{"id":1153,"date":"2013-05-03T23:19:25","date_gmt":"2013-05-03T15:19:25","guid":{"rendered":"http:\/\/www.hzaumycology.com\/chenlianfu_blog\/?p=1153"},"modified":"2013-05-04T12:05:28","modified_gmt":"2013-05-04T04:05:28","slug":"genome-guided-trinity-for-gene-structure-annotation","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=1153","title":{"rendered":"Genome-guided Trinity for Gene Structure Annotation"},"content":{"rendered":"

\u4f7f\u7528genome\u6765\u5f15\u5bfcTrinity\u8fdb\u884c\u57fa\u56e0\u7ed3\u6784\u6ce8\u91ca\u3002<\/a><\/strong><\/p>\n

RNA-seq\u7684\u4e00\u4e2a\u4e3b\u8981\u7528\u9014\u662f\u8bc6\u522b\u57fa\u56e0\u7ec4\u7684\u8f6c\u5f55\u533a\uff0c\u91cd\u6784\u8f6c\u5f55\u5b50\u7ed3\u6784\uff0c\u540c\u65f6\uff0c\u9274\u5b9a\u8f6c\u5f55\u5b50\u7684\u53ef\u53d8\u526a\u5207\u3002<\/p>\n

\u73b0\u5728\u6700\u65b0\u7684\u57fa\u4e8egenome\u7684\u8f6c\u5f55\u5b50\u9884\u6d4b\u65b9\u6cd5\u662f\u5c06RNA-seq\u7684reads\u4f7f\u7528\u526a\u63a5\u6bd4\u5bf9\u7684\u65b9\u6cd5\u6bd4\u5bf9\u5230\u57fa\u56e0\u7ec4\uff0c\u7136\u540e\u7ec4\u88c5\u6bd4\u5bf9\u7ed3\u679c\u4ece\u800c\u5f97\u5230\u8f6c\u5f55\u5b50\u7684\u7ed3\u6784\u3002(eg. cufflinks, scripture)\u3002\u6211\u4eec\u5c06\u8fd9\u79cd\u65b9\u6cd5\u79f0\u4e3a\uff1aalign-reads then assemble-alignments<\/p>\n

Trinity\u53ef\u4ee5\u8fdb\u884c\u4e0d\u9700\u8981\u53c2\u8003\u57fa\u56e0\u7ec4\u7684de novo\u7ec4\u88c5\uff0c\u89c1\uff1aTrinity\u7684\u5b89\u88c5\u4e0e\u4f7f\u7528<\/a>\uff1b\u4e5f\u80fd\u8fdb\u884c\u6709\u53c2\u8003\u57fa\u56e0\u7ec4\u652f\u6301\u7684\u7ec4\u88c5\uff1a\u5373\u5c06RNA-Seq\u6bd4\u5bf9\u5230genome\u3001RNA-Seq read\u7684de novo\u7ec4\u88c5 \u548c \u8f6c\u5f55\u5b50\u6bd4\u5bf9 \u7ed3\u5408\u8d77\u6765\u3002<\/p>\n

1. \u6b65\u9aa4<\/h1>\n

1.1 align-reads<\/h2>\n

\u4f7f\u7528GSNAP\u6765\u5c06reads\u6bd4\u5bf9\u5230\u57fa\u56e0\u7ec4\u3002\u5c06\u57fa\u56e0\u7ec4\u5206\u6210\u5404\u4e2a\u88abreads\u8986\u76d6\u7684\u533a\u3002<\/p>\n

1.2 assemble-reads<\/h2>\n

\u5bf9\u6bcf\u4e2a\u533a\u4f7f\u7528Trinity\u5bf9\u76f8\u5e94\u7684reads\u8fdb\u884c\u7ec4\u88c5\u3002<\/p>\n

1.3 align-transcripts<\/h2>\n

\u4f7f\u7528PASA\u8f6f\u4ef6\u8c03\u7528GMAP\u6765\u5c06Trinity-assembled transcripts\u6bd4\u5bf9\u5230genome.<\/p>\n

1.4 assemble-transcript_alignments<\/h2>\n

\u4f7f\u7528PASA\u8f6f\u4ef6\u6765\u7ec4\u88c5\u4e0a\u4e00\u6b65\u9aa4\u7684\u6bd4\u5bf9\u7ed3\u679c\uff0c\u5f97\u51fa\u5b8c\u6574\u7684\u8f6c\u5f55\u5b50\u7ed3\u6784\uff0c\u540c\u65f6\uff0c\u4e5f\u80fd\u89e3\u6790\u53ef\u53d8\u526a\u63a5\u7684\u8f6c\u5f55\u5b50\u7ed3\u6784\u3002\u8be5\u6b65\u9aa4\u548c\u4e0a\u4e00\u6b65\u9aa4\u5176\u5b9e\u662f\u5728\u540c\u4e00\u4e2aPASA\u7a0b\u5e8f\u4e2d\u6267\u884c\u5f97\u5230\u7684\u3002<\/p>\n

2. \u9700\u8981\u7684\u8f6f\u4ef6<\/h1>\n

Trinity<\/a>
\nGSNAP & GMAP<\/a>
\nPASA<\/a><\/p>\n

3. \u8fd0\u884c<\/h1>\n

Below, we describe the steps required for running the genome-guided Trinity-based transcript reconstruction pipeline. \u9002\u5408\u4e8e\u771f\u83cc\u7269\u79cd\uff0c\u5176\u57fa\u56e0\u5bc6\u5ea6\u8f83\u5927\u3002<\/p>\n

3.1 Align RNA-Seq reads to the genome<\/h2>\n
$ $TRINITY_HOME\/util\/alignReads.pl --seqType fq --left reads.left.fq --right reads.right.fq --target genome.fasta --aligner gsnap -- -t 8\r\n$ samtools view gsnap_out\/gsnap.coordSorted.bam > gsnap.coordSorted.sam<\/pre>\n
3.2 Assemble the aligned reads using Trinity<\/h2>\n
$ % $TRINITY_HOME\/util\/prep_rnaseq_alignments_for_genome_assisted_assembly.pl --SS_lib_type FR --coord_sorted_SAM gsnap.coordSorted.sam -I 1000000\r\n$ find Dir_* -name \"*reads\" > read_files.list\r\n$ $TRINITY_HOME\/util\/GG_write_trinity_cmds.pl --reads_list_file read_files.list --paired --SS --jaccard_clip > trinity_GG.cmds\r\n$ $TRINITY_HOME\/Inchworm\/bin\/ParaFly -c trinity_GG.cmds -CPU 6 -failed_cmds trinity_GG.cmds.failed -v\r\n$ find Dir_*  -name \"*inity.fasta\" -exec cat {} + | $TRINITY_HOME\/util\/inchworm_accession_incrementer.pl > Trinity_GG.fasta<\/pre>\n
3.3 Align and assemble the Trinity-reconstructed transcripts using the PASA pipeline<\/h2>\n
$ cp $PASA_HOME\/pasa_conf\/pasa.alignAssembly.Template.txt alignAssembly.config\r\n$ perl -p -i -e 's\/MYSQLDB=.*\/MYSQLDB=sample_mysql_database\/' alignAssembly.config\r\n$ $PASA_HOME\/scripts\/Launch_PASA_pipeline.pl -c alignAssembly.config -C -R -g genome.fasta -t Trinity_GG.fasta --ALIGNERS blat,gmap --transcribed_is_aligned_orient --stringent_alignment_overlap 30.0<\/pre>\n","protected":false},"excerpt":{"rendered":"
\u4f7f\u7528genome\u6765\u5f15\u5bfcTrinity\u8fdb\u884c\u57fa\u56e0\u7ed3\u6784\u6ce8\u91ca\u3002 RNA-seq\u7684\u4e00\u4e2a\u4e3b\u8981 … \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":[30,35],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1153"}],"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=1153"}],"version-history":[{"count":8,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1153\/revisions"}],"predecessor-version":[{"id":1176,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1153\/revisions\/1176"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1153"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1153"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1153"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}