{"id":1456,"date":"2013-06-09T08:40:53","date_gmt":"2013-06-09T00:40:53","guid":{"rendered":"http:\/\/www.hzaumycology.com\/chenlianfu_blog\/?p=1456"},"modified":"2013-06-09T10:44:01","modified_gmt":"2013-06-09T02:44:01","slug":"ngs%e6%95%b0%e6%8d%ae%e7%9a%84%e8%b4%a8%e9%87%8f%e8%af%84%e4%bc%b0%e5%92%8creads%e7%9a%84%e5%a4%84%e7%90%86","status":"publish","type":"post","link":"http:\/\/www.chenlianfu.com\/?p=1456","title":{"rendered":"NGS\u6570\u636e\u7684\u8d28\u91cf\u8bc4\u4f30\u548creads\u7684\u5904\u7406"},"content":{"rendered":"

1. \u57fa\u56e0\u7ec4\u6d4b\u5e8f\u548c\u8f6c\u5f55\u6d4b\u5e8f\u7684NGS\u6570\u636e\u5904\u7406\u7b56\u7565<\/h1>\n

\u4ece\u6d4b\u5e8f\u516c\u53f8\u62ff\u5230\u6570\u636e\u540e\uff0c\u9996\u5148\u9700\u8981\u5bf9\u6570\u636e\u8fdb\u884c\u9884\u5904\u7406\uff0c\u4e3b\u8981\u5206\u4e24\u6b65\u8d70\uff1a<\/p>\n

1.1 QC(reads\u7684\u8d28\u91cf\u63a7\u5236)<\/h2>\n

Quality Control,\u5373\u8fc7\u6ee4\u4f4e\u8d28\u91cfreads,\u4f4e\u8d28\u91cf\u7684reads\u6709\u5982\u4e0b\u51e0\u79cd\uff1a
\n\u542b\u6709Primer\/Adaptor\u7684reads
\n\u542b\u6709\u8fc7\u591anon-ATCG\u78b1\u57faN\u7684reads
\n\u6d4b\u5e8f\u8d28\u91cf\u8f83\u4f4e\u7684\u78b1\u57fa\u6570\u5360\u7684\u6bd4\u4f8b\u8fc7\u9ad8\u7684reads<\/p>\n

\u9700\u8981\u5c06\u8fd9\u4e9breads\u5b8c\u5168\u8fc7\u6ee4\u6389\uff0c\u624d\u80fd\u7528\u4e8e\u4e0b\u4e00\u6b65\u7684\u5206\u6790\u3002<\/p>\n

1.2 \u5bf9reads\u8fdb\u884ctrim\u5904\u7406<\/h2>\n

\u5982\u679c\u8fdb\u884c\u57fa\u56e0\u7ec4\u7ec4\u88c5\uff0c\u5219\u4e0d\u9700\u8981\u8fdb\u884c\u8be5\u6b65\u9aa4\u3002\u5982\u679c\u662f\u9700\u8981\u8fdb\u884c\u8f6c\u5f55\u7ec4\u7684\u5206\u6790\uff0c\u5219\u5fc5\u987b\u8981\u8be5\u6b65\u9aa4\u3002<\/p>\n

\u672c\u6b65\u9aa4\u4ece3’\u7aef\u6765\u5bf9reads\u8fdb\u884ctrim\uff0c\u6765\u63a7\u5236reads\u4e2d\u4f4e\u8d28\u91cf\u78b1\u57fa\u7684\u6bd4\u4f8b\u3002\u76f4\u5230trim\u7684read\u957f\u5ea6\u4f4e\u4e8e\u4e00\u5b9a\u7684\u6570\u65f6\uff0c\u5219\u5b8c\u5168\u820d\u5f03\u8be5read\u3002<\/p>\n

2. NGS\u6570\u636e\u7684QC\u8f6f\u4ef6<\/h1>\n

2.1 NGSQC toolkit<\/h2>\n

\u8be5\u8f6f\u4ef6\u7684citation:Patel RK, Jain M (2012). NGS QC Toolkit: A toolkit for quality control of next generation sequencing data. PLoS ONE, 7(2): e30619. <\/a><\/p>\n

\u8be5\u8f6f\u4ef6\u7684\u5b98\u7f51\uff1ahttp:\/\/www.nipgr.res.in\/ngsqctoolkit.html<\/a><\/p>\n

\u8be5\u8f6f\u4ef6\u89e3\u538b\u7f29\u540e\u5305\u62ec4\u4e2a\u6587\u4ef6\u5939\u548c1\u4e2aPDF\u683c\u5f0f\u7684manual\u6587\u4ef6\u3002manual\u6587\u4ef6\u662f\u8be6\u7ec6\u7684\u8bf4\u660e\uff1b4\u4e2a\u6587\u4ef6\u5939\u4e2d\u90fd\u662f\u4f7f\u7528perl\u7f16\u5199\u7684\u7528\u4e8eQC\u7684\u7a0b\u5e8f\u3002\u6309\u5176\u91cd\u8981\u7a0b\u5ea6\u51b3\u5b9a\u5148\u540e\uff0c\u5176\u4ecb\u7ecd\u5982\u4e0b\uff1a<\/p>\n

2.1.1 QC\u6587\u4ef6\u5939\u4e2d\u5305\u542b\u4e864\u652fperl\u7a0b\u5e8f\uff0c\u7528\u4e8e454 reads\u6216Illumina reads\u7684QC\uff0c\u5206\u522b\u4e3a\uff1a<\/span><\/h3>\n

IlluQC.pl<\/span> \u7528\u4e8eIllumina reads\u7684QC\u3002\u9ed8\u8ba4\u60c5\u51b5\u4e0b\u53bb\u9664\u6389\u542b\u6709primer\/adaptor\u7684reads\u548c\u4f4e\u8d28\u91cf\u7684reads\uff0c\u5e76\u7ed9\u51fa\u7edf\u8ba1\u7ed3\u679c\u548c6\u79cd\u56fe\u5f62\u7ed3\u679c\u3002\u9ed8\u8ba4\u8bbe\u7f6e (‘-s’ \u53c2\u6570) \u78b1\u57fa\u8d28\u91cf\u4f4e\u4e8e20\u7684\u4e3a\u4f4e\u8d28\u91cf\u78b1\u57fa\uff1b\u9ed8\u8ba4\u8bbe\u7f6e ( ‘-l’ \u53c2\u6570)\u4f4e\u8d28\u91cf\u78b1\u57fa\u5728reads\u4e2d\u6bd4\u4f8b >30% \u7684\u4e3a\u4f4e\u8d28\u91cfreads\u3002\u7a0b\u5e8f\u8fd0\u884c\u4f8b\u5b50\uff1a<\/p>\n

$ perl $NGSQCHome\/QC\/IlluQC_PRLL.pl -pe r1.fq r2.fq 2 5 -p 8 -l 70 -s 20<\/pre>\n
IlluQC_PRLL.pl<\/span> \u548c\u4e0a\u4e00\u4e2a\u7a0b\u5e8f\u6ca1\u6709\u591a\u5927\u533a\u522b\uff0c\u53ea\u662f\u591a\u4e86 ‘-c’ \u53c2\u6570\u6765\u8fdb\u884c\u5e76\u884c\u8ba1\u7b97\uff0c\u589e\u52a0\u7a0b\u5e8f\u901f\u5ea6\u3002<\/p>\n
454QC.pl<\/span> \u5bf9454 reads\u8fdb\u884cQC\u3002
\n454QC_PRLL.pl<\/span> \u548c\u4e0a\u4e00\u4e2a\u7a0b\u5e8f\u4e00\u773c\u4e2a\uff0c\u53ea\u662f\u591a\u4e86 ‘-c’ \u53c2\u6570\u6765\u8fdb\u884c\u5e76\u884c\u8ba1\u7b97\uff0c\u589e\u52a0\u7a0b\u5e8f\u901f\u5ea6\u3002
\n454QC_PE.pl <\/span>\u5bf9paired-end\u6d4b\u5e8f\u7684454 reads\u8fdb\u884cQC\u3002<\/p>\n
2.1.2 TrimingReads\u6587\u4ef6\u5939\u5305\u542b3\u652f\u7a0b\u5e8f\uff0c\u7528\u4e8ereads\u7684trimming\uff0c\u5206\u522b\u4e3a\uff1a<\/span><\/h3>\n
AmbiguityFiltering.pl<\/span> \u5bf9\u542b\u6709non-ATCG\u7684reads\u8fdb\u884ctrimming\u7684\u7a0b\u5e8f\u3002\u67094\u79cd(4\u90091)trim\u65b9\u6cd5\uff1a\u5141\u8bb8\u6700\u5927non-ATCG\u6570\u76ee\uff1b\u5141\u8bb8\u6700\u5927\u7684non-ATCG\u6bd4\u4f8b(\u4f8b\u5b50\u5982\u4e0b)\uff1b\u4ece5’\u7aeftrim\u6389\u542bN\u7684\u5e8f\u5217\uff1b\u4ece3’\u7aeftrim\u6389\u542bN\u7684\u5e8f\u5217\u3002\u52a0\u4e0a\u4e2a\u901a\u7528\u7684\u53c2\u6570\uff1a\u4f4e\u4e8e\u4e00\u5b9a\u957f\u5ea6\u7684reads\u88abcutoff\u6389\u3002<\/p>\n
$ perl $NGSQCHome\/Trimming\/AmbiguityFiltering.pl -i r1.fq -irev r2.fq -p 2 -n 50<\/pre>\n
TrimmingReads.pl<\/span> \u67093\u79cd(3\u90091)trim\u65b9\u6cd5\uff1a\u5bf9\u6240\u6709read\u4ece5’\u7aeftrim\u6389\u5236\u5b9a\u6570\u76ee\u7684\u78b1\u57fa\uff1b\u5bf9\u6240\u6709reads\u4ece3’\u7aeftrim\u6389\u6307\u5b9a\u6570\u76ee\u7684\u78b1\u57fa\uff1b\u4ece3’\u7aeftrim\u6389\u8d28\u91cf\u4f4e\u4e8e\u6307\u5b9a\u503c\u7684\u78b1\u57fa(\u4f8b\u5b50\u5982\u4e0b\uff09\u3002\u52a0\u4e0a\u4e2a\u901a\u7528\u7684\u53c2\u6570\uff1a\u4f4e\u4e8e\u4e00\u5b9a\u957f\u5ea6\u7684reads\u88abcutoff\u6389\u3002<\/p>\n
$ perl $NGSQCHome\/Trimming\/TrimmingReads.pl  -i r1.fq -irev r2.fq -q 13 -n 50<\/pre>\n
HomopolymerTrimming.pl<\/span><\/p>\n
2.1.3 Statistics\u6587\u4ef6\u5939\u4e2d2\u652f\u7a0b\u5e8f\uff0c\u7528\u4e8e\u8fdb\u884cN50\u7edf\u8ba1\u7b49<\/span><\/h3>\n
N50Stat.pl<\/span> \u7528\u4e8e\u7edf\u8ba1fasta\u6587\u4ef6\u7684N50
\nAvgQuality.pl<\/span> \u7528\u4e8e\u7edf\u8ba1454\u6587\u4ef6\u7684reads\u8d28\u91cf<\/p>\n
2.1.4 Formt-converter\u6587\u4ef6\u5939\u4e2d\u7a0b\u5e8f\u8fd0\u7528\u4e8e\u4e0d\u540c\u683c\u5f0f\u6587\u4ef6\u7684\u8f6c\u6362\uff0c\u5176\u4e2d\u542b\u67094\u4e2aperl\u7a0b\u5e8f\uff0c\u5206\u522b\u4e3a\uff1a<\/span><\/h3>\n
FastqTo454.pl\u3001FastqToFasta.pl\u3001SangerFastqToIlluFastq.pl\u3001SolexaFastqToIlluFastq.pl<\/span>\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"
1. \u57fa\u56e0\u7ec4\u6d4b\u5e8f\u548c\u8f6c\u5f55\u6d4b\u5e8f\u7684NGS\u6570\u636e\u5904\u7406\u7b56\u7565 \u4ece\u6d4b\u5e8f\u516c\u53f8\u62ff\u5230\u6570\u636e\u540e\uff0c\u9996\u5148\u9700\u8981\u5bf9 … \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":[],"tags":[],"_links":{"self":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1456"}],"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=1456"}],"version-history":[{"count":13,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1456\/revisions"}],"predecessor-version":[{"id":1458,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=\/wp\/v2\/posts\/1456\/revisions\/1458"}],"wp:attachment":[{"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1456"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1456"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.chenlianfu.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1456"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}