\u786e\u6027\u548c\u8fd0\u884c\u65f6\u95f4\u3002\u9ed8\u8ba4\u4e0b\r\n\u8be5\u53c2\u6570\u6bd4\u4e0a\u4e00\u4e2a\u53c2\u6570\u7684\u503c\u5927\uff0c\u5219\u8868\u793a\u5bf9reads\u8fdb\u884c\u91cd\u65b0\u6bd4\u5bf9\u3002\r\n\r\n--bowtie1  default: bowtie2<\/span>\r\n    \u4f7f\u7528Bowtie1\u6765\u4ee3\u66ffBowtie2\u8fdb\u884c\u6bd4\u5bf9\u3002\u7279\u522b\u662f\u4f7f\u7528colorspace reads\u65f6\uff0c\u56e0\u4e3a\u53ea\r\n\u6709Bowtie1\u652f\u6301\uff0c\u800cBowtie2\u4e0d\u652f\u6301\u3002\r\n\r\n-o | --output <string>  default: .\/tophat_out<\/span>\r\n    \u8f93\u51fa\u7684\u6587\u4ef6\u5939\u8def\u5f84\r\n\r\n-r | --mate-inner-dist <int>  default: 50<\/span>\r\n    \u6210\u5bf9\u7684reads\u4e4b\u95f4\u7684\u5e73\u5747inner\u8ddd\u79bb\u3002\u4f8b\u5982\uff1afragments\u957f\u5ea6300bp\uff0creads\u957f\u5ea650bp\r\n\uff0c \u5219\u5176inner\u8ddd\u79bb\u4e3a200bp\uff0c\u8be5\u503c\u8be5\u8bbe\u4e3a200\u3002\r\n\r\n--mate-std-dev <int>  default:20<\/span>\r\n    inner\u8ddd\u79bb\u7684\u6807\u51c6\u504f\u5dee\u3002\r\n\r\n-a | --min-anchor-length <int>  default: 8<\/span>\r\n    read\u7684\u951a\u5b9a\u957f\u5ea6\uff1a\u8be5\u53c2\u6570\u80fd\u8bbe\u5b9a\u7684\u6700\u5c0f\u503c\u4e3a3\uff1b\u951a\u5b9a\u5728junction\u4e24\u8fb9\u7684reads\u957f\u5ea6\u53ea\r\n\u6709\u90fd\u5927\u4e8e\u6b64\u503c\uff0c\u624d\u80fd\u7528\u4e8ejunction\u7684\u9a8c\u8bc1\u3002\r\n\r\n-m | --splice-mismatches <int>  default: 0<\/span>\r\n    \u5bf9\u4e8e\u4e00\u4e2a\u526a\u5207\u6bd4\u5bf9\uff0c\u5176\u5728\u951a\u5b9a\u533a\u80fd\u51fa\u73b0\u7684\u6700\u5927\u7684\u4e0d\u5339\u914d\u78b1\u57fa\u6570\u3002\r\n\r\n-i | --min-intron-length <int>  default:70<\/span>\r\n    \u6700\u5c0f\u7684intron\u957f\u5ea6\u3002Tophat\u4f1a\u5ffd\u7565\u6bd4\u8be5\u957f\u5ea6\u8981\u5c0f\u7684donor\/acceptor pairs\uff0c\u8ba4\r\n\u4e3a\u8be5\u533a\u5c5e\u4e8eexon\u3002\r\n\r\n--I | --max-intron-length <int>  default:500000<\/span>\r\n    \u6700\u5927\u7684intron\u957f\u5ea6\u3002Tophat\u4f1a\u5ffd\u7565\u957f\u5ea6\u5927\u4e8e\u8be5\u503c\u7684donor\/acceptor pairs\uff0c\u9664\r\n\u975e\u6709long read\u652f\u6301\u3002\r\n\r\n--max-insertion-length <int>  defautl: 3<\/span>\r\n    \u6700\u5927\u7684\u63d2\u5165\u957f\u5ea6\r\n\r\n--max-deletion-length <int>  default: 3<\/span>\r\n    \u6700\u5927\u7684\u7f3a\u5931\u957f\u5ea6\r\n\r\n--solexa-quals<\/span>\r\n    fastq\u6587\u4ef6\u4f7f\u7528Solexa\u7684\u78b1\u57fa\u8d28\u91cf\u683c\u5f0f\r\n\r\n--solexa1.3-quals | --phred64-quals<\/span>\r\n    \u4f7f\u7528Illumina GA pipeline version 1.3\u7684\u78b1\u57fa\u8d28\u91cf\u683c\u5f0f\uff0c\u5373Phred64.\r\n\r\n-Q | --quals<\/span>\r\n    \u8bf4\u660e\u662f\u4f7f\u7528\u5355\u72ec\u7684\u78b1\u57fa\u8d28\u91cf\u6587\u4ef6\r\n\r\n--inter-quals<\/span>\r\n    \u6709\u7a7a\u683c\u9694\u5f00\u7684\u6574\u6570\u503c\u6765\u4ee3\u8868\u78b1\u57fa\u8d28\u91cf\u3002\u5f53\u4f7f\u7528 -C \u53c2\u6570\u65f6\uff0c\u8be5\u53c2\u6570\u4e3a\u9ed8\u8ba4\u53c2\u6570\u3002\r\n\r\n-C | --color<\/span>\r\n    Colorspace reads\u3002\u4f7f\u7528\u8fd9\u4e00\u79cdreads\u7684\u65f6\u5019\u547d\u4ee4\u5982\u4e0b\uff1a\r\n$ tophat --color --quals --bowtie1 [other options]* <colorspac\r\ne_index_base> <reads1_1[,...,readsN_1]> <reads2_1[,...,readN_2]>\r\n <quals1_1[,...,qualsN_1]> <quals1_2[,...,qualsN_2]>\r\n\r\n-p | --num-threads <int>  default: 1<\/span>\r\n    \u6bd4\u5bf9reads\u7684\u7ebf\u7a0b\u6570\r\n\r\n-g | --max-multihits <int>  default: 20<\/span>\r\n    \u5bf9\u4e8e\u4e00\u4e2areads\uff0c\u53ef\u80fd\u4f1a\u6709\u591a\u4e2a\u6bd4\u5bf9\u7ed3\u679c\uff0c\u4f46tophat\u6839\u636e\u6bd4\u5bf9\u5f97\u5206\uff0c\u6700\u591a\u4fdd\u7559\u7684\u6bd4\u5bf9\u7ed3\r\n\u679c\u6570\u76ee\u3002\u5982\u679c\u6ca1\u6709 --report-secondary-alignments \u53c2\u6570\uff0c\u5219\u53ea\u4f1a\u62a5\u544a\u51fa\u6700\u4f73\u7684\u6bd4\u5bf9\r\n\u7ed3\u679c\u3002\u82e5\u6700\u4f73\u6bd4\u5bf9\u7ed3\u679c\u6570\u76ee\u8d85\u8fc7\u8be5\u53c2\u6570\u503c\uff0c\u5219\u53ea\u968f\u673a\u62a5\u544a\u51fa\u8be5\u6570\u76ee\u7684\u6700\u4f73\u6bd4\u5bf9\u7ed3\u679c\uff1b\u82e5\u6709 --\r\nreport-secondary-alignments \u53c2\u6570\uff0c\u5219\u6309\u5f97\u5206\u987a\u5e8f\u62a5\u544a\u51fa\u6bd4\u5bf9\u7ed3\u679c\uff0c\u76f4\u81f3\u8fbe\u5230\u9ed8\u8ba4\r\n\u7684\u6570\u76ee\u4e3a\u6b62\u3002\r\n\r\n--report-secondary-alignments<\/span>\r\n    \u662f\u5426\u62a5\u544aadditional or secondary alignments\uff08\u57fa\u4e8e\u6bd4\u5bf9\u5206\u503cAS\u6765\u786e\u5b9a\u7684\uff09\u3002\r\n\r\n--no-discordant<\/span>\r\n    \u5bf9\u4e8epaired reads\uff0c\u4ec5\u4ec5\u62a5\u544aconcordant mappings\u3002\r\n\r\n--no-mixed<\/span>\r\n    \u5bf9\u4e8epaired reads\uff0c\u53ea\u62a5\u544aconcordant mappings \u548c discordant mappi\r\nngs\u3002\u9ed8\u8ba4\u4e0a\uff0c\u662f\u6240\u6709\u7684\u6bd4\u5bf9\u7ed3\u679c\u90fd\u62a5\u544a\u3002\r\n\r\n--no-coverage-search<\/span>\r\n    \u53d6\u6d88\u4ee5\u8986\u76d6\u5ea6\u4e3a\u57fa\u7840\u6765\u641c\u5bfbjunctions\uff0c\u548c\u4e0b\u4e00\u4e2a\u53c2\u6570\u5bf9\u7acb\uff0c\u8be5\u53c2\u6570\u4e3a\u9ed8\u8ba4\u53c2\u6570\u3002\r\n--coverage-search<\/span>\r\n    \u786e\u5b9a\u4ee5\u8986\u76d6\u5ea6\u4e3a\u57fa\u7840\u6765\u641c\u5bfbjunctions\u3002\u8be5\u53c2\u6570\u80fd\u589e\u5927\u654f\u611f\u6027\u3002\r\n\r\n--microexon-search<\/span>\r\n    \u4f7f\u7528\u8be5\u53c2\u6570\uff0cpipeline\u4f1a\u5c1d\u8bd5\u5bfb\u627emicro-exons\u3002\u4ec5\u4ec5\u5728reads\u957f\u5ea6>=50bp\u65f6\u6709\u6548\u3002\r\n\r\n--library-type<\/span>\r\n    Tophat\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\r\n\u636e\u7684library-type\u4e3a fr-unstranded\u3002<\/pre>\n
3. \u9ad8\u7ea7\u53c2\u6570<\/h2>\n
 --keep-tmp<\/span>\r\n    \u4fdd\u7559\u4e2d\u95f4\u6587\u4ef6\u548c\u4e34\u65f6\u6587\u4ef6\uff0c\u5bf9\u4e8edebug\u6709\u7528\r\n\r\n--keep-fasta-order<\/span>\r\n    \u5bf9\u6bd4\u5bf9\u7ed3\u679c\u6309\u57fa\u56e0\u7ec4fasta\u6587\u4ef6\u8fdb\u884c\u6392\u5e8f\u3002\u8be5\u53c2\u6570\u4f1a\u4f7f\u8f93\u51fa\u7684SAM\/BAM\u6587\u4ef6\u548ctophat\u7684\r\n1.41\u6216\u4ee5\u524d\u7248\u672c\u4e0d\u517c\u5bb9\r\n\r\n--no-sort-bam<\/span>\r\n    \u8f93\u51fa\u7684BAM\u6587\u4ef6\u4e0d\u662fcoordinate-sorted.\r\n\r\n--no-convert-bam<\/span>\r\n    \u4e0d\u8981\u8f6c\u6362\u6210bam\u683c\u5f0f\u3002\u8f93\u51fa\u7ed3\u679c\u4e3asam\u683c\u5f0f\u3002\r\n\r\n-R | --resume <string><\/span>\r\n    \u4ece\u6700\u672b\u5c3e\u7684\u6210\u529f\u5b8c\u6210\u70b9\u5904\uff0c\u63a5\u7740\u8fd0\u884cTophat\u3002\u4f7f\u7528\u65b9\u6cd5\u4e3a\uff1a\r\n$ tophat -R tophat_out\r\n\r\n-z | --zpacker  default:gzip<\/span>\r\n    \u7528\u6765\u5bf9\u4e34\u65f6\u6587\u4ef6\u8fdb\u884c\u538b\u7f29\u7684\u7684\u538b\u7f29\u7a0b\u5e8f<\/pre>\n
4. Bowtie2\u7684\u7279\u522b\u53c2\u6570<\/h2>\n
\u4f7f\u7528tophat2\u7684\u65f6\u5019\uff0c\u5176\u4e2d\u7684\u4e00\u4e9b\u53c2\u6570\u4f20\u9012\u4e3abowtie2\u7684\u53c2\u6570\uff0c\u8fd9\u4e9b\u53c2\u6570\u90fd\u4ee5’b2’\u5f00\u5934\u3002\u5176\u5b9e\uff0c\u8fd9\u4e9b\u53c2\u6570\u4f7f\u7528\u9ed8\u8ba4\u7684\u5373\u53ef\u3002<\/p>\n
end-to-end\u6a21\u5f0f(Tophat2\u4e0d\u80fd\u4f7f\u7528local alignment):\r\n--b2-very-fast\r\n--b2-fast\r\n--b2-sensitive\r\n--b2-very-sensitive\r\n\r\n\u6bd4\u5bf9\u53c2\u6570\uff1a\r\n--b2-N  default: 0\r\n--b2-L  default: 20\r\n--b2-i  default: S,1,1.25\r\n--b2-n-ceil  default: L,0,0.15\r\n--b2-gbar  defaut: 4\r\n\r\n\u5f97\u5206\u53c2\u6570\uff1a\r\n--b2-mp  default: 6,2\r\n--b2-np  default: 1\r\n--b2-rdg  default: 5,3\r\n--b2-rfg  default: 5,3\r\n--b2-score-min  default: L,-0.6,-0.6\r\n\r\nEffort\u53c2\u6570\r\n--b2-D  default: 15\r\n--b2-R  default: 2<\/pre>\n
5. \u878d\u5408\u8f6c\u5f55\u5b50mapping<\/h2>\n
\u5982\u679c\u8bbe\u5b9a –fusion-search \u53c2\u6570\uff0c\u5219\u6709\u4e9breads\u80fd\u6bd4\u5bf9\u5230\u6f5c\u5728\u7684\u878d\u5408\u8f6c\u5f55\u5b50(fusion transcripts)\u4e0a\u3002\u989d\u5916\u878d\u5408\u4fe1\u606f\u4fdd\u5b58\u5728 fusions.out \u4e2d\u3002<\/p>\n
--fusion-search<\/span> \r\n    \u5f00\u542f\u878d\u5408\u8f6c\u5f55\u5b50\u7684\u6bd4\u5bf9\r\n--fusion-anchor-length  default: 20<\/span>\r\n    read\u6bd4\u5bf9\u5230\u878d\u5408\u5b50\u7684\u4e24\u8fb9\uff0c\u6bcf\u4ee5\u8fb9\u81f3\u5c11\u5339\u914d\u7684\u78b1\u57fa\u6570\u3002<\/pre>\n
6. \u63d0\u4f9b\u7684\u8f6c\u5f55\u5b50\u7684\u7ed3\u6784\u6ce8\u91ca\u6570\u636e<\/h2>\n
\u503c\u5f97\u6ce8\u610f\u7684\u63d0\u4f9b\u7684GTF\u6587\u4e2d\u4e2d\u7684\u67d3\u8272\u4f53\u540d\u79f0\u548cBowtie index\u4e2d\u7684\u4e00\u81f4\u3002\u8fd9\u4e9b\u540d\u79f0\u662f\u533a\u5206\u5927\u5c0f\u5199\u7684\u3002<\/p>\n
-j | --raw-juncs <.juncs file><\/span>\r\n    \u63d0\u4f9bjunctions\u6587\u4ef6\u3002\u8be5\u6587\u4ef6\u53ef\u4ee5\u4f7f\u7528tophat\u540c\u4e00\u76ee\u5f55\u4e0b\u7684\u7a0b\u5e8fbed_to_juncs\u7a0b\u5e8f\r\n\u6765\u5904\u7406tophat\u7684\u7ed3\u679c\u6587\u4ef6junctions.bed\u751f\u6210\u3002\r\n$ bed_to_juncs <junctions.bed>\r\n    junctions\u6587\u4ef6\u662ftab\u5206\u9694\u7684\u6587\u4ef6\uff0c\u5185\u5bb9\u4e3a\uff1a\r\n<chrom> <leftgt> <rigth> <+\/->\r\n\u5176\u4e2dleft\u548cright\u6570\u503c\u662f0-based\u7684junction\u4e24\u7aef\u7684\u503c\u3002\r\n\r\n--no-novel-juncs<\/span>\r\n\u53ea\u641c\u5bfb\u548cGFF\u6216junctions\u6587\u4ef6\u4e2d\u63d0\u4f9b\u7684junctions\u60f3\u5339\u914d\u7684reads\u3002\u5982\u679c\u6ca1\u6709 -G \u6216 -j\r\n \u53c2\u6570\uff0c\u5219\u8be5\u53c2\u6570\u65e0\u6548\u3002\r\n\r\n-G | --GTF <GTF\/GFF3 file><\/span>\r\n\u63d0\u4f9b\u57fa\u56e0\u6a21\u578b\u7684\u6ce8\u91ca\u6587\u4ef6\uff0cGTF 2.2 \u6216\u8005 GFF 3 \u683c\u5f0f\u7684\u6587\u4ef6\u3002\u5982\u679c\u8bbe\u7f6e\u4e86\u8be5\u53c2\u6570\uff0cTophat\r\n\u5219\u5148\u63d0\u53d6\u51fa\u8f6c\u5f55\u5b50\u5e8f\u5217\uff0c\u7136\u540e\u4f7f\u7528Bowtie2\u5c06reads\u6bd4\u5bf9\u5230\u63d0\u53d6\u7684\u8f6c\u5f55\u7ec4\u4e2d\uff1b\u53ea\u6709\u4e0d\u80fd\u6bd4\u5bf9\u4e0a\r\n\u7684reads\u518d\u6bd4\u5bf9\u5230genome\uff1b\u6bd4\u5bf9\u4e0a\u7684reads\u518d\u6253\u65ad\u8f6c\u53d8\u6210genomic mappings\uff1b\u518d\u878d\u5408\u65b0\r\n\u7684mappings\u548cjunctions\u4f5c\u4e3a\u6700\u540e\u7684\u8f93\u51fa\u3002\r\n\u503c\u5f97\u6ce8\u610f\u7684\u662fGTF\/GFF\u6587\u4ef6\u4ee3\u8868chromosome\u548ccontig\u7684\u7b2c\u4e00\u5217\u8981\u548cbowtie index\u4e2d\u7684\r\n\u53c2\u8003\u5e8f\u5217\u540d\u4e00\u81f4\u3002 `$ bowtie-inspect --names your_index` \u547d\u4ee4\u53ef\u4ee5\u83b7\u5f97bowt\r\nie\u7684index\u3002\r\n\r\n--transcriptome-index <dir\/prefix><\/span>\r\n\u662f\u4f7f\u7528\u4e86 -G \u53c2\u6570\u540e\uff0cTophat\u63d0\u53d6\u8f6c\u5f55\u5b50\u5e8f\u5217\uff0c\u7136\u540e\u4f7f\u7528bowtie2-build\u6765\u5efa\u7acbindex\uff0c\r\n\u8fd9\u4e2a\u8fc7\u7a0b\u4f1a\u6d88\u8017\u4e0d\u5c11\u65f6\u95f4\u3002\u4e8e\u662f\uff0c\u4f7f\u7528\u8be5\u53c2\u6570\uff0c\u4f1a\u5c06index\u6587\u4ef6\u751f\u6210\u5230\u6307\u5b9a\u6587\u4ef6\u5939\u3002\u5219\u540e\u7eed\u7684\r\n\u8fd0\u7528\u540c\u6837\u7684index\u5219\u4e0d\u518d\u9700\u8981\u989d\u5916\u8017\u65f6\u4e86\u3002<\/pre>\n
7. \u63d0\u4f9binsertions\/deletions<\/h2>\n
\u4ee5\u4e0b\u53c2\u6570\u662f\u4f7f\u7528RNA-seq\u6570\u636e\u6765\u9a8c\u8bc1indels\u3002<\/p>\n
--insertions | --deletions <.juncs file><\/span>\r\n    juncs\u6587\u4ef6\u4f8b\u5b50\uff1a\r\nchr1 20564 20567\r\n0-based\u6570\u503c\u3002\u8868\u793a\u670920565\u548c20566\u8fd92\u4e2a\u78b1\u57fa\u7f3a\u5931\r\nchr1 17491 17491 CA\r\n\u8868\u793a\u572817491\u5904\u63d2\u5165\u4e862\u4e2a\u78b1\u57faCA\r\n\r\n--no-novel-indels<\/span>\r\n    \u4ec5\u4ec5\u53ea\u641c\u5bfb\u5728\u5df2\u7ed9\u7684\u4f4d\u70b9\u7684reads\u3002<\/pre>\n
\u56db. Tophat\u7684\u8f93\u51fa\u7ed3\u679c<\/h1>\n
\u4e3b\u8981\u7684\u7ed3\u679c\u6587\u4ef6\u662f\uff1a
\n1. accepted_hits.bam
\n2. junctions.bed UCSC BED<\/a>\u683c\u5f0f
\n3. insertions.bed \u548c deletions.bed<\/p>\n
\u4e94. \u601d\u8003\u9898<\/h1>\n
\u5bf9\u4e00\u7269\u79cd\u7684\u4e24\u4e2a\u6837\u672cA\u548cB\u4f7f\u7528Illumina Hiseq2000\u5206\u522b\u8fdb\u884c\u4e86\u8f6c\u5f55\u7ec4\u6d4b\u5e8f\uff0c\u5f97\u5230\u4e86\u7ed3\u679c\u6587\u4ef6A.reads1_1.fastq, A.reads1_2.fastq, B.reads1_1.fastq \u548c B.reads1_2fastq\u3002\u6d4b\u5e8f\u6587\u5e93\u7684\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\u4e3a200bp\uff0creads\u957f\u5ea6\u4e3a90bp\u3002\u7269\u79cd\u7684\u57fa\u56e0\u7ec4\u6587\u4ef6\u4e3aspecies.fasta\u3002\u8bf7\u7528Tophat\u5206\u6790\u8be5\u7269\u79cd\u7684\u8f6c\u5f55\u7ed3\u5408\u4f4d\u70b9\uff0cindel\u4fe1\u606f?<\/p>\n
$ bowtie2-build species.fastq species\r\n$ tophat \\\r\n\u00a0 --read-realign-edit-dist 0 \\\r\n\u00a0 -o .\/tophat_out \\\r\n\u00a0 -r 20 \\\r\n  --mate-std-dev 20 \\\r\n  --coverage-search \\\r\n  --microexon-search \\\r\n  -p 24 \\\r\n  --library-type fr-unstranded \\\r\n  species \\\r\n  A.reads1_1.fastq,B.reads1_1.fastq A.reads1_2.fastq B.reads1_2fastq<\/pre>\n","protected":false},"excerpt":{"rendered":"
Tophat\u4f7f\u7528RNA-seq\u7684reads\u6570\u636e\u6765\u5bfb\u627e\u57fa\u56e0\u7684\u526a\u5207\u70b9(splice junction\uff09\u3002\u8be5\u8f6f\u4ef6\u8c03\u7528Bowtie,\u6216Bowtie2\u6765\u5c06reads\u6bd4\u5bf9\u5230\u53c2\u8003\u57fa\u56e0\u7ec4\u4e0a\uff0c\u5206\u6790\u6bd4\u5bf9\u7ed3\u679c\uff0c\u4ece\u800c\u5bfb\u627e\u51fa\u5916\u663e\u5b50\u4e4b\u95f4\u7684\u7ed3\u5408\u4f4d\u70b9\u3002\u672c\u6587\u91cd\u70b9\u4ecb\u7ecdTophat\u7684\u4f7f\u7528\u53c2\u6570\u3002 \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":[3],"tags":[20,41],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/910"}],"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=910"}],"version-history":[{"count":24,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/910\/revisions"}],"predecessor-version":[{"id":1498,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/910\/revisions\/1498"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=910"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=910"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=910"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}