{"id":2290,"date":"2015-08-25T16:39:39","date_gmt":"2015-08-25T08:39:39","guid":{"rendered":"http:\/\/www.chenlianfu.com\/?p=2290"},"modified":"2015-08-25T16:39:39","modified_gmt":"2015-08-25T08:39:39","slug":"%e4%bd%bf%e7%94%a8-splicegrapher-%e8%bf%9b%e8%a1%8c%e5%8f%af%e5%8f%98%e5%89%aa%e6%8e%a5%e5%88%86%e6%9e%90","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=2290","title":{"rendered":"\u4f7f\u7528 SpliceGrapher \u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u5206\u6790"},"content":{"rendered":"<h1>1. SpliceGrapher \u7b80\u4ecb<\/h1>\n<p><a href=\"http:\/\/splicegrapher.sourceforge.net\/index.html\" target=\"_blank\">SpliceGrapher<\/a> \u4e3b\u8981\u7528\u4e8e\u4f7f\u7528 RNA-Seq \u6570\u636e\u5bf9\u5df2\u6709\u7684\u57fa\u56e0\u6a21\u578b\u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u5206\u6790\uff0c\u5e76\u80fd\u7ed9\u51fa\u56fe\u5f62\u7ed3\u679c\u3002\u6b64\u5916\uff0c SpliceGrapher \u4e5f\u80fd\u63a5\u53d7 EST \u6570\u636e\u4f5c\u4e3a\u8f93\u5165\u3002\u7531\u4e8e\u4f7f\u7528 Sam \u6587\u4ef6\u4f5c\u4e3a\u8f93\u5165\uff0c\u5176\u53ef\u53d8\u526a\u63a5\u5206\u6790\u7ed3\u679c\u6bd4\u8f83\u5168\u9762\u51c6\u786e\u3002<br \/>\nSpliceGraper \u7684\u4f7f\u7528\u8bf4\u660e\u975e\u5e38\u8be6\u7ec6\uff0c\u5177\u4f53\u8bf7\u89c1\uff1a<a href=\"http:\/\/splicegrapher.sourceforge.net\/userguide.html\" target=\"_blank\">http:\/\/splicegrapher.sourceforge.net\/userguide.html<\/a>\u3002<\/p>\n<h1>2. SpliceGrapher \u4e0b\u8f7d\u4e0e\u5b89\u88c5<\/h1>\n<p>\u5b89\u88c5 SpliceGrapher<\/p>\n<pre>\r\n$ wget http:\/\/cznic.dl.sourceforge.net\/project\/splicegrapher\/SpliceGrapher-0.2.4.tgz\r\n$ tar zxf SpliceGrapher-0.2.4.tgz -C \/opt\/biosoft\/\r\n$ cd \/opt\/biosoft\/SpliceGrapher-0.2.4\r\n$ python setup.py build\r\n$ sudo python setup.py install\r\n\r\n\u68c0\u6d4b SpliceGrapher \u662f\u5426\u5b89\u88c5\u6210\u529f\u4ee5\u53ca\u80fd\u5426\u6b63\u5e38\u8fd0\u884c\r\n$ python\r\n>>> import SpliceGrapher\r\n>>> SpliceGrapher.__version__\r\n'0.2.4'\r\n$ cd examples\r\n$ sh run_tests.sh\r\n<\/pre>\n<p>\u6b64\u5916\uff0cSpliceGrapher\u6709\u8f83\u591a\u7684\u6b65\u9aa4\uff0c\u6839\u636e\u9700\u8981\u6765\u51b3\u5b9a\u662f\u5426\u8fd0\u884c\u76f8\u5e94\u7684\u6b65\u9aa4\u3002\u8fd9\u4e9b\u6b65\u9aa4\u7684\u6b63\u5e38\u8fd0\u884c\u9700\u8981\u5b89\u88c5\u4e00\u4e9b\u5176\u4ed6\u8f6f\u4ef6\u3002<br \/>\n\u521b\u5efa\u526a\u63a5\u4f4d\u70b9\uff08splice sites\uff09\u6a21\u578b\u6587\u4ef6\u65f6\uff0c\u9700\u8981\u5b89\u88c5<a href=\"http:\/\/http:\/\/pyml.sourceforge.net\/\" target=\"_blank\"> PyML <\/a>0.7.9\u6216\u4ee5\u4e0a\u7248\u672c\u3002<\/p>\n<pre>\r\n$ wget http:\/\/downloads.sourceforge.net\/project\/pyml\/PyML-0.7.13.3.tar.gz\r\n$ tar zxf PyML-0.7.13.3.tar.gz\r\n$ cd PyML-0.7.13.3\r\n$ python setup.py build\r\n$ sudo python setup.py install\r\n<\/pre>\n<p>\u5982\u679c\u9700\u8981\u4f7f\u7528 Bam \u6587\u4ef6\uff0c\u5219\u9700\u8981\u5b89\u88c5<a href=\"http:\/\/code.google.com\/p\/pysam\/\" target=\"_blank\"> Pysam <\/a>0.5\u6216\u4ee5\u4e0a\u7248\u672c\u3002\u7531\u4e8e Pysam \u5c06\u4ee3\u7801\u653e\u7f6e\u4e8e google \u4e0a\uff0c\u56e0\u6b64\u56fd\u5185\u65e0\u6cd5\u4e0b\u8f7d\u3002Pysam \u53ea\u662f python \u7248\u672c\u7684 samtools\uff0c\u56e0\u6b64\uff0c\u81ea\u884c\u4f7f\u7528 samtools \u5c06 Bam \u6587\u4ef6\u8f6c\u6362\u6210 Sam \u6587\u4ef6\u5373\u53ef\u3002<\/p>\n<p>\u5f53\u4f7f\u7528 PSGInfer pipeline \u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u8f6c\u5f55\u5b50\u9884\u6d4b\u7684\u65f6\u5019\uff0c\u9700\u8981\u5b89\u88c5<a href=\"http:\/\/deweylab.biostat.wisc.edu\/psginfer\/psginfer-1.1.3.tar.gz\" target=\"_blank\"> PSGInfer<\/a> 1.1.3\u6216\u4ee5\u4e0a\u7248\u672c<\/p>\n<pre>\r\n$ wget deweylab.biostat.wisc.edu\/psginfer\/psginfer-1.1.3.tar.gz\r\n$ tar zxf psginfer-1.1.3.tar.gz -C \/opt\/biosoft\r\n$ echo 'PATH=$PATH:\/opt\/biosoft\/psginfer-1.1.3' >> ~\/.bashrc\r\n<\/pre>\n<p>\u5f53\u4f7f\u7528 IsoLasso Pipeline \u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u8f6c\u5f55\u5b50\u9884\u6d4b\u7684\u65f6\u5019\uff0c\u9700\u8981 UCSC \u7684 <a href=\"http:\/\/hgdownload.cse.ucsc.edu\/admin\/exe\/linux.x86_64\/gtfToGenePred\" target=\"_blank\">gtfToGenePred <\/a>\u548c <a href=\"http:\/\/hgdownload.cse.ucsc.edu\/admin\/exe\/linux.x86_64\/genePredToBed\" target=\"_blank\">genePredToBed<\/a> \u7a0b\u5e8f\uff0c\u4ee5\u53ca <a href=\"http:\/\/alumni.cs.ucr.edu\/~liw\/isolasso.html\" target=\"_blank\">IsoLasso<\/a> 2.6.1\u6216\u4ee5\u4e0a\u7248\u672c\u3002<\/p>\n<pre>\r\n$ wget http:\/\/hgdownload.cse.ucsc.edu\/admin\/exe\/linux.x86_64\/gtfToGenePred\r\n$ wget http:\/\/hgdownload.cse.ucsc.edu\/admin\/exe\/linux.x86_64\/genePredToBed\r\n\r\n\u5b89\u88c5 Isolasso \u524d\u9700\u8981\u4f9d\u8d56 GSL(http:\/\/www.gnu.org\/s\/gsl\/) and CGAL(http:\/\/www.cgal.org\/) library\u652f\u6301\r\n\u4f7f\u7528 yum \u5b89\u88c5 gsl\r\n$ sudo yum install gsl*\r\n\u5728 Isolasso \u4e2d\u5e26\u6709 CGAL\uff0c\u4f46\u662f\u9700\u8981 libboost_thread.so.1.42.0 \u652f\u6301\uff0c\u9700\u8981\u5b89\u88c5 boost C++ library\r\n$ wget http:\/\/sourceforge.net\/projects\/boost\/files\/latest\/download?source=files\r\n$ tar zxf boost_1_57_0.tar.gz\r\n$ cd boost_1_57_0\r\n$ .\/bootstrap.sh \r\n$ .\/b2 -j 8\r\n$ sudo .\/b2 install\r\n$ sudo ln -s \/usr\/local\/lib\/libboost_thread.so.1.57.0 \/usr\/local\/lib\/libboost_thread.so.1.42.0\r\n\u5b89\u88c5 Isolasso\r\n$ wget http:\/\/alumni.cs.ucr.edu\/~liw\/isolasso-2.6.1.tar.gz\r\n$ tar zxf isolasso-2.6.1.tar.gz -C \/opt\/biosoft\r\n$ cd \/opt\/biosoft\/isolasso\/src\r\n$ export CXXFLAGS=\"$CXXFLAGS -I\/opt\/biosoft\/isolasso\/src\/isolassocpp\/CGAL\/include -L\/opt\/biosoft\/isolasso\/src\/isolassocpp\/CGAL\/lib\"\r\n$ export LD_LIBRARY_PATH=$LD_LIBRARY_PATH:\/opt\/biosoft\/isolasso\/src\/isolassocpp\/CGAL\/lib\/\r\n$ make\r\n$ echo 'PATH=$PATH:\/opt\/biosoft\/isolasso\/bin\/' >> ~\/.bashrc\r\n\u7a0b\u5e8f\u8fd0\u884c\u65f6\u9700\u8981 CGAL library \u652f\u6301\uff0c\u5c06\u4e4bcopy\u5230\u7cfb\u7edf\u80fd\u8bc6\u522b\u7684\u8def\u5f84\u4e2d\u3002\r\n$ sudo cp isolassocpp\/CGAL\/lib\/libCGAL.so* \/usr\/local\/lib\/\r\n$ sudo cp isolassocpp\/CGAL\/include\/* \/usr\/local\/include\/ -r\r\n<\/pre>\n<h1>3. SpliceGrapher \u7684\u4f7f\u7528<\/h1>\n<p>SpliceGrapher \u662f\u7684\u4f7f\u7528\u5176\u5b9e\u662f\u8fd0\u884c\u4e00\u4e9b python \u7a0b\u5e8f\uff0c\u8fd9\u4e9b python \u7a0b\u5e8f\u7684\u8f93\u5165\u6587\u4ef6\u5927\u90fd\u5305\u542b\u4e24\u4e2a\u6587\u4ef6\uff1a \u57fa\u56e0\u7ec4fasta\u6587\u4ef6(genome.fasta)\u548c\u57fa\u56e0\u6a21\u578b\u6587\u4ef6(geneModels.gff3)\u3002<br \/>\n\u6b64\u5904\u8bb2\u89e3 SpliceGrapher \u7684\u4f7f\u7528\uff0c\u4f7f\u7528\u4e86 RNA-Seq \u6570\u636e\u7528\u4e8e\u53ef\u53d8\u526a\u63a5\u9884\u6d4b\uff0c\u5728\u5f53\u524d\u5de5\u4f5c\u76ee\u5f55\u4e0b\u6709\u5982\u4e0b\u6587\u4ef6\u4f5c\u4e3a\u8f93\u5165\uff1a<\/p>\n<pre>\r\n$ ls\r\ngenome.fasta geneModels.gff3 reads.1.fastq reads.2.fastq tophat.bam\r\n\r\n\u6709\u4e9b\u8f6c\u5f55\u7ec4\u6570\u636e\u91cf\u8f83\u5927\uff0c\u63a8\u8350\u4f7f\u7528 Trinity \u8fdb\u884c\u6807\u51c6\u5316\u540e\u7684\u6570\u636e\u3002\r\n<\/pre>\n<p>\u7531\u4e8e\u591a\u4e2a python \u7a0b\u5e8f\u90fd\u9700\u8981 genome.fasta \u548c geneModels.gff3 \u4f5c\u4e3a\u8f93\u5165\uff0c\u56e0\u6b64\uff0c\u8f6f\u4ef6\u63a5\u53d7 Linux \u73af\u5883\u53d8\u91cf\u6307\u5b9a\u8fd9\u4e24\u4e2a\u6587\u4ef6\u7684\u8def\u5f84\uff0c\u6216\u5355\u72ec\u4f7f\u7528 -f \u548c -m \u53c2\u6570\u63a5\u53d7\u57fa\u56e0\u7ec4\u6587\u4ef6\u548c\u57fa\u56e0\u6a21\u578b\u6587\u4ef6\u7684\u8f93\u5165\u3002<br \/>\n\u4f7f\u7528\u5982\u4e0b\u547d\u4ee4\u6765\u8fdb\u884c\u73af\u5883\u53d8\u91cf\u8bbe\u7f6e\uff0c\u6709\u5229\u4e8e\u540e\u7eed\u7a0b\u5e8f\u7684\u7b80\u5355\u8fd0\u884c\u3002<\/p>\n<pre>\r\n$ export SG_FASTA_REF=$PWD\/genome.fasta\r\n$ export SG_GENE_MODEL=$PWD\/geneModels.gff3\r\n<\/pre>\n<h2>3.1  \u521b\u5efa\u526a\u63a5\u4f4d\u70b9\u6a21\u578b\u6587\u4ef6 (Create Splice Site Classifiers)<\/h2>\n<p>\u4ee5\u4e0b\u547d\u4ee4\u662f\u8fd0\u884c build_classifiers.py \u6765\u521b\u5efa\u526a\u63a5\u4f4d\u70b9\u6a21\u578b\uff0c\u7528\u4e8e\u9274\u5b9a GT-AG\/GC-AG\/AT-AC \u4f4d\u70b9\u3002\u4e00\u822c\u7269\u79cd\u7684\u526a\u63a5\u4f4d\u70b9\u4e3b\u8981\u662f GT-AG\/GC-AG\uff0c\u4f7f\u7528\u53c2\u6570 -d gt,gc \u5373\u53ef\u3002<\/p>\n<pre>\r\n$ mkdir 1.create_classifiers\r\n$ cd 1.create_classifiers\r\n$ build_classifiers.py -d gt,gc,at -a ag,ac -l create_classifiers.log\r\n\r\n\u7a0b\u5e8f\u8fd0\u884c\u5b8c\u6bd5\uff0c\u67e5\u770b\u6a21\u578b\u7684 ROC scores\r\n$ grep roc *.cfg\r\n\u5f97\u5230\u7684 donor \u7684\u5206\u6570\u4e00\u822c\u662f 0.9 \u4ee5\u4e0a\uff0c\u6bd4 acceptor \u7684\u5206\u6570\u8981\u9ad8\u3002\r\n<\/pre>\n<p>build_classifiers.py \u7a0b\u5e8f\u9996\u5148\u8c03\u7528 generate_splice_site_data.py \u751f\u6210\u7528\u4e8e training \u7684\u5e8f\u5217\uff0c\u518d\u8c03\u7528 select_model_parameters.py \u751f\u6210\u6a21\u578b\u6587\u4ef6\u3002<br \/>\n\u672c\u6b65\u9aa4\u7684\u4e3b\u8981\u7ed3\u679c\u6587\u4ef6\u662f classifiers.zip \u3002<br \/>\n\u8fd0\u884c build_classifiers.py \u547d\u4ee4\u4e00\u6b65\u5230\u4f4d\uff0c\u751f\u6210\u6a21\u578b\u6587\u4ef6  classifiers.zip\u3002\u82e5\u9700\u8981\u66f4\u52a0\u51c6\u786e\u7684\u6a21\u578b\u6587\u4ef6\uff0c\u5219\u53c2\u8003\u8f6f\u4ef6\u7684\u4f7f\u7528\u8bf4\u660e\u5206\u6b65\u9009\u62e9\u66f4\u597d\u7684\u53c2\u6570\u8fd0\u884c\u3002<\/p>\n<h2>3.2 \u8fc7\u6ee4 RNA-Seq reads \u7684\u6bd4\u5bf9\u7ed3\u679c (Filter Alignment)<\/h2>\n<p>\u4f7f\u7528\u4e0a\u4e00\u6b65\u7684\u6a21\u578b\u6587\u4ef6\uff0c\u5bf9 RNA-Seq \u7684\u6bd4\u5bf9\u7ed3\u679c\u8fdb\u884c\u8fc7\u6ee4\u3002<\/p>\n<pre>\r\n$ mkdir ..\/2.filter_alignments\r\n$ cd ..\/2.filter_alignments\r\n$ samtools view -h ..\/tophat.bam > tophat.sam\r\n$ ln -s ..\/1.create_classifiers\/classifiers.zip .\/\r\n$ sam_filter.py tophat.sam classifiers.zip -o filtered.sam -v\r\n<\/pre>\n<p>\u672c\u6b65\u9aa4\u5f97\u5230\u8fc7\u6ee4\u540e\u7684\u6bd4\u5bf9\u7ed3\u679c\u6587\u4ef6 filtered.sam\u3002<\/p>\n<h2>3.3 \u9884\u6d4b\u53ef\u53d8\u526a\u63a5 Graph (Predict splice graphs)<\/h2>\n<pre>\r\n$ mkdir ..\/3.predict_graphs\r\n$ cd ..\/3.predict_graphs\r\n$ predict_graphs.py ..\/2.filter_alignments\/filtered.sam -v\r\n<\/pre>\n<p>\u672c\u6b65\u9aa4\u5f97\u5230\u53ef\u53d8\u526a\u63a5 Graph\u3002\u7ed3\u679c\u8868\u73b0\u65b9\u5f0f\u4e3a\uff1a \u9ed8\u8ba4\u518d\u5f53\u524d\u76ee\u5f55\u4e0b\u751f\u6210\u8bb8\u591a\u7684\u6587\u4ef6\u5939\uff0c\u6bcf\u4e2a\u6587\u4ef6\u5939\u4ee5 chromosome \u540d\u79f0\u7684\u5c0f\u5199\u5b57\u6bcd\u4f5c\u4e3a\u6587\u4ef6\u540d\uff1b\u6bcf\u4e2a\u6587\u4ef6\u5939\u4e0b\u6709\u5f88\u591a GFF \u6587\u4ef6\uff0c\u6bcf\u4e2a GFF \u6587\u4ef6\u4ee5 gene model \u7684\u5927\u5199\u5b57\u6bcd\u4f5c\u4e3a\u6587\u4ef6\u540d\u524d\u7f00\uff1b \u6bcf\u4e2a GFF \u7684\u5185\u5bb9\u5373\u4e3a Graph \u7684\u6587\u672c\u5f62\u5f0f\uff0c\u6ce8\u610f\u5176\u683c\u5f0f\u4e0d\u662f\u771f\u6b63\u7684 GFF \u6587\u4ef6\u683c\u5f0f\u3002<\/p>\n<p>\u7531\u4e8e predict_graphs.py \u4e3a\u5355\u7ebf\u7a0b\u8fd0\u884c\u3002\u5982\u679c\u57fa\u56e0\u7ec4\u8f83\u5927\uff0c\u57fa\u56e0\u6a21\u578b\u8f83\u591a\uff0c\u6216 Sam \u6587\u4ef6\u8f83\u5927\uff0c\u5219\u8fd0\u884c\u65f6\u95f4\u6781\u957f\u3002\u53ef\u4ee5\u8003\u8651\u5c06\u6587\u4ef6\u4ee5\u67d3\u8272\u4f53\u4e3a\u5355\u5143\u8fdb\u884c\u5206\u5272\uff0c\u7136\u540e\u5e76\u884c\u8fd0\u7b97\uff1a<\/p>\n<pre>\r\n$ sam_collate.py ..\/2.filter_alignments\/filtered.sam\r\n$ for i in `ls *.sam`\r\ndo\r\n    echo \"predict_graphs.py $i\" >> command.predict_graphs.list\r\ndone\r\n$ ParaFly -c command.predict_graphs.list -CPU 24\r\n<\/pre>\n<p>\u6b64\u5916\uff0c \u5feb\u901f\u8fd0\u884c\u7684\u53e6\u5916\u4e00\u79cd\u65b9\u6cd5\u662f\uff1a\u5c06 sam \u6587\u4ef6\u8f6c\u53d8\u6210 depths \u6587\u4ef6\uff0c\u518d\u7528\u4e8e\u53ef\u53d8\u526a\u63a5\u9884\u6d4b\u3002<\/p>\n<pre>\r\n$ sam_to_depths.py ..\/2.filter_alignments\/filtered.sam -o filtered.depths\r\n$ predict_graphs.py filtered.depths -v\r\n<\/pre>\n<h2>3.3 \u91cd\u65b0\u6bd4\u5bf9 RNA-Seq reads \u6765\u5bf9\u53ef\u53d8\u526a\u63a5 Graphs \u8fdb\u884c\u66f4\u65b0 (Realignment RNA-seq reads)<\/h2>\n<p>SpliceGraper \u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u9884\u6d4b\u53ef\u80fd\u5f97\u5230\u4e00\u4e9b\u65b0\u7684 exons\uff0c \u5176\u4e2d\u53ef\u80fd\u5305\u542b\u4e00\u4e9b\u9519\u8bef\u7684 exon\u3002\u56e0\u6b64\uff0c\u6839\u636e\u4e0a\u4e00\u6b65\u7684\u7ed3\u679c\u63d0\u53d6 putative transcripts\uff0c\u7136\u540e\u4f7f\u7528 BWA \u5c06 RNA-Seq reads \u6bd4\u5bf9\u4e0a\u53bb\uff0c\u9a8c\u8bc1 realigned reads \u80fd\u5426\u5b8c\u5168\u8986\u76d6\u65b0\u7684 exon \u53ca\u5176\u8fb9\u754c\uff0c\u4ece\u800c\u5bf9\u53ef\u53d8\u526a\u63a5\u7684\u9884\u6d4b\u7ed3\u679c\u8fdb\u884c\u4e86\u66f4\u65b0\u3002<\/p>\n<pre>\r\n$ mkdir ..\/4.realignment_pipeline\r\n$ cd ..\/4.realignment_pipeline\r\n$ realignment_pipeline.py ..\/3.predict_graphs\/ -1 ..\/reads.1.fatsq -2 ..\/reads.2.fastq -v\r\n<\/pre>\n<p>\u672c\u6b65\u9aa4\u5f97\u5230\u7684\u4e3b\u8981\u7ed3\u679c\u4e3a\u66f4\u65b0\u540e\u7684\u53ef\u53d8\u526a\u63a5 Graphs\uff0c\u5176\u7ed3\u679c\u7684\u5448\u73b0\u65b9\u5f0f\u548c\u4e0a\u4e00\u6b65\u9aa4\u4e00\u6837\u3002<\/p>\n<h2>3.4 \u53ef\u53d8\u526a\u63a5\u8f6c\u5f55\u5b50\u9884\u6d4b (Transcript Prediction Pipelines)<\/h2>\n<p>SpliceGrapher \u63d0\u4f9b\u4e86 2 \u79cd\u65b9\u6cd5\u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u8f6c\u5f55\u5b50\u7684\u9884\u6d4b\uff1a PSGInfer Pipeline \u6216 IsoLasso Pipeline\u3002\u524d\u8005\u7684\u8f93\u5165\u6587\u4ef6\u662f fastq \u6587\u4ef6\uff0c\u540e\u8005\u7684\u8f93\u5165\u6587\u4ef6\u662f Sam \u6587\u4ef6\u3002\u8fd9\u4e24\u79cd\u65b9\u6cd5\u90fd\u9700\u8981\u4e0a\u4e00\u6b65\u9aa4\u7684\u7ed3\u679c\u6587\u4ef6\u5939\u4f5c\u4e3a\u8f93\u5165\u3002<\/p>\n<h3>3.4.1 PSGInfer Pipeline<\/h3>\n<p>\u4f7f\u7528 PSGInfer pipeline \u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u8f6c\u5f55\u5b50\u9884\u6d4b\u7684\u65f6\u5019\uff0c\u9700\u8981\u5b89\u88c5<a href=\"http:\/\/deweylab.biostat.wisc.edu\/psginfer\/psginfer-1.1.3.tar.gz\" target=\"_blank\"> PSGInfer<\/a> 1.1.3\u6216\u4ee5\u4e0a\u7248\u672c\u3002<\/p>\n<pre>\r\n$ mkdir ..\/5.Transcript_Prediction\r\n$ psginfer_pipeline.py ..\/4.realignment_pipeline ..\/reads.1.fatsq ..\/reads.2.fastq -d psginfer -l psginfer.log\r\n<\/pre>\n<h3>3.4.2 Isoasso Pipeline<\/h3>\n<p>\u4f7f\u7528 IsoLasso Pipeline \u8fdb\u884c\u53ef\u53d8\u526a\u63a5\u8f6c\u5f55\u5b50\u9884\u6d4b\u7684\u65f6\u5019\uff0c\u9700\u8981 UCSC \u7684 <a href=\"http:\/\/hgdownload.cse.ucsc.edu\/admin\/exe\/linux.x86_64\/gtfToGenePred\" target=\"_blank\">gtfToGenePred <\/a>\u548c <a href=\"http:\/\/hgdownload.cse.ucsc.edu\/admin\/exe\/linux.x86_64\/genePredToBed\" target=\"_blank\">genePredToBed<\/a> \u7a0b\u5e8f\uff0c\u4ee5\u53ca <a href=\"http:\/\/alumni.cs.ucr.edu\/~liw\/isolasso.html\" target=\"_blank\">IsoLasso<\/a> 2.6.1\u6216\u4ee5\u4e0a\u7248\u672c\u3002<\/p>\n<pre>\r\nIsolasso \u63d0\u4f9b\u4e86\u4e24\u79cd\u7b97\u6cd5\uff0c\u4f7f\u7528 CEM \u7b97\u6cd5\u5219\u5f00\u542f -C \u53c2\u6570\u3002\r\n$ isolasso_pipeline.py ..\/4.realignment_pipeline ..\/2.filter_alignments\/filtered.sam -t 1.00 -d isolasso_PSG -v\r\n$ isolasso_pipeline.py ..\/4.realignment_pipeline ..\/2.filter_alignments\/filtered.sam -t 1.00 -Cd isolasso_CEM -v\r\n\r\n\u503c\u5f97\u6ce8\u610f\u7684\u662f\uff0c\u8f6f\u4ef6\u5728chromosome\u540d\u79f0\u7684\u5927\u5c0f\u5199\u8f6c\u6362\u4e0a\u5b58\u5728bug\uff0c\u5bfc\u81f4\u751f\u6210\u7684 bed \u683c\u5f0f\u6587\u4ef6\u7684 chromosome \u540d\u79f0\u9519\u8bef\uff0c\u4ece\u800c\u4f7f\u7ed3\u679c\u4e0d\u6b63\u786e\u3002\u6700\u597d\u4e86\u89e3 Isolasso \u8f6f\u4ef6\u7684\u4f7f\u7528\u4ece\u800c\u5206\u6b65\u8fd0\u884c\u3002\r\n$ generate_putative_sequences.py ..\/4.realignment_pipeline\/.lis -A -f $SG_FASTA_REF -M _forms.gtf -m _forms.map\r\n$ gtfToGenePred _forms.gtf stdout | genePredToBed | sort -k1,1 -k2,2n > _forms.bed\r\n\u4fee\u6539 bed \u6587\u4ef6\u4e2d\u7684 chromosome \u540d\u79f0\uff0c\u4f8b\u5982\uff1a\r\n$ perl -p -i -e 's\/Le01scaffold\/LE01Scaffold\/' _forms.bed\r\n$ runlasso.py -x _forms.bed --forceref  -o isolasso_PSG ..\/2.filter_alignments\/filtered.sam\r\n$ isolasso_update_graphs.py ..\/4.realignment_pipeline\/.lis _forms.map isolasso_PSG.pred.gtf -t 1.00 -d isolasso_PSG\r\n\r\n<\/pre>\n<h3>3.4.3 GTF \u8f6c\u6362\u4e3a GFF3<\/h3>\n<p>\u4ee5\u4e0a\u5f97\u5230 GTF \u7684\u7ed3\u679c\u6587\u4ef6\uff0c\u4e00\u822c\u9700\u8981\u5c06\u4e4b\u8f6c\u6362\u4e3a GFF3 \u683c\u5f0f\u6587\u4ef6\uff1a<\/p>\n<pre>\r\n$ convert_models.py psginfer\/putative_forms.gtf -gff3=spliceGrapher.gff3\r\n<\/pre>\n<h2>3.5 \u53ef\u53d8\u526a\u63a5\u56fe\u5f62\u6587\u4ef6\u5236\u4f5c<\/h2>\n<p>\u6b64\u90e8\u5206\u6682\u65f6\u63a0\u8fc7<\/p>\n","protected":false},"excerpt":{"rendered":"<p>1. SpliceGrapher \u7b80\u4ecb SpliceGrapher \u4e3b\u8981\u7528\u4e8e\u4f7f\u7528 &hellip; <a href=\"http:\/\/www.chenlianfu.com\/?p=2290\">\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":[72,30],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2290"}],"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=2290"}],"version-history":[{"count":1,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2290\/revisions"}],"predecessor-version":[{"id":2291,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2290\/revisions\/2291"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=2290"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=2290"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=2290"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}