{"id":2237,"date":"2023-04-14T15:46:08","date_gmt":"2023-04-14T06:46:08","guid":{"rendered":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/?p=2237"},"modified":"2023-06-17T03:13:05","modified_gmt":"2023-06-16T18:13:05","slug":"bioinfonanopore-v14-kit%e3%81%aeduplex%e3%83%aa%e3%83%bc%e3%83%89%e3%82%92basecall%e3%81%99%e3%82%8b%e3%80%82","status":"publish","type":"post","link":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/?p=2237","title":{"rendered":"[bioinfo]Nanopore V14 kit\u306eDuplex\u30ea\u30fc\u30c9\u3092basecall\u3059\u308b\u3002"},"content":{"rendered":"\n<p>Nanopore\u306eFlongle v10.4.1\u30d5\u30ed\u30fc\u30bb\u30eb\u3068Ligation Sequencing Kit V14 (LSK-SQK114)\u3092\u4f7f\u3063\u3066\u30b7\u30fc\u30af\u30a8\u30f3\u30b9\u3057\u305f\u306e\u3067\u3001Dorado\u3068duplex-tools\u3092\u4f7f\u3063\u3066Duplex\u30ea\u30fc\u30c9\u3092\u51fa\u529b\u3059\u308b\uff082023\/04\/14\u7248\uff1a\u5185\u5bb9\u306f\u3059\u3050\u306b\u66f4\u65b0\u3055\u308c\u308b\u53ef\u80fd\u6027\u304c\u9ad8\u3044\u306e\u3067\u66ab\u5b9a\u7684\u306a\u3084\u308a\u65b9\u306b\u306a\u308b\u3068\u601d\u308f\u308c\u308b\uff09\u3002<\/p>\n\n\n\n<p>Dorado\u306b\u3064\u3044\u3066\u306fNanopore\u306e\u30b5\u30a4\u30c8\u304b\u3089precompile\u3055\u308c\u305f\u3082\u306e\u304c\u30c0\u30a6\u30f3\u30ed\u30fc\u30c9\u3067\u304d\u308b\u304c\u3001\u30d0\u30fc\u30b8\u30e7\u30f3\u304c\u6700\u65b0\u3067\u306f\u306a\u3044\u306e\u3067github\u304b\u3089\u30c0\u30a6\u30f3\u30ed\u30fc\u30c9\u3057\u305f\u65b9\u304c\u826f\u3044\u3002<br>https:\/\/github.com\/nanoporetech\/dorado<br><\/p>\n\n\n\n<p>\u307e\u305a\u3001fast5\u304b\u3089POD5\u306b\u751f\u30c7\u30fc\u30bf\u3092\u5909\u63db\u3059\u308b\u3002<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">#pip\u3067pod5\u30d1\u30c3\u30b1\u30fc\u30b8\u3092\u30a4\u30f3\u30b9\u30c8\u30fc\u30eb\npip install pod5\n\n#pod5\u306e\u30d5\u30a9\u30eb\u30c0\u3092\u4f5c\u3063\u3066\u304a\u3044\u3066\u3001fast5_pass\u30d5\u30a9\u30eb\u30c0\u5185\u306e\u3059\u3079\u3066\u306e\u30d5\u30a1\u30a4\u30eb\u3092\u5909\u63db\nmkdir pod5_pass\npod5 convert fast5 .\/fast5_pass\/*.fast5 --output pod5_pass\/<\/pre>\n\n\n\n<p><mark style=\"background-color:#fcb900\" class=\"has-inline-color has-black-color\">*20230617\u66f4\u65b0\u3000\u65b0\u3057\u3044guppy\u3067\u30d9\u30fc\u30b9\u30b3\u30fc\u30eb\u3057\u305f\u5834\u5408\u306f\u3059\u3067\u306bpod5_pass\u304c\u751f\u6210\u3055\u308c\u3066\u3044\u308b\u3002<\/mark><\/p>\n\n\n\n<p>\u6b21\u306bdorado\u3067model\u3092\u30c0\u30a6\u30f3\u30ed\u30fc\u30c9\u3057\u3066\u3001\u307e\u305a\u3001simplex basecall\u3092\u884c\u3046\u3002\u305d\u306e\u969b\u3001&#8211;emit-moves\u30aa\u30d7\u30b7\u30e7\u30f3\u3092\u4ed8\u3051\u308b\uff08\u3088\u308a\u591a\u304f\u306e\u30da\u30a2\u3092\u8ffd\u52a0\u3067\u304d\u308b\u3089\u3057\u3044\uff09\u3002<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">dorado download --model dna_r10.4.1_e8.2_400bps_hac@v4.1.0\ndorado basecaller dna_r10.4.1_e8.2_400bps_hac@v4.1.0 pod5_pass\/ --emit-moves &gt; unmapped_reads_with_moves.sam<\/pre>\n\n\n\n<p><mark style=\"background-color:#fcb900\" class=\"has-inline-color has-black-color\">*20230617\u66f4\u65b0\u3000\u65b0\u3057\u3044guppy\u3067\u30d9\u30fc\u30b9\u30b3\u30fc\u30eb\u3057\u305f\u5834\u5408\u306f5khz\u30e2\u30c7\u30eb\u306a\u306e\u3067\u3001\u6b21\u306e\u30b3\u30de\u30f3\u30c9\u3067\u3002\u540c\u3058\u30e9\u30a4\u30d6\u30e9\u30ea\u3067\u8907\u6570\u306e\u30d5\u30ed\u30fc\u30bb\u30eb\u3067\u30e9\u30f3\u3057\u3066\u3044\u308b\u5834\u5408\u3001\u3042\u308b\u3044\u306f\u540c\u3058\u30d5\u30ed\u30fc\u30bb\u30eb\u3067\u3082\u8907\u6570\u56de\u30e9\u30f3\u3057\u3066\u3044\u308b\u5834\u5408\u306f\u3001-r\u3092\u4ed8\u3051\u3066\u4e0b\u4f4d\u306e\u30d5\u30a9\u30eb\u30c0\u306epod5\u3092\u3059\u3079\u3066\u30b3\u30fc\u30eb\u3059\u308b\u3088\u3046\u306b\u3059\u308b\u3002\u307e\u305f\u305d\u306e\u969b\u3001pod5_fail\u3082\u30b3\u30fc\u30eb\u3057\u3066\u3057\u307e\u3046\u306e\u3067\u3001&#8211;min-qscore 10\u3092\u4ed8\u3051\u308b\u3002<\/mark><\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">#var20230617\ndorado download --model dna_r10.4.1_e8.2_400bps_hac@v4.2.0\ndorado download --model dna_r10.4.1_e8.2_400bps_sup@v4.2.0\ndorado basecaller dna_r10.4.1_e8.2_400bps_hac@v4.2.0 --min-qscore 10 -r ..\/ --emit-moves > unmapped_reads_with_moves.sam<\/pre>\n\n\n\n<p>duplex-tools\u3092\u4f7f\u3063\u3066duplex read\u3092\u5224\u5225\u3059\u308b\u3002<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">#\u4eee\u60f3\u74b0\u5883\u306bduplex_tools\u3092\u30a4\u30f3\u30b9\u30b3\u3002\npython -m venv venv --prompt duplex\n. venv\/bin\/activate\npip install duplex_tools\n\n#duplex read\u3092\u5224\u5225\nduplex_tools pair --output_dir pairs_from_sam unmapped_reads_with_moves.sam<\/pre>\n\n\n\n<p>duplex_tools\u3067\u5224\u5225\u3057\u305fpair_ids_filtered.txt\u3092\u4f7f\u3063\u3066dorado\u3067stereo basecall\u3059\u308b\u3002<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">dorado duplex dna_r10.4.1_e8.2_400bps_sup@v4.1.0 pod5_pass\/ --pairs pairs_from_sam\/pair_ids_filtered.txt > duplex_orig.sam<\/pre>\n\n\n\n<p><mark style=\"background-color:#fcb900\" class=\"has-inline-color has-black-color\">*20230617\u66f4\u65b0\u3000\u6700\u5f8c\u306eduplex\u30b3\u30fc\u30eb\u30825khz\u30e2\u30c7\u30eb\u3067\u30b3\u30fc\u30eb\u3059\u308b\u3002<\/mark>\u3000<\/p>\n\n\n\n<pre class=\"wp-block-preformatted\">#var20230617\ndorado duplex dna_r10.4.1_e8.2_400bps_sup@v4.2.0 pod5_pass\/ --pairs pairs_from_sam\/pair_ids_filtered.txt > duplex_orig.sam<\/pre>\n\n\n\n<p>\u51fa\u529b\u3055\u308c\u305fduplex_orig.sam\u304cduplex reads\u3067\u3042\u308b\u3002<br>2023\/04\/14\u73fe\u5728\u30015kbp amplicon\u306e\u30c7\u30fc\u30bf\u306716%\u7a0b\u5ea6\u3001\u30b2\u30ce\u30e0\u306e\u30c7\u30fc\u30bf\u30678\uff05\u7a0b\u5ea6\u304cduplex reads\u3067\u3001Q-score\u306f\u3001<br>&gt;= Q20: 82.4%<br>&gt;= Q30: 65.8%<br>&gt; = Q40: 21.7%<br>Ave. 31.4<br>\u3068\u306a\u3063\u3066\u3044\u3066\u3001\u304b\u306a\u308a\u6b63\u78ba\u6027\u306e\u9ad8\u3044\u30c7\u30fc\u30bf\u304c\u5f97\u3089\u308c\u308b\u3002\u30ea\u30fc\u30c9\u6570\u306f\u9650\u5b9a\u3055\u308c\u308b\u304c\u3001amplicon\u306e\u30c7\u30fc\u30bf\u3068\u3057\u3066\u306f\u5341\u5206\u3067\u3042\u308b\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Nanopore\u306eFlongle v10<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"colormag_page_container_layout":"default_layout","colormag_page_sidebar_layout":"default_layout","_locale":"ja","_original_post":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/?p=2237","footnotes":""},"categories":[7],"tags":[],"class_list":["post-2237","post","type-post","status-publish","format-standard","hentry","category-protocol","ja"],"_links":{"self":[{"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=\/wp\/v2\/posts\/2237","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=2237"}],"version-history":[{"count":4,"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=\/wp\/v2\/posts\/2237\/revisions"}],"predecessor-version":[{"id":2307,"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=\/wp\/v2\/posts\/2237\/revisions\/2307"}],"wp:attachment":[{"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=2237"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=2237"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/home.hiroshima-u.ac.jp\/~tigawa\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=2237"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}