{"id":688,"date":"2013-03-19T09:47:09","date_gmt":"2013-03-19T01:47:09","guid":{"rendered":"http:\/\/www.hzaumycology.com\/chenlianfu_blog\/?p=688"},"modified":"2013-05-06T08:22:16","modified_gmt":"2013-05-06T00:22:16","slug":"trinity%e7%9a%84%e5%ae%89%e8%a3%85%e4%b8%8e%e4%bd%bf%e7%94%a8","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=688","title":{"rendered":"Trinity\u7684\u5b89\u88c5\u4e0e\u4f7f\u7528"},"content":{"rendered":"

\u4e00\u3001 Trinity\u7b80\u4ecb<\/h1>\n

Trinity<\/a>\uff0c\u662f\u7531 the Broad Institute<\/a> \u5f00\u53d1\u7684\u8f6c\u5f55\u7ec4de novo<\/em>\u7ec4\u88c5\u8f6f\u4ef6\uff0c\u7531\u4e09\u4e2a\u72ec\u7acb\u7684\u8f6f\u4ef6\u6a21\u5757\u7ec4\u6210\uff1aInchworm,Chrysalis\u548cButterfly\u3002\u4e09\u4e2a\u8f6f\u4ef6\u4f9d\u6b21\u6765\u5904\u7406\u5927\u89c4\u6a21\u7684RNA-seq\u7684reads\u6570\u636e\u3002Trinity\u7684\u7b80\u8981\u5de5\u4f5c\u6d41\u7a0b\u4e3a\uff1a
\nInchworm: <\/span>\u5c06RNA-seq\u7684\u539f\u59cbreads\u6570\u636e\u7ec4\u88c5\u6210Unique\u5e8f\u5217\uff1b
\nChrysalis:<\/span> \u5c06\u4e0a\u4e00\u6b65\u751f\u6210\u7684contigs\u805a\u7c7b\uff0c\u7136\u540e\u5bf9\u6bcf\u4e2a\u7c7b\u6784\u5efaBruijn\u56fe\uff1b
\nButterfly:<\/span> \u5904\u7406\u8fd9\u4e9bBruijn\u56fe\uff0c\u4f9d\u636e\u56fe\u4e2dreads\u548c\u6210\u5bf9\u7684reads\u6765\u5bfb\u627e\u8def\u5f84\uff0c\u4ece\u800c\u5f97\u5230\u5177\u6709\u53ef\u53d8\u526a\u63a5\u7684\u5168\u957f\u8f6c\u5f55\u5b50\uff0c\u540c\u65f6\u5c06\u65c1\u7cfb\u540c\u6e90\u57fa\u56e0\u7684\u8f6c\u5f55\u5b50\u5206\u5f00\u3002
\nTrinity\u53d1\u8868\u5728 Nature Biotechnology<\/a>\u3002<\/p>\n

\u4e8c\u3001 Trinity\u7684\u5b89\u88c5<\/h1>\n

1. \u4e0b\u8f7dTrinity<\/a>\u3002
\n2. \u5b89\u88c5Trinity\u3002<\/p>\n

$ tar zxvf trinityrnaseq_r2013-02-25.tgz\r\n$ cd trinityrnaseq_r2013-02-25\r\n$ make\r\n\u4ec5\u9700\u8981\u5728\u5b89\u88c5\u76ee\u5f55\u4e0b\u8fdb\u884cmake\u5373\u53ef\u3002\u8be5\u547d\u4ee4\u7f16\u8bd1\u4e86\u7531C++\u7f16\u5199\u7684Inchworm\u548cChrysalis\uff0c\u800c\r\n\u4f7f\u7528Java\u7f16\u5199\u7684Butterfly\u5219\u4e0d\u9700\u8981\u7f16\u8bd1\uff0c\u53ef\u4ee5\u76f4\u63a5\u4f7f\u7528\u3002<\/pre>\n
\u4e09\u3001Trinity\u7684\u4f7f\u7528<\/h1>\n
1. \u76f4\u63a5\u8fd0\u884c\u5b89\u88c5\u76ee\u5f55\u4e0b\u7684\u7a0b\u5e8fTrinity.pl\u6765\u4f7f\u7528\u8be5\u8f6f\u4ef6\uff0c\u4e0d\u5e26\u53c2\u6570\u5219\u7ed9\u51fa\u4f7f\u7528\u5e2e\u52a9\u3002\u5176\u5178\u578b\u7528\u6cd5\u4e3a\uff1a<\/p>\n
Trinity.pl --seqType fq --JM 50G --left reads_1.fq  --right reads_2.fq --CPU 6<\/pre>\n
2. Trinity\u53c2\u6570<\/p>\n
\u5fc5\u987b\u7684\u53c2\u6570\uff1a\r\n--seqType<\/span>     reads\u7684\u7c7b\u578b\uff1a(cfa, cfq, fa, or fq)\r\n--JM <\/span>         jellyfish\u4f7f\u7528\u591a\u5c11G\u5185\u5b58\u7528\u6765\u8fdb\u884ck-mer\u7684\u8ba1\u7b97\uff0c\u5305\u542b\u2018G\u2019\u8fd9\u4e2a\u5b57\u7b26\r\n--left<\/span>        \u5de6\u8fb9\u7684reads\u7684\u6587\u4ef6\u540d\r\n--rigth <\/span>      \u53f3\u8fb9\u7684reads\u7684\u6587\u4ef6\u540d\r\n--single <\/span>     \u4e0d\u6210\u5bf9\u7684reads\u7684\u6587\u4ef6\u540d\r\n\r\n\u53ef\u9009\u53c2\u6570\uff1a\r\n\r\nMisc\uff1a\r\n--SS_lib_type<\/span>        reads\u7684\u65b9\u5411\u3002\u6210\u5bf9\u7684reads: RF or FR; \u4e0d\u6210\u5bf9\u7684reads\r\n: F or R\u3002\u5728\u6570\u636e\u5177\u6709\u94fe\u7279\u5f02\u6027\u7684\u65f6\u5019\uff0c\u8bbe\u7f6e\u6b64\u53c2\u6570\uff0c\u5219\u6b63\u4e49\u548c\u53cd\u4e49\u8f6c\u5f55\u5b50\u80fd\u5f97\u5230\u533a\u5206\u3002\u9ed8\u8ba4\r\n\u60c5\u51b5\u4e0b\uff0c\u4e0d\u8bbe\u7f6e\u6b64\u53c2\u6570\uff0creads\u88ab\u5f53\u4f5c\u975e\u94fe\u7279\u5f02\u6027\u5904\u7406\u3002FR: \u5339\u914d\u65f6\uff0cread1\u57285'\u7aef\u4e0a\u6e38, \r\n\u548c\u524d\u5bfc\u94fe\u4e00\u81f4, read2\u57283'\u4e0b\u6e38, \u548c\u524d\u5bfc\u94fe\u53cd\u5411\u4e92\u8865. \u6216\u8005read2\u5728\u4e0a\u6e38, read1\u5728\u4e0b\u6e38\u53cd\r\n\u5411\u4e92\u8865; RF: read1\u57285'\u7aef\u4e0a\u6e38, \u548c\u524d\u5bfc\u94fe\u53cd\u5411\u4e92\u8865, read2\u57283'\u7aef\u4e0b\u6e38, \u548c\u524d\u5bfc\u94fe\u4e00\u81f4;\r\n--output <\/span>            \u8f93\u51fa\u7ed3\u679c\u6587\u4ef6\u5939\u3002\u9ed8\u8ba4\u60c5\u51b5\u4e0b\u751f\u6210trinity_out_dir\u6587\u4ef6\u5939\u5e76\r\n\u5c06\u8f93\u51fa\u7ed3\u679c\u4fdd\u5b58\u5230\u6b64\u6587\u4ef6\u5939\u4e2d\u3002\r\n--CPU<\/span>                \u4f7f\u7528\u7684CPU\u7ebf\u7a0b\u6570\uff0c\u9ed8\u8ba4\u4e3a2\r\n--min_contig_length<\/span>  \u62a5\u544a\u51fa\u7684\u6700\u77ed\u7684contig\u957f\u5ea6\u3002\u9ed8\u8ba4\u4e3a200\r\n--jaccard_clip<\/span>       \u5982\u679c\u4e24\u4e2a\u8f6c\u5f55\u5b50\u4e4b\u95f4\u6709UTR\u533a\u91cd\u53e0\uff0c\u5219\u8fd9\u4e24\u4e2a\u8f6c\u5f55\u5b50\u5f88\u6709\u53ef\u80fd\u5728\r\nde novo<\/em>\u7ec4\u88c5\u7684\u65f6\u5019\u88ab\u62fc\u63a5\u6210\u4e00\u6761\u5e8f\u5217\uff0c\u79f0\u4e3a\u878d\u5408\u8f6c\u5f55\u5b50(Fusion Transcript)\u3002\u5982\u679c\u6709\r\nfastq\u683c\u5f0f\u7684paired reads\uff0c\u5e76\u5c3d\u53ef\u80fd\u51cf\u5c11\u6b64\u7c7b\u7ec4\u88c5\u9519\u8bef\uff0c\u5219\u9009\u7528\u6b64\u53c2\u6570\u3002\u503c\u5f97\u8bf4\u660e\u7684\u662f\uff1a\r\n1. \u9002\u5408\u4e8e\u57fa\u56e0\u5728\u57fa\u56e0\u7ec4\u6bd4\u8f83\u7a20\u5bc6\uff0c\u8f6c\u5f55\u5b50\u7ecf\u5e38\u5728UTR\u533a\u57df\u91cd\u53e0\u7684\u7269\u79cd\uff0c\u6bd4\u5982\u771f\u83cc\u57fa\u56e0\u7ec4\u3002\u800c\u5bf9\r\n\u4e8e\u810a\u690e\u52a8\u7269\u548c\u690d\u7269\uff0c\u5219\u4e0d\u63a8\u8350\u4f7f\u7528\u6b64\u53c2\u6570; 2. \u8981\u6c42fastq\u683c\u5f0f\u7684paired reads\u6587\u4ef6(\u6587\u4ef6\r\n\u4e2dreads\u540d\u5206\u522b\u4ee5\/1\u548c\/2\u7ed3\u5c3e\uff0c\u4ee5\u5229\u4e8e\u8f6f\u4ef6\u8bc6\u522b)\uff0c\u540c\u65f6\u8fd8\u9700\u8981\u5b89\u88c5bowtie\u8f6f\u4ef6\u7528\u4e8ereads\r\n\u7684\u6bd4\u5bf9; 3. \u5355\u72ec\u4f7f\u7528\u5177\u6709\u94fe\u7279\u5f02\u6027\u7684RNA-seq\u6570\u636e\u7684\u65f6\u5019\uff0c\u80fd\u6781\u5927\u5730\u51cf\u5c11UTR\u91cd\u53e0\u533a\u5f88\u5c0f\u7684\r\n\u878d\u5408\u8f6c\u5f55\u5b50; 4. \u6b64\u9009\u9879\u8017\u8d39\u8fd0\u7b97\uff0c\u82e5\u6ca1\u5fc5\u8981\uff0c\u5219\u4e0d\u7528\u6b64\u53c2\u6570\u3002\r\n--prep<\/span>               \u4ec5\u4ec5\u51c6\u5907\u4e00\u4e9b\u6587\u4ef6(\u5229\u4e8eI\/O\uff09\u5e76\u5728kmer\u8ba1\u7b97\u524d\u505c\u6b62\u7a0b\u5e8f\u8fd0\u884c\r\n--no_cleanup<\/span>         \u4fdd\u7559\u6240\u6709\u7684\u4e2d\u95f4\u8f93\u5165\u6587\u4ef6\r\n--full_cleanup<\/span>       \u4ec5\u4fdd\u7559Trinity fasta\u6587\u4ef6\uff0c\u5e76\u91cd\u547d\u540d\u6210${output_dir}.\r\nTrinity.fasta\r\n--cite<\/span>               \u663e\u793aTrinity\u6587\u732e\u5f15\u8bc1\u548c\u4e00\u4e9b\u53c2\u4e0e\u7684\u8f6f\u4ef6\u5de5\u5177\r\n--version<\/span>            \u62a5\u544aTrinity\u7248\u672c\u5e76\u63a8\u51fa\r\n\r\nInchworm \u548c K-mer \u8ba1\u7b97\u76f8\u5173\u9009\u9879\uff1a\r\n--min_kmer_cov<\/span>      \u4f7f\u7528Inchworm\u6765\u8ba1\u7b97K-mer\u6570\u91cf\u65f6\u5019\uff0c\u8bbe\u7f6e\u7684Kmer\u7684\u6700\u5c0f\u503c\u3002\r\n\u9ed8\u8ba4\u4e3a1\r\n--inchworm_cpu<\/span>      Inchworm\u4f7f\u7528\u7684CPU\u7ebf\u7a0b\u6570\uff0c\u9ed8\u8ba4\u4e3a6\u548c--CPU\u8bbe\u7f6e\u7684\u503c\u4e2d\u7684\r\n\u5c0f\u503c\u3002\r\n\r\nChrysalis\u76f8\u5173\u9009\u9879\uff1a\r\n--max_reads_per_graph<\/span>   \u5728\u4e00\u4e2aBruijn\u56fe\u4e2d\u951a\u5b9a\u7684\u6700\u5927\u7684reads\u6570\u76ee\uff0c\u9ed8\u8ba4\u4e3a200\r\n000\r\n--no_run_chrysalis<\/span>      \u8fd0\u884cInchworm\u5b8c\u6bd5\uff0c\u5728\u8fd0\u884cchrysalis\u4e4b\u524d\u505c\u6b62\u8fd0\u884c\r\nTrinity\r\n--no_run_quantifygraph<\/span>  \u5728\u5e73\u884c\u5316\u8fd0\u7b97quantifygrahp\u524d\u505c\u6b62\u8fd0\u884cTrinity\r\n\r\nButterfly\u76f8\u5173\u9009\u9879\uff1a\r\n--bfly_opts<\/span>                    Butterfly\u989d\u5916\u7684\u53c2\u6570\r\n--max_number_of_paths_per_node<\/span> \u4ecenode A -> B,\u6700\u591a\u5141\u8bb8\u591a\u5c11\u6761\u8def\u5f84\u3002\u9ed8\u8ba4\r\n\u4e3a10\r\n--group_pairs_distance<\/span>         \u6700\u5927\u63d2\u5165\u7247\u8bfb\u957f\u5ea6\uff0c\u9ed8\u8ba4\u4e3a500\r\n--path_reinforcement_distance<\/span>  \u5ef6\u957f\u8f6c\u5f55\u5b50\u8def\u5f84\u65f6\u5019\uff0creads\u95f4\u6700\u5c0f\u7684\u91cd\u53e0\u78b1\u57fa\r\n\u6570\u3002\u9ed8\u8ba4PE:75; SE\uff1a25\r\n--no_triplet_lock<\/span>              \u4e0d\u9501\u5b9atriplet-supported nodes<\/code>\r\n--bflyHeapSpaceMax<\/span>             \u8fd0\u884cButterfly\u65f6java\u6700\u5927\u7684\u5806\u79ef\u7a7a\u95f4\uff0c\u9ed8\u8ba4\r\n\u4e3a20G\r\n--bflyHeapSpaceInit<\/span>            java\u521d\u59cb\u7684\u5806\u79ef\u7a7a\u95f4\uff0c\u9ed8\u8ba4\u4e3a1G\r\n--bflyGCThreads<\/span>                java\u8fdb\u884c\u65e0\u7528\u4fe1\u606f\u7684\u6574\u7406\u65f6\u4f7f\u7528\u7684\u7ebf\u7a0b\u6570\uff0c\u9ed8\r\n\u8ba4\u7531java\u6765\u51b3\u5b9a\r\n--bflyCPU<\/span>                      \u8fd0\u884cButterfly\u65f6\u4f7f\u7528\u7684CPU\u7ebf\u7a0b\u6570\uff0c\u9ed8\u8ba4\u4e3a2\r\n--bflyCalculateCPU<\/span>             \u8ba1\u7b97Butterfly\u6240\u8fd0\u884c\u7684CPU\u7ebf\u7a0b\u6570\uff0c\u7531\u516c\u5f0f\r\n\u00a080% * max_memory \/ maxbflyHeapSpaceMax \u5f97\u5230\r\n--no_run_butterfly<\/span>             \u5728Chrysalis\u8fd0\u884c\u5b8c\u6bd5\u540e\uff0c\u505c\u6b62\u8fd0\u884cButterfly\r\n\r\nGrid-computing\u9009\u9879\uff1a\r\n--grid_computing_module<\/span>   \u9009\u5b9aPerl\u6a21\u5757\uff0c\u5728\/Users\/bhaas\/SVN\/trinityr\r\nnaseq\/trunk\/PerlLibAdaptors\/\u3002<\/pre>\n
3. \u9002\u5408\u4e8eillumina\u6d4b\u5e8f\u6570\u636e\u7684\u771f\u83cc\u7269\u79cd\u8f6c\u5f55\u7ec4\u7ec4\u88c5\u7684Trinity\u547d\u4ee4\u4e3a\uff1a<\/p>\n
Trinity.pl --seqType fq --JM 50G --left reads_1.fq  --right reads_2.fq --SS_lib_type FR --output transcriptome_tissue --CPU 24 --jaccard_clip --inchworm_cpu 24 --group_pairs_distance 500 --bflyCPU 24<\/pre>\n
4. Trinity\u751f\u6210\u7684\u7ed3\u679c\u6587\u4ef6
\n\u8fd0\u884c\u7a0b\u5e8f\u7ed3\u675f\u540e\uff0c\u8f6c\u5f55\u7ec4\u7ed3\u679c\u4e3atrinity_out_dir\/Trinity.fasta\u3002\u53ef\u4ee5\u4f7f\u7528\u8f6f\u4ef6\u6240\u5e26\u7684\u4e00\u652f\u7a0b\u5e8f\u5206\u6790\u8f6c\u5f55\u7ec4\u7edf\u8ba1\u4fe1\u606f\u3002<\/p>\n
$ $TRINITY_HOME\/util\/TrinityStats.pl trinity_out_dir\/Trinity.fasta\r\nTotal trinity transcripts:\t30706\r\nTotal trinity components:\t26628\r\nContig N50: 554\r\n<\/pre>\n
\u4e09. Trinity\u8fd0\u884c\u539f\u7406\u4e0e\u8fc7\u7a0b<\/h1>\n
1. \u68c0\u6d4bjava\u7684\u53ef\u8fd0\u884c\u6027\uff0c\u56e0\u4e3abuttfly\u4f1a\u7528\u5230<\/h2>\n
2. \u8fd0\u884cjellyfish\uff0c\u4f7f\u7528\u5176dump\u547d\u4ee4\u5f97\u5230jellyfish.kmers.fa\u6587\u4ef6<\/h2>\n
3. Inchworm(Linear contig construction from k-mers)<\/h2>\n
assembles the RNA-seq data into the unique sequences of transcripts, often generating full-length transcripts for a dominant isoform, but then reports just the unique portions of alternatively spliced transcripts.<\/p>\n
4. Chrysalis<\/h2>\n
clusters the Inchworm contigs into clusters and constructs complete de Bruijn graphs for each cluster. Each cluster represents the full transcriptonal complexity for a given gene (or sets of genes that share sequences in common). Chrysalis then partitions the full read set among these disjoint graphs. <\/p>\n
5. Butterfly<\/h2>\n
then processes the individual graphs in parallel, tracing the paths that reads and pairs of reads take within the graph, ultimately reporting full-length transcripts for alternatively spliced isoforms, and teasing apart transcripts that corresponds to paralogous genes. <\/p>\n
\u56db. \u6ce8\u610f\u4e8b\u9879<\/h1>\n
3.1 Trinity\u5206\u6b65\u8fd0\u884c<\/h2>\n
\u5f53\u6570\u636e\u91cf\u6bd4\u8f83\u5927\u7684\u65f6\u5019\uff0ctrinity\u8fd0\u884c\u7684\u65f6\u95f4\u4f1a\u5f88\u957f\uff0c\u540c\u65f6\uff0c\u5185\u5b58\u4e0d\u591f\u7b49\u60c5\u51b5\u51fa\u73b0\u7684\u65f6\u5019\u6709\u53ef\u80fd\u7a0b\u5e8f\u8fd0\u884c\u5d29\u6e83\u3002\u6700\u597d\u662f\u5206\u6b65\u8fd0\u884c\u3002\u4e0b\u4e00\u6b65\u4f1a\u63a5\u7740\u524d\u4e00\u6b65\u8fdb\u884c\u4e0b\u53bb\u3002<\/p>\n
Stage 1: generate the kmer-catalog and run Inchworm: –no_run_chrysalis<\/p>\n
Stage 2: Chrysalis clustering of inchworm contigs and mapping reads: –no_run_quantifygraph<\/p>\n
Stage 3: Chrysalis deBruijn graph construction: –no_run_butterfly<\/p>\n
Stage 4: Run butterfly, generate final Trinity.fasta file. (exclude –no_ options)<\/p>\n
3.2 \u8ba1\u7b97\u8d44\u6e90<\/h2>\n
Ideally, you will have access to a large-memory server, ideally having ~1G of RAM per 1M reads to be assembled (but often, much less memory may be required). <\/p>\n
The assembly from start to finish can take anywhere from ~1\/2 hour to 1 hour per million reads (your mileage may vary). \u4e2a\u4eba\u8bb0\u5f55\u4e86\u4e00\u6b21\uff0c\u4f7f\u7528dell\u670d\u52a1\u5668\uff0c64GB RAM\uff0c24 threads : 53M \u7684reads\uff0c\u8fd0\u884c\u4e8616.5h\uff08\u5e73\u57473.2M\/h\uff09\uff0c\u5185\u5b58\u4f7f\u7528\u5cf0\u503c\u4e3a43G.<\/p>\n","protected":false},"excerpt":{"rendered":"
\u4e00\u3001 Trinity\u7b80\u4ecb Trinity\uff0c\u662f\u7531 the Broad Instit … \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":[35],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/688"}],"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=688"}],"version-history":[{"count":36,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/688\/revisions"}],"predecessor-version":[{"id":693,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/688\/revisions\/693"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=688"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=688"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=688"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}