{"id":2286,"date":"2015-08-25T16:35:31","date_gmt":"2015-08-25T08:35:31","guid":{"rendered":"http:\/\/www.chenlianfu.com\/?p=2286"},"modified":"2015-11-16T14:42:47","modified_gmt":"2015-11-16T06:42:47","slug":"%e5%af%b9%e8%bd%ac%e5%bd%95%e7%bb%84%e6%95%b0%e6%8d%ae%e8%bf%9b%e8%a1%8c%e8%b4%a8%e9%87%8f%e6%8e%a7%e5%88%b6%e5%88%86%e6%9e%90","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=2286","title":{"rendered":"\u5bf9\u8f6c\u5f55\u7ec4\u6570\u636e\u8fdb\u884c\u8d28\u91cf\u63a7\u5236\u5206\u6790"},"content":{"rendered":"<h1>1. \u8f6c\u5f55\u7ec4\u6570\u636e\u8d28\u91cf\u63a7\u5236\u5206\u6790\u7684\u5185\u5bb9<\/h1>\n<p>\u4e3b\u8981\u5305\u542b\u51e0\u4e2a\u65b9\u9762\uff1a<\/p>\n<pre>\r\n1. \u6d4b\u5e8f\u6570\u636e\u7684\u8d28\u91cf\u63a7\u5236\uff0c\u63a8\u8350\u4f7f\u7528 Trimmomatic \u5bf9\u4f4e\u8d28\u91cf\u6570\u636e\u8fdb\u884c\u622a\u9664\uff1b\r\n2. \u662f\u5426\u662f\u94fe\u7279\u5f02\u6027\u6d4b\u5e8f\uff0c\u4ee5\u53ca\u94fe\u7279\u5f02\u6027\u6d4b\u5e8f\u7684\u79cd\u7c7b\uff1b\r\n3. \u8f6c\u5f55\u7ec4\u6570\u636e\u5bf9\u53c2\u8003\u57fa\u56e0\u7ec4\u7684\u5339\u914d\u60c5\u51b5\uff0c\u5305\u62ec\u5206\u6790\u5339\u914d\u5230\u57fa\u56e0\u5185\u6216\u57fa\u56e0\u95f4\u533a\u7684\u6570\u636e\u91cf\uff1b\r\n4. \u8f6c\u5f55\u7ec4\u6570\u636e\u4e2d rRNA \u6570\u636e\u542b\u91cf\uff1b\r\n5. \u8f6c\u5f55\u5b50\u4ece 5' \u5230 3' \u7aef\u7684\u8986\u76d6\u5ea6\u5206\u6790\uff1b\r\n6. \u8f6c\u5f55\u7ec4\u6d4b\u5e8f\u6570\u636e\u91cf\u7684\u9971\u548c\u5ea6\u5206\u6790\u3002\r\n<\/pre>\n<p>\u672c\u6587\u4e3b\u8981\u8bb2\u89e3\u540e\u9762 4 \u4e2a\u5185\u5bb9\u7684\u5206\u6790\u65b9\u6cd5\u3002<\/p>\n<h1>2. \u4f7f\u7528 RNA-SeQC<\/h1>\n<p>\u4e0b\u8f7d RNA-SeQC \u53ca\u5176\u8bf4\u660e\u6587\u6863<\/p>\n<pre>\r\n$ wget http:\/\/www.broadinstitute.org\/cancer\/cga\/tools\/rnaseqc\/RNA-SeQC_v1.1.8.jar\r\n$ wget http:\/\/www.broadinstitute.org\/cancer\/cga\/sites\/default\/files\/data\/tools\/rnaseqc\/RNA-SeQC_Help_v1.1.2.pdf\r\n<\/pre>\n<p>\u7a0b\u5e8f\u8fd0\u884c\u9700\u6c42\u7684\u8f93\u5165\u6587\u4ef6\uff1a<\/p>\n<pre>\r\ngenome.fasta        \u57fa\u56e0\u7ec4\u7684\u5e8f\u5217\u6587\u4ef6\uff0c\u540c\u65f6\u9700\u8981\u6709\u76f8\u5e94\u7684 .fai \u548c .dict \u6587\u4ef6\u3002\r\ngenome.gtf          \u57fa\u56e0\u7ec4\u6ce8\u91ca\u6587\u4ef6\uff0c\u8be5\u8f6f\u4ef6\u4ec5\u652f\u6301 GTF \u683c\u5f0f\u3002\u6807\u51c6\u7684 gtf \u6587\u4ef6\u5fc5\u987b\u6709 start_codon, stop_codon \u548c CDS \u5185\u5bb9\uff0c\u4f46\u672c\u8f6f\u4ef6\u8981\u6c42 gtf \u6587\u4ef6\u5fc5\u987b\u8fd8\u542b\u6709 exon \u5185\u5bb9\u3002\r\naccepted_hits.bam   \u8f6c\u5f55\u7ec4\u6570\u636e\u6bd4\u5bf9\u5230\u57fa\u56e0\u7ec4\u7684\u7ed3\u679c\uff0c\u4f8b\u5982 tophat \u7684\u7ed3\u679c\u3002\u6b64 bam \u6587\u4ef6\u5fc5\u987b\u542b\u6709 RG \u53c2\u6570\u3002\r\nrRNA.fasa           rRNA \u5e8f\u5217\uff0c\u7528\u4e8e\u8ba1\u7b97 rRNA \u6d4b\u5e8f\u6570\u636e\u542b\u91cf\u3002\u6b64\u6587\u4ef6\u4e0d\u662f\u5fc5\u987b\u7684\u3002\r\nrRNA_intervals.list rRNA \u7684\u7ed3\u6784\u4fe1\u606f\u6587\u4ef6\uff0c\u6bcf\u884c\u4ee5 \"chromosome_id:start-end\" \u4fe1\u606f\u8868\u793a rRNA \u7ed3\u6784\u3002\u6b64\u6587\u4ef6\u4e0d\u662f\u5fc5\u987b\u7684\u3002\u4f46\u548c\u4e0a\u4e00\u4e2a\u6587\u4ef6\u5fc5\u987b\u8981\u4e8c\u9009\u4e00\u3002\r\n<\/pre>\n<p>\u51c6\u5907\u8f93\u5165\u6587\u4ef6<\/p>\n<pre>\r\n\u5f97\u5230\u57fa\u56e0\u7ec4\u7684 .fai \u548c .dict \u6587\u4ef6\r\n$ samtools faidx genome.fasta\r\n$ java -jar \/opt\/biosoft\/picard-tools-1.95\/CreateSequenceDictionary.jar R=genome.fasta O=genome.dict\r\n\r\n\u82e5\u8fd0\u884c tophat \u6ca1\u6709\u8f93\u5165 RG \u53c2\u6570\uff0c\u5219\u8fd0\u884c\u4e0b\u9762\u547d\u4ee4\u5f97\u5230 RG \u53c2\u6570\r\n$ java -jar \/opt\/biosoft\/picard-tools-1.95\/AddOrReplaceReadGroups.jar I=accepted_hits.bam O=accepted_hits.RG.bam ID=A1 LB=A1 PL=ILLUMINA SM=A1 PU=run\r\n\r\n\u82e5\u4ec5\u6709 GFF3 \u6587\u4ef6\uff0c\u6ca1\u6709\u6807\u51c6\u7684 GTF \u6587\u4ef6\uff0c\u9700\u8981\u81ea\u5df1\u7f16\u5199\u7a0b\u5e8f\u8fdb\u884c\u8f6c\u6362\r\n$ gff3ToGtf.pl genome.fasa genome.gff3 > genome.gtf\r\n$ perl -p -i -e 's\/^\\s*$\/\/' genome.gtf\r\n\u82e5\u4e0d\u53bb\u9664 genome.gtf \u6587\u4ef6\u4e2d\u7684\u7a7a\u884c\uff0c\u5219\u4f1a\u51fa\u73b0\u9519\u8bef\u63d0\u793a\uff1a No attributes were found in the GTF file on line \u3002\r\n<\/pre>\n<p>\u8fd0\u884c RNA-SeQC \u547d\u4ee4<\/p>\n<pre>\r\n\u5bf9\u4e00\u4e2a\u6570\u636e\u8fd0\u884c RNA-SeQC\uff0c\u5e76\u7ed3\u679c\u8f93\u51fa\u5230\u5236\u5b9a\u6587\u4ef6\u5939\r\n$ java -jar RNA-SeQC_v1.1.8.jar -o A1 -r genome.fasta -s \"A1|accepted_hits.RG.bam|A1_note\" -t genome.gtf -BWArRNA rRNA.fasta\r\n\r\n\u5bf9\u591a\u4e2a\u6570\u636e\u8fd0\u884c RNA-SeQC\r\n$ echo -e \"A1\\tA1.bam\\tA1_note\r\nA2\\tA2.bam\\tA2_note\r\nB1\\tB1.bam\\tB1_note\r\nB2\\tB2.bam\\tB2_note\" > samples.txt\r\n$ java -jar RNA-SeQC_v1.1.8.jar -o rna-seqc_out -r genome.fasta -s samples.txt -t genome.gtf -BWArRNA rRNA.fasta\r\n\r\n\u6ce8\u610f\uff0c\u591a\u4e2a\u6570\u636e\u8fd0\u884c RNA-SeQC\uff0c\u8f93\u5165\u7684 samples.txt \u6587\u4ef6\u5185\u5bb9\u4e3a\uff1a\r\n\u7b2c\u4e00\u884c\u4fe1\u606f\u4e3a\uff1a \"Sample ID\\tBam File\\tNotes\", \u8fd9\u884c\u5934\u90e8\u4f1a\u88ab\u5ffd\u7565\uff0c\u4ec5\u8d77\u6ce8\u91ca\u4f5c\u7528\u3002\r\n\u540e\u9762\u591a\u884c\u5219\u4e3a\u76f8\u5173\u7684\u4fe1\u606f\uff0c\u75283\u5217\u8868\u793a\uff0c\u7528tab\u5206\u5272\uff0c\u6ce8\u610fBam\u6587\u4ef6\u5fc5\u987b\u4e3a\u7edd\u5bf9\u8def\u5f84\u3002\r\n<\/pre>\n","protected":false},"excerpt":{"rendered":"<p>1. \u8f6c\u5f55\u7ec4\u6570\u636e\u8d28\u91cf\u63a7\u5236\u5206\u6790\u7684\u5185\u5bb9 \u4e3b\u8981\u5305\u542b\u51e0\u4e2a\u65b9\u9762\uff1a 1. \u6d4b\u5e8f\u6570\u636e\u7684\u8d28\u91cf\u63a7\u5236 &hellip; <a href=\"http:\/\/www.chenlianfu.com\/?p=2286\">\u7ee7\u7eed\u9605\u8bfb <span class=\"meta-nav\">&rarr;<\/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":[30],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2286"}],"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=2286"}],"version-history":[{"count":3,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2286\/revisions"}],"predecessor-version":[{"id":2394,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2286\/revisions\/2394"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=2286"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=2286"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=2286"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}