{"id":485,"date":"2020-04-15T00:11:45","date_gmt":"2020-04-14T16:11:45","guid":{"rendered":"http:\/\/www.wuchangsong.com\/?p=485"},"modified":"2020-04-28T19:57:30","modified_gmt":"2020-04-28T11:57:30","slug":"iso-seq3%e5%ae%89%e8%a3%85%e4%b8%8e%e4%bd%bf%e7%94%a8%ef%bc%88%e5%85%a8%e9%95%bf%e8%bd%ac%e5%bd%95%e7%bb%84%e5%88%86%e6%9e%90%ef%bc%89","status":"publish","type":"post","link":"http:\/\/www.wuchangsong.com\/?p=485","title":{"rendered":"Iso-seq3\u5b89\u88c5\u4e0e\u4f7f\u7528\uff08\u5168\u957f\u8f6c\u5f55\u7ec4\u5206\u6790\uff09"},"content":{"rendered":"<p>\u4f7f\u7528conda\u5b89\u88c5Iso-seq3<\/p>\n<pre>conda create -n anaCogent5.2 python=2.7 anaconda\r\nsource activate anaCogent5.2\r\nconda install -n anaCogent5.2 biopython\r\nconda install -n anaCogent5.2 -c http:\/\/conda.anaconda.org\/cgat bx-python\r\nconda install -n anaCogent5.2 -c bioconda isoseq3\r\nconda install -n anaCogent5.2 -c bioconda pbccs\r\nconda install -n anaCogent5.2 -c bioconda lima\r\n#The packages below are optional:\r\nconda install -n anaCogent5.2 -c bioconda pbcoretools # for manipulating PacBio datasets\r\nconda install -n anaCogent5.2 -c bioconda bamtools # for converting BAM to fasta\r\nconda install -n anaCogent5.2 -c bioconda pysam # for making CSV reports<\/pre>\n<p>Running IsoSeq<br \/>\nTypical workflow:<br \/>\n1. Generate consensus sequences from your raw subread data<br \/>\n$ ccs movie.subreads.bam movie.ccs.bam &#8211;noPolish &#8211;minPasses 1<\/p>\n<p>2. Generate full-length reads by primer removal and demultiplexing<br \/>\n$ cat primers.fasta<br \/>\n&gt;primer_5p<br \/>\nAAGCAGTGGTATCAACGCAGAGTACATGGGG<br \/>\n&gt;primer_3p<br \/>\nAAGCAGTGGTATCAACGCAGAGTAC<br \/>\n$ lima movie.ccs.bam primers.fasta movie.fl.bam &#8211;isoseq &#8211;no-pbi<\/p>\n<p>3. Remove noise from FL reads<br \/>\n$ isoseq3 refine movie.fl.P5&#8211;P3.bam primers.fasta movie.flnc.bam<\/p>\n<p>4. Cluster consensus sequences to generate unpolished transcripts<br \/>\n$ isoseq3 cluster movie.flnc.bam unpolished.bam &#8211;verbose<\/p>\n<p>5. Optionally, polish transcripts using subreads<br \/>\n$ isoseq3 polish unpolished.bam movie.subreads.bam polished.bam<\/p>\n<p>6. Map unpolished or polished transcripts to genome and collapse transcripts based on genomic mapping<br \/>\n$ pbmm2 align unpolished.bam reference.fasta aligned.sorted.bam &#8211;preset ISOSEQ &#8211;sort<br \/>\n$ isoseq3 collapse aligned.sorted.bam out.gff<br \/>\nor $ isoseq3 collapse aligned.sorted.bam movie.ccs.bam out.gff<\/p>\n<p>\u53c2\u8003\uff1a<a href=\"https:\/\/github.com\/PacificBiosciences\/IsoSeq\/blob\/master\/isoseq-clustering.md#step-2---primer-removal-and-demultiplexing\">https:\/\/github.com\/PacificBiosciences\/IsoSeq\/blob\/master\/isoseq-clustering.md#step-2&#8212;primer-removal-and-demultiplexing<\/a><\/p>\n<p><a href=\"https:\/\/github.com\/PacificBiosciences\/IsoSeq_SA3nUP\/wiki\/Tutorial:-Installing-and-Running-Iso-Seq-3-using-Conda\">https:\/\/github.com\/PacificBiosciences\/IsoSeq_SA3nUP\/wiki\/Tutorial:-Installing-and-Running-Iso-Seq-3-using-Conda<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>\u4f7f\u7528conda\u5b89\u88c5Iso-seq3 conda create -n anaCogent5.2 python=2.7 anaconda source activate anaCogent5.2 conda install -n anaCogent5.2 biopython conda install -n anaCogent5.2 -c http:\/\/conda.anaconda.org\/cgat bx-python conda install -n anaCogent5.2 -c bioconda isoseq3 conda install -n anaCogent5.2 -c bioconda pbccs conda install -n anaCogent5.2 -c bioconda lima #The packages below are optional: conda install -n anaCogent5.2 -c bioconda pbcoretools [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[4,1],"tags":[],"_links":{"self":[{"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=\/wp\/v2\/posts\/485"}],"collection":[{"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=485"}],"version-history":[{"count":7,"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=\/wp\/v2\/posts\/485\/revisions"}],"predecessor-version":[{"id":500,"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=\/wp\/v2\/posts\/485\/revisions\/500"}],"wp:attachment":[{"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=485"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=485"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.wuchangsong.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=485"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}