{"id":1039,"date":"2013-04-19T08:36:39","date_gmt":"2013-04-19T00:36:39","guid":{"rendered":"http:\/\/www.hzaumycology.com\/chenlianfu_blog\/?p=1039"},"modified":"2013-04-19T10:25:25","modified_gmt":"2013-04-19T02:25:25","slug":"%e5%8f%aa%e6%a0%b9%e6%8d%ae%e4%b8%a4%e4%b8%aa%e8%bd%ac%e5%bd%95%e7%bb%84%e6%95%b0%e6%8d%ae%e5%88%86%e6%9e%90%e5%b7%ae%e5%bc%82%e8%a1%a8%e8%be%be%e5%9f%ba%e5%9b%a0","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=1039","title":{"rendered":"\u53ea\u6839\u636e\u4e24\u4e2a\u8f6c\u5f55\u7ec4\u6570\u636e\u5206\u6790\u5dee\u5f02\u8868\u8fbe\u57fa\u56e0"},"content":{"rendered":"<h2>1. \u6570\u636e<\/h2>\n<p>\u624b\u5934\u6709\u4e24\u4e2a\u8f6c\u5f55\u7ec4\u6570\u636e\uff0c\u5206\u522b\u662f\u67d0\u4e00\u771f\u83cc\u7269\u79cd(\u8be5\u7269\u79cd\u6ca1\u6709\u57fa\u56e0\u7ec4\u6570\u636e)\u7684\u53cc\u6838\u83cc\u4e1d\u9636\u6bb5\u548c\u5b50\u5b9e\u4f53\u9636\u6bb5\u7684\u8f6c\u5f55\u7ec4\u6570\u636e\u3002\u6d4b\u5e8f\u5e73\u53f0\u662fIllumina Hiseq2000\uff1b\u63d2\u5165\u7247\u6bb5\u957f\u5ea6200bp\uff0c\u6d4b\u5e8f\u7684reads\u957f\u5ea690bp\u3002\u5f97\u5230\u7684\u6570\u636e\u6587\u4ef6\u4e3a\uff1a<\/p>\n<pre>\/home\/user\/RNA-seq\/mycelium_reads1.fastq\r\n\/home\/user\/RNA-seq\/mycelium_reads2.fastq\r\n\/home\/user\/RNA-seq\/fruitingbody_reads1.fastq\r\n\/home\/user\/RNA-seq\/fruitingbody_reads2.fastq<\/pre>\n<h2>2. \u6570\u636e\u7684\u9884\u5904\u7406<\/h2>\n<p>2.1. \u53bb\u9664N\u7684\u6bd4\u4f8b\u5927\u4e8e5%\u7684reads;\u53bb\u9664\u4f4e\u8d28\u91cfreads\uff08\u8d28\u91cf\u503cQ\u226420\u7684\u78b1\u57fa\u6570\u5360\u6574\u4e2aread\u768450\uff05\u4ee5\u4e0a\uff09\uff1b<br \/>\n2.2. \u6839\u636eFastQC\u5bf9\u4e0a\u8ff0\u8fc7\u6ee4\u540e\u7684reads\u7684\u8d28\u91cf\u68c0\u6d4b\uff0c\u53bb\u9664reads\u9996\u5c3e\u540410bp\u7684\u78b1\u57fa\uff0c\u5f97\u5230\u7684\u9884\u5904\u7406\u6570\u636e\u4e3a\uff1a<\/p>\n<pre>\/home\/user\/RNA-seq\/clean_reads\/mycelium_reads1.fastq\r\n\/home\/user\/RNA-seq\/clean_reads\/mycelium_reads2.fastq\r\n\/home\/user\/RNA-seq\/clean_reads\/fruitingbody_reads1.fastq\r\n\/home\/user\/RNA-seq\/clean_reads\/fruitingbody_reads2.fastq<\/pre>\n<h2>3. \u4f7f\u7528Trinity\u548cTGICL\u8fdb\u884c\u8f6c\u5f55\u7ec4\u7684\u7ec4\u88c5<\/h2>\n<p>3.1. \u5bf9mycelium\u6570\u636e\u548cfruitingbody\u6570\u636e\u5206\u522b\u8fdb\u884c\u8f6c\u5f55\u7ec4\u7684\u7ec4\u88c5<\/p>\n<pre>$ pwd\r\n\/home\/user\/RNA-seq\/\r\n\r\n$ mkdir assembly\r\n$ cd assembly\r\n\r\n$ Trinity.pl --jaccard_clip --seqType fq --JM 50G --SS_lib_type FR --CPU 24 --inchworm_cpu 24 --bflyCPU 24 --group_pairs_distance 500 -min_contig_length 200 --output mycelium_contig --left \/home\/user\/RNA-seq\/clean_reads\/mycelium_reads1.fastq --right \/home\/user\/RNA-seq\/clean_reads\/mycelium_reads2.fastq\r\n$ Trinity.pl --jaccard_clip --seqType fq --JM 50G --SS_lib_type FR --CPU 24 --inchworm_cpu 24 --bflyCPU 24 --group_pairs_distance 500 -min_contig_length 200 --output fruitingbody_contig --left \/home\/user\/RNA-seq\/clean_reads\/fruitingbody_reads1.fastq --right \/home\/user\/RNA-seq\/clean_reads\/fruitingbody_reads2.fastq\r\n\r\n\u5f53\u7136\uff0c\u9700\u8981\u5c06\u751f\u6210\u7684\u4e24\u4e2a\u8f6c\u5f55\u7ec4\u7684Trinity.fasta\u5e8f\u5217\u6309\u957f\u5ea6\u8fdb\u884c\u6392\u5e8f\uff1b\u7edf\u4e00\u66f4\u6539\u7684fasta\r\n\u5934\u540d\u79f0\uff1b\u66f4\u6539fasta\u6587\u4ef6\u540d\r\n\r\n$ cp mycelium_contig\/Trinity.fasta mycelium_contigs.fasta\r\n$ cp fruitingbody_contig\/Trinity.fasta fruitingbody_contigs.fasta<\/pre>\n<p>3.2. \u4f7f\u7528TGICL\u5c06\u4e24\u4e2a\u8f6c\u5f55\u7ec4\u5e8f\u5217\u5408\u5e76<\/p>\n<pre>$ pwd\r\n\/home\/user\/RNA-seq\/assembly\r\n\r\n$ mkdir all_tissue_contig\r\n$ cd all_tissue_contig\r\n\r\n$ cat ..\/mycelium_contigs.fasta ..\/fruitingbody_contigs.fasta &gt; all.contigs.fasta\r\n$ tgicl -F all.contigs.fasta\r\n\r\ntgicl\u751f\u6210\u4e86\u4e24\u4e2a\u6709\u7528\u7684\u6587\u4ef6\uff1a asm_1\/contigs  \u548c all.contigs.fasta.single\r\ntons\u3002\u5176\u4e2d\u524d\u8005\u662f\u805a\u7c7b\u540e\u7684contigs\u7ed3\u679c\uff1b\u540e\u8005\u662f\u6ca1\u6709\u805a\u7c7b\u7684\u5355\u72ec\u7684contigs\u7684\u5e8f\u5217\u540d\uff0c\u9700\r\n\u8981\u5206\u522b\u5230..\/mycelium_contigs.fasta \u548c ..\/fruitingbody_contigs.fasta\u6587\r\n\u4ef6\u4e2d\u63d0\u53d6\u51fa\u76f8\u5e94\u7684\u5e8f\u5217: all.contigs.fasta.singletons.mycelium.fasta \u548c \r\nall.contigs.fasta.singletons.fruitingbody.fasta\r\n\r\n$ cat asm_1\/contigs all.contigs.fasta.singletons.mycelium.fasta all.contigs.fasta.singletons.fruitingbody.fasta &gt; all_contigs.fasta\r\n\r\n\u5728\u5bf9all_contigs.fasta\u8fdb\u884cfasta\u5934\u7684\u91cd\u547d\u540d\uff0c\u5e76\u5c06\u5e8f\u5217\u6309\u957f\u5ea6\u6392\u5e8f\uff1b\u540c\u65f6\u8981\u5f97\u5230all_\r\ncontigs.fasta\u4e2d\u7684\u5e8f\u5217\u548cmycelium_contigs.fasta\uff0cfruitingbody_contigs.\r\nfasta\u4e2d\u5e8f\u5217\u7684\u5bf9\u5e94\u5173\u7cfb\u3002\r\n\r\n$ cp all_contigs.fasta ..\/<\/pre>\n<p>3.3. \u81f3\u6b64\u5f97\u51fa\u8f6c\u5f55\u7ec4\u7684\u7ec4\u88c5\u7ed3\u679c\uff1a<\/p>\n<pre>\/home\/user\/RNA-seq\/assembly\/all_contigs.fasta<\/pre>\n<h2>4. \u4f7f\u7528cufflinks\u6765\u5206\u6790\u5dee\u5f02\u8868\u8fbe\u57fa\u56e0<\/h2>\n<p>4.1 \u4f7f\u7528tophat\u6765\u5c06\u9884\u5904\u7406\u540e\u7684reads\u6bd4\u5bf9\u5230\u8f6c\u5f55\u7ec4\u5e8f\u5217\u4e0a<\/p>\n<pre>\r\n$ pwd\r\n\/home\/user\/RNA-seq\/\r\n\r\n$ mkdir cufflinks\r\n$ cd cufflinks\r\n\r\n$ bowtie2-build ..\/all_contigs.fasta all_contigs\r\n\r\n$ tophat -o tophat_out_mycelium -r 60 --mate-std-dev 80 -p 24 --library-type fr-unstranded all.contigs \/home\/user\/RNA-seq\/clean_reads\/mycelium_reads1.fastq \/home\/user\/RNA-seq\/clean_reads\/mycelium_reads2.fastq\r\n$ tophat -o tophat_out_fruitingbody -r 60 --mate-std-dev 80 -p 24 --library-type fr-unstranded all.contigs \/home\/user\/RNA-seq\/clean_reads\/fruitingbody_reads1.fastq \/home\/user\/RNA-seq\/clean_reads\/fruitingbody_reads2.fastq\r\n<\/pre>\n<p>4.2 \u81ea\u5236\u4e00\u4e2atranscripts.gtf\u6587\u4ef6<br \/>\n\u7531\u4e8ecuffdiff\u8fd0\u884c\u9700\u8981\u4e00\u4e2a\u53c2\u8003\u5e8f\u5217\u7684transcripts.gtf\u6587\u4ef6: \u8be5\u6587\u4ef6\u67099\u5217\uff0c\u4f7f\u7528tab\u5206\u9694\uff1b\u4f7fexon\u7684\u8303\u56f4\u4e3a\u6574\u4e2acontig\u3002\u5176\u683c\u5f0f\u5982\uff1a<\/p>\n<pre>\r\nAA.auricula_all_contig_1     chenlianfu  exon  1  9401  .  .  .  gene_id \"A.auricula_all_contig_1\"; transcript_id \"A.auricula_all_contig_1\";\r\nA.auricula_all_contig_10     chenlianfu  exon  1  4464  .  .  .  gene_id \"A.auricula_all_contig_10\"; transcript_id \"A.auricula_all_contig_10\";\r\nA.auricula_all_contig_100    chenlianfu  exon  1  3090  .  .  .  gene_id \"A.auricula_all_contig_100\"; transcript_id \"A.auricula_all_contig_100\";\r\nA.auricula_all_contig_1000   chenlianfu  exon  1  1768  .  .  .  gene_id \"A.auricula_all_contig_1000\"; transcript_id \"A.auricula_all_contig_1000\";\r\nA.auricula_all_contig_10000  chenlianfu  exon  1  586   .  .  .  gene_id \"A.auricula_all_contig_10000\"; transcript_id \"A.auricula_all_contig_10000\"; \r\nA.auricula_all_contig_10001  chenlianfu  exon  1  586   .  .  .  gene_id \"A.auricula_all_contig_10001\"; transcript_id \"A.auricula_all_contig_10001\";\r\n<\/pre>\n<p>4.3 \u4f7f\u7528cuffdiff\u6765\u5206\u6790\u8f6c\u5f55\u5b50\u7684\u8868\u8fbe\u91cf\u548c\u5dee\u5f02\u8868\u8fbe\u57fa\u56e0<\/p>\n<pre>\r\n$ pwd\r\n\/home\/user\/RNA-seq\/cufflinks\r\n\r\n$ cuffdiff -L mycelium,fruitingbody --library-type fr-unstranded -p 8 -o cuffdiff .\/transcriptome.gtf .\/tophat_out_mycelium\/accepted_hits.bam .\/tophat_out_fruitingbody\/accepted_hits.bam\r\n<\/pre>\n<p>\u81f3\u6b64\u5f97\u51fa\u8f6c\u5f55\u5b50\u7684\u8868\u8fbe\u91cf\u6570\u636e\u548c\u5dee\u5f02\u8868\u8fbe\u5206\u6790<\/p>\n<pre>\r\n\/home\/user\/RNA-seq\/cufflinks\/cuffdiff\/gene_exp.diff\r\n<\/pre>\n","protected":false},"excerpt":{"rendered":"<p>1. \u6570\u636e \u624b\u5934\u6709\u4e24\u4e2a\u8f6c\u5f55\u7ec4\u6570\u636e\uff0c\u5206\u522b\u662f\u67d0\u4e00\u771f\u83cc\u7269\u79cd(\u8be5\u7269\u79cd\u6ca1\u6709\u57fa\u56e0\u7ec4\u6570\u636e)\u7684\u53cc &hellip; <a href=\"http:\/\/www.chenlianfu.com\/?p=1039\">\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":[39,20,29,35],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1039"}],"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=1039"}],"version-history":[{"count":13,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1039\/revisions"}],"predecessor-version":[{"id":1054,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1039\/revisions\/1054"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1039"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1039"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1039"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}