{"id":2266,"date":"2015-08-25T16:21:49","date_gmt":"2015-08-25T08:21:49","guid":{"rendered":"http:\/\/www.chenlianfu.com\/?p=2266"},"modified":"2015-08-25T16:23:05","modified_gmt":"2015-08-25T08:23:05","slug":"%e4%bd%bf%e7%94%a8pacbiotoca%e4%bf%ae%e6%ad%a3pacbio%e6%95%b0%e6%8d%ae","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=2266","title":{"rendered":"\u4f7f\u7528PacBioToCA\u4fee\u6b63Pacbio\u6570\u636e"},"content":{"rendered":"

1. PacBioToCA \u7b80\u4ecb<\/h1>\n

PacBioToCA \u662f Celera Assembler<\/a> \u7684\u4e00\u4e2a\u6a21\u5757\uff0c\u7528\u4e8ePacbio\u6570\u636e\u7684\u6821\u6b63\u3002\u56e0\u6b64\uff0c\u4f7f\u7528 PacBioToCA \u9700\u8981\u5b89\u88c5 Celera Assembler\u3002
\n\u5229\u7528 PacBioToCA \u8fdb\u884c\u6821\u6b63\u65f6\uff1a\u5982\u679c Pacbio \u6570\u636e\u91cf > 20x \u65f6\uff0c\u53ef\u4ee5\u8fdb\u884c\u81ea\u6211\u6821\u6b63\uff0c\u800c\u4e0d\u9700\u8981\u5176\u5b83\u66f4\u4e3a\u7cbe\u786e\u7684\u6570\u636e\uff1b\u63a8\u8350 Pacbio \u6570\u636e\u91cf > 50x \u65f6\u8fdb\u884c\u81ea\u6211\u6821\u6b63\uff1b\u901a\u5e38 > 15x \u7684 Pacbio \u6570\u636e\u5373\u53ef\u5f97\u5230\u5f88\u597d\u7684\u7ec4\u88c5\u7ed3\u679c\u3002<\/p>\n

2. \u5b89\u88c5 Celera Assembler<\/p>\n

\n\u63a8\u8350\u4f7f\u7528\u6e90\u7801\u5b89\u88c5\uff0c\u76f4\u63a5\u4e0b\u8f7d\u4e8c\u8fdb\u5236\u7248\u672c\uff0c\u53ef\u80fd\u63d0\u793a\u9519\u8bef\uff1a\/lib64\/libc.so.6: version `GLIBC_2.14′ not found<\/p>\n
\r\n$ wget http:\/\/sourceforge.net\/projects\/wgs-assembler\/files\/wgs-assembler\/wgs-8.3\/wgs-8.3rc2.tar.bz2\r\n$ tar jxf wgs-8.3rc2.tar.bz2 -C \/opt\/biosoft\/\r\n$ cd \/opt\/biosoft\/wgs-8.3rc2\/kmer\r\n\u4ee5\u4e0b\u4f7f\u7528 make\uff0c\u4e0d\u8981\u4f7f\u7528 -j \u53c2\u6570\u8fdb\u884c\u591a\u7ebf\u7a0b\u7f16\u8bd1\uff0c\u4f1a\u51fa\u9519\u3002\r\n$ make && make install && cd ..\r\n$ cd src && make && cd ..\r\n$ echo 'PATH=$PATH:\/opt\/biosoft\/wgs-8.3rc2\/Linux-amd64\/bin\/' >> ~\/.bashrc\r\n\r\n\u5982\u679c\u8fdb\u884c Pacbio \u6570\u636e\u7684\u81ea\u6211\u6821\u6b63\uff0c\u5219\u9700\u8981 JAVA 1.8 \u7248\u672c\r\n$ wget http:\/\/javadl.sun.com\/webapps\/download\/AutoDL?BundleId=106240 -O jre1.8.0_45.tar.gz\r\n$ tar zxf jre1.8.0_45.tar.gz -C \/opt\/biosoft\r\n$ echo 'PATH=\/opt\/biosoft\/jre1.8.0_45\/bin\/:$PATH' >> ~\/.bashrc\r\n$ source ~\/.bashrc\r\n<\/pre>\n
3. PacBioToCA \u7684\u4f7f\u7528<\/h1>\n
3.1 \u5c06\u9519\u8bef\u7387\u4f4e\u7684\u6d4b\u5e8f\u6570\u636e\u8f6c\u6362\u4e3a FRG \u6570\u636e<\/h2>\n
Celera Assembler \u8f6f\u4ef6\u8f93\u5165\u7684\u6d4b\u5e8f\u6570\u636e\u7684\u6587\u4ef6\u683c\u5f0f\u662f FRG \u683c\u5f0f<\/a>\u3002PacBioToCA \u8f93\u5165\u7684\u9519\u8bef\u7387\u4f4e\u7684\u6570\u636e\u9700\u8981 FRG \u683c\u5f0f\u3002
\n1. \u4f7f\u7528 fastqToCA<\/a> \u5c06 fastq \u6570\u636e\u8f6c\u6362\u4e3a FRG \u683c\u5f0f<\/b>
\n\u4e00\u822c\u60c5\u51b5\u4e0b\u662f\u5229\u7528\u9519\u8bef\u7387\u8f83\u4f4e\u7684 Illumina \u6570\u636e\u6765\u5bf9 Pacbio \u6570\u636e\u8fdb\u884c\u6821\u6b63\u3002 \u56e0\u6b64\u9700\u8981\u5c06 Illumina \u7684 Fastq \u6587\u4ef6\u8f6c\u6362\u6210 FRG \u683c\u5f0f\u3002 FRG \u6587\u4ef6\u662f\u6587\u672c\u6587\u4ef6\uff0c\u5176\u4e2d\u5305\u542b\u6709 fastq \u6587\u4ef6\u7684\u8def\u5f84\u548c\u4e00\u4e9b\u6d4b\u5e8f\u4fe1\u606f\uff0c\u6bd4\u5982\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\u3001\u6d4b\u5e8f\u65b9\u5411\u548c\u78b1\u57fa\u8d28\u91cf\u683c\u5f0f\u7b49\u3002
\nfastqToCA \u7684\u5e38\u7528\u793a\u4f8b\u548c\u5e38\u7528\u53c2\u6570\uff1a<\/p>\n
\r\n$ fastqToCA -insertsize 500 50 -libraryname pe500 -mates reads.1.fastq,reads.2.fastq > pe500.frg\r\n\r\n-insertsize\r\n    \u8bbe\u7f6e\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\uff0c\u4e3a2\u4e2a\u503c\uff0c\u524d\u8005\u4e3a\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\uff0c\u540e\u8005\u4e3a\u6807\u51c6\u5dee\u3002\r\n-libraryname\r\n    \u6587\u5e93\u7684ID\r\n-reads\r\n    \u5355\u7aef\u6d4b\u5e8f\u7684 fastq \u6587\u4ef6\u8def\u5f84\u3002\r\n-mates\r\n    \u53cc\u7aef\u6d4b\u5e8f\u7684 fastq \u6587\u4ef6\u8def\u5f84\u3002\u82e5\u53cc\u7aef\u6570\u636e\u4ea4\u53c9\u5728\u4e00\u4e2a\u6587\u4ef6\u4e2d\uff0c\u5219\u8be5\u53c2\u6570\u540e\u53ea\u6709\u4e00\u4e2a\u8def\u5f84\uff1b\u82e5\u53cc\u7aef\u6570\u636e\u57282\u4e2afastq\u6587\u4ef6\u4e2d\uff0c\u52192\u4e2a\u8def\u5f84\u7528\u9017\u53f7\u5206\u5272\u3002\r\n-technology  default:illumina\r\n    \u6d4b\u5e8f\u6280\u672f\u3002\u53ef\u9009\u7684\u503c\u6709\uff1anone, sanger, 454, illumina(\u8bfb\u957f < 160bp), illumina-long(\u4efb\u610f\u8bfb\u957f), moleculo, pacbio-ccs, pacbio-corrected, pacbio-raw\u3002\r\n-type  default:sanger\r\n    \u78b1\u57fa\u8d28\u91cf\u683c\u5f0f\u3002\u53ef\u9009\u7684\u503c\u6709\uff1asanger, solexa, illumina\u3002\r\n-innie\r\n    \u6d4b\u5e8f\u65b9\u5411\u4e3a 5'-3' <-> 3'-5'\uff0c\u9002\u5408\u5c0f\u7247\u6bb5\u6d4b\u5e8f\u6587\u5e93\u3002\r\n-outtie\r\n    \u6d4b\u5e8f\u65b9\u5411\u4e3a 3'-5' <-> 5'-3'\uff0c\u9002\u5408\u5927\u7247\u6bb5\u6587\u5e93\u3002\r\n<\/pre>\n
2. \u4f7f\u7528 sffToCA<\/a> \u5c06 SFF \u6570\u636e\u4e3a FRG \u683c\u5f0f<\/b>
\nRoche\/454 \u6570\u636e\u683c\u5f0f\u4e3a SFF\u3002
\nsffToCA \u7684\u5e38\u7528\u793a\u4f8b\u548c\u5e38\u7528\u53c2\u6570\uff1a<\/p>\n
\r\n$ sffToCA  -libraryname LIB -output NAME IN.SFF ...\r\n\r\n-insertsize\r\n    \u8bbe\u7f6e\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\uff0c\u4e3a2\u4e2a\u503c\uff0c\u524d\u8005\u4e3a\u63d2\u5165\u7247\u6bb5\u957f\u5ea6\uff0c\u540e\u8005\u4e3a\u6807\u51c6\u5dee\u3002\r\n-libraryname\r\n    \u6587\u5e93\u7684ID\u3002\r\n-output\r\n    \u8f93\u51fa\u6587\u4ef6\u7684\u524d\u7f00\u3002\r\n<\/pre>\n
3.2 \u4f7f\u7528 pacBioToCA\/PBcR<\/a> \u8fdb\u884c Pacbio \u6570\u636e\u7684\u4fee\u6b63<\/h2>\n
pacBioToCA \u80fd\u5bf9 Pacbio \u6570\u636e\u8fdb\u884c\u6821\u6b63\uff0c\u4e5f\u80fd\u8fdb\u884c\u8c03\u7528 runCA \u8fdb\u884c\u57fa\u56e0\u7ec4\u7ec4\u88c5\u3002
\npacBioToCA \u7684\u8f93\u5165\u6587\u4ef6\u4e3a\uff1a<\/p>\n
\r\n1. pacbio.spec   \u8be5\u6587\u4ef6\u5305\u542b\u4e00\u4e9b\u53c2\u6570\u4fe1\u606f\uff0c\u4ee5 \u53c2\u6570=\u503c \u8868\u793a\u3002\u5fc5\u987b\u63d0\u4f9b\u8be5\u6587\u4ef6\uff0c\u8be5\u6587\u4ef6\u5185\u5bb9\u4e3a\u7a7a\uff0c\u5219\u5168\u90e8\u4f7f\u7528\u9ed8\u8ba4\u53c2\u6570\u3002\r\n2. pacbio.fastq  Pacbio\u6d4b\u5e8f\u7684fastq\u6587\u4ef6\uff0c\u5bf9\u8be5\u6587\u4ef6\u4e2d\u7684\u6570\u636e\u8fdb\u884c\u6821\u6b63\u3002\r\n3. seq.frg        Illumina\u6216454\u6570\u636e\u7531 fatsqToCA \u8f6c\u6362\u5f97\u5230\u7684 FRG \u683c\u5f0f\u6587\u4ef6\u3002\u5982\u679c\u4e0d\u63d0\u4f9b\u8be5\u6587\u4ef6\uff0c\u5219\u8fdb\u884c\u81ea\u6211\u6821\u6b63\u3002\r\n<\/pre>\n
pacBioToCA \u7684\u5e38\u7528\u4f8b\u5b50(\u793a\u4f8b\u6570\u636e<\/a>)\u548c\u53c2\u6570\uff1a<\/p>\n
\r\n$ pacBioToCA -libraryname out_prefix -s pacbio.spec -fastq pacbio.fastq seq1.frg seq2.frg\r\n\r\n\u5fc5\u987b\u53c2\u6570\uff1a\r\n-libraryname STRING\r\n    \u8bbe\u5b9a\u8f93\u51fa\u6587\u4ef6\u7684\u524d\u7f00\u3002\r\n-s FILE\r\n    \u4ece\u6307\u5b9a\u7684\u53c2\u6570\u6587\u4ef6\u83b7\u53d6\u989d\u5916\u7684\u53c2\u6570\u3002\u82e5\u8be5\u6587\u4ef6\u5185\u5bb9\u4e3a\u7a7a\uff0c\u5219\u4f7f\u7528\u9ed8\u8ba4\u7684\u53c2\u6570\u3002\r\n-fastq FILE\r\n    \u8f93\u5165\u9700\u8981\u6821\u6b63\u7684 Pacbio \u6570\u636e\u3002\r\n\r\n\u53ef\u9009\u53c2\u6570\uff1a\r\n-length INT    default:500\r\n    pacBioToCA \u5bf9\u957f\u5ea6\u5927\u4e8e\u6b64\u503c\u7684\u6d4b\u5e8f reads \u8fdb\u884c\u4fee\u6b63\u3002\r\n-partitions INT    default:200\r\n    \u5c06\u7528\u4e8e\u6821\u6b63\u7684\u6570\u636e\u5206\u6210\u6307\u5b9a\u4efd\u6570\uff0c\u8fdb\u884c\u8ba1\u7b97\u3002\u5185\u5b58\u4e0d\u591f\uff0c\u5219\u9700\u8981\u589e\u5927\u8be5\u503c\u3002\u82e5\u5185\u5b58\u8db3\u591f\uff0c\u51cf\u5c0f\u8be5\u503c\uff0c\u53ef\u4ee5\u51cf\u5c11\u8ba1\u7b97\u65f6\u95f4\u3002\u82e5\u8be5\u503c\u592a\u5c0f(< \u603b\u7ebf\u7a0b\u6570)\uff0c\u8f6f\u4ef6\u4f1a\u4e2d\u9014\u8fd0\u884crunCorrection.sh\u62a5\u9519\u5e76\u505c\u6b62\u8fd0\u884c\u3002\r\n-threads INT    default\uff1amax\r\n    \u4f7f\u7528\u7684\u7ebf\u7a0b\u6570\u3002\u9ed8\u8ba4\u4e3a\u6240\u6709\u53ef\u7528\u7684\u7ebf\u7a0b\u6570\u3002\r\n-shortReads\r\n    \u5982\u679c\u7528\u4e8e\u8f85\u52a9\u6821\u51c6\u7684\u5e8f\u5217\u957f\u5ea6\u90fd <= 100bp\uff0c\u5219\u4f7f\u7528\u8be5\u53c2\u6570\u3002\r\n-genomeSize INT    default:0\r\n    \u8bbe\u7f6e\u57fa\u56e0\u7ec4\u5927\u5c0f\u3002\u8bbe\u7f6e\u6539\u503c\u540e\uff0c\u5728\u5176\u5b83\u9ed8\u8ba4\u6761\u4ef6\u4e0b\uff0c\u5982\u679c\u6821\u6b63\u540e\u7684\u6570\u636e\u91cf >25x\uff0c\u5219\u4f1a\u8fdb\u884c\u57fa\u56e0\u7ec4\u7ec4\u88c5\u3002\u4e0d\u8bbe\u7f6e\u8be5\u503c\uff0c\u53ef\u80fd\u7b49\u540c\u4e8e\u8bbe\u7f6e\u8be5\u503c\u4e3a 5000000 \uff08\u4ece\u8f6f\u4ef6\u8f93\u51fa\u4fe1\u606f\u4e2d\u67e5\u77e5\uff09\u3002\r\n-maxCoverage INT    default:40\r\n    \u9ed8\u8ba4\u4e0b\uff0c\u6700\u591a\u6821\u6b63 40x \u7684\u6570\u636e\u91cf\u3002\r\n-maxGap INT\r\n    \u9ed8\u8ba4\u4e0b\uff0c\u5982\u679c\u6709\u4e00\u6bb5 pacibo-read \u533a\u57df\u6ca1\u6709 short-read \u8986\u76d6\uff0c\u5219\u4f1a\u6253\u65ad pacbio-read\u3002\u6b64\u503c\u8bbe\u5b9a\u6240\u80fd\u5141\u8bb8\u7684\u6ca1\u6709 short-read \u8986\u76d6\u7684\u6700\u5927\u957f\u5ea6\u3002\u5f53\u7136\uff0c\u82e5\u6b64\u6bb5\u533a\u57df\u6ca1\u6709\u5176\u5b83 pacbio-reads \u91cd\u53e0\uff0c\u5219\u4e5f\u4f1a\u8fdb\u884c\u6253\u65ad\u3002\r\n<\/pre>\n
\u6b64\u5916\uff0c\u5728 pacbio.spec \u6587\u4ef6\u4e2d\u5e38\u8bbe\u7f6e\u7684\u53c2\u6570\u4e3a\uff1a<\/p>\n
\r\n\u82e5\u4e0d\u8fdb\u884c\u57fa\u56e0\uff0c\u5219\u9700\u8981\u8bbe\u7f6e\uff1a\r\nassemble=0\r\n\r\n\u8f6f\u4ef6\u9ed8\u8ba4\u8bbe\u7f6e\u6700\u5927\u5185\u5b58\u4f7f\u7528\u91cf\u8bbe\u7f6e\u4e3a\u7cfb\u7edf\u6700\u5927\u53ef\u4f7f\u7528\u7684\u5185\u5b58\u91cf\uff0c\u82e5\u8bbe\u5b9a\u6700\u5927\u4f7f\u7528\u5185\u5b58\u4e3a 64G\uff0c\u5219\u8bbe\u7f6e\uff1a\r\novlMemory=64\r\n\r\n\u4f7f\u7528 CA 8 \u7248\u672c\u5bf9\u5927\u57fa\u56e0\u7ec4\uff08>100Mbp\uff09\u8fdb\u884c\u6821\u6b63\uff0c\u8bbe\u7f6e\uff1a\r\nmaxGap=1500\r\novlHashBlockLength=1000000000\r\novlRefBlockLength=1000000000\r\n\u4f7f\u7528Illumina\u6570\u636e\u8fdb\u884c\u6821\u6b63\uff1a\r\nblasr=-noRefineAlign -advanceHalf -noSplitSubreads -minMatch 10 -minPctIdentity 70 -bestn 24 -nCandidates 24\r\n\u81ea\u6211\u6821\u6b63\uff1a\r\nblasr=-minReadLength 200 -maxScore -1000 -maxLCPLength 16 -bestn 24 -nCandidates 24\r\novlThreads=16\r\n\u4e0b\u9762\u4e00\u4e2a\u53c2\u6570\u8bbe\u7f6e\u5e76\u884c\u6570\u3002\u5176\u503c\u63a8\u8350\u8bbe\u7f6e\u4e3a \u603b\u7ebf\u7a0b\u6570 \/ ovlThreads\u503c \u3002\u8bbe\u7f6e\u4e3a2\uff0c\u8868\u793a\u6bcf\u4e2a blasr \u7a0b\u5e8f\u8fd0\u884c\u6d88\u8017 16 \u7ebf\u7a0b\uff0c\u5e76\u884c\u8fd0\u884c 2 \u4e2a blasr \u7a0b\u5e8f\u3002\r\novlConcurrency=2\r\n\r\n\u82e5\u8fdb\u884c\u57fa\u56e0\u7ec4\u7ec4\u88c5\u3002\u9ed8\u8ba4\u53c2\u6570\u9002\u7528\u4e8e\u5355\u500d\u4f53\u6570\u636e\u6216\u8fd1\u4eb2\u6742\u4ea4\u6570\u636e\u3002\u5bf9\u4e8e\u5927\u57fa\u56e0\u7ec4\u6216\u53cc\u500d\u4f53\u57fa\u56e0\u7ec4\uff0c\u5219\u9700\u8981\u8bbe\u7f6e\uff1a\r\nasmUtgErrorRate=0.10\r\nasmCnsErrorRate=0.10\r\nasmCgwErrorRate=0.10\r\nasmOBT=1\r\nasmObtErrorRate=0.08\r\nasmObtErrorLimit=4.5\r\nutgGraphErrorRate=0.05\r\nutgMergeErrorRate=0.05\r\novlHashBits=24\r\novlHashLoad=0.80\r\n<\/pre>\n","protected":false},"excerpt":{"rendered":"
1. PacBioToCA \u7b80\u4ecb PacBioToCA \u662f Celera Ass … \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":[66],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2266"}],"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=2266"}],"version-history":[{"count":1,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2266\/revisions"}],"predecessor-version":[{"id":2267,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/2266\/revisions\/2267"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=2266"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=2266"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=2266"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}