"use strict"; const {Fragment: _Fragment, jsx: _jsx, jsxs: _jsxs} = arguments[0]; const {useMDXComponents: _provideComponents} = arguments[0]; function _createMdxContent(props) { const _components = { a: "a", annotation: "annotation", blockquote: "blockquote", code: "code", div: "div", h1: "h1", h2: "h2", h3: "h3", img: "img", li: "li", math: "math", mfrac: "mfrac", mi: "mi", mn: "mn", mo: "mo", mover: "mover", mrow: "mrow", msqrt: "msqrt", msub: "msub", msup: "msup", mtext: "mtext", ol: "ol", p: "p", path: "path", pre: "pre", semantics: "semantics", span: "span", svg: "svg", table: "table", tbody: "tbody", td: "td", th: "th", thead: "thead", tr: "tr", ul: "ul", ..._provideComponents(), ...props.components }; return _jsxs(_Fragment, { children: [_jsxs(_components.h1, { id: "background", children: [_jsx(_components.a, { className: "heading-link", href: "#background", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "Background"] }), "\n", _jsxs(_components.p, { children: ["关于这篇", _jsx(_components.a, { href: "https://www.pnas.org/doi/10.1073/pnas.1815441116#data-availability", target: "_blank", rel: "noopener noreferrer", children: "Paper" })] }), "\n", _jsxs(_components.p, { children: ["各部分数据：", _jsx(_components.a, { href: "http://ftp.genek.cn:8888/20220919_ZXdwxls/", target: "_blank", rel: "noopener noreferrer", children: "http://ftp.genek.cn:8888/20220919_ZXdwxls/" })] }), "\n", _jsx(_components.p, { children: "参考Data：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# from甜橙数据库：http://citrus.hzau.edu.cn/download.php" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "wget" }), " http://citrus.hzau.edu.cn/data/Genome_info/SWO.v3.0/SWO.v3.0.genome.fa\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "wget" }), " http://citrus.hzau.edu.cn/data/Genome_info/SWO.v3.0/SWO.v3.0.gene.model.gff3\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "wget" }), " http://citrus.hzau.edu.cn/data/Genome_info/SWO.v3.0/SWO.v3.0.protein.fa ", _jsx(_components.span, { className: "token comment", children: "#蛋白，可选下载" }), "\n"] })] }) }), "\n", _jsxs(_components.h1, { id: "参考数据处理seqtkseqkitgffreadgtftk", children: [_jsx(_components.a, { className: "heading-link", href: "#参考数据处理seqtkseqkitgffreadgtftk", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "参考数据处理（seqtk、seqkit、gffread、gtftk）"] }), "\n", _jsx(_components.p, { children: "如果序列不符合标准格式，可以用seqtk转为fasta格式规范：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsx(_components.code, { className: "language-bash code-highlight", children: _jsxs(_components.span, { className: "code-line", children: ["seqtk ", _jsx(_components.span, { className: "token function", children: "seq" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-l" }), " ", _jsx(_components.span, { className: "token number", children: "60" }), " SWO.v3.0.genome.fa ", _jsx(_components.span, { className: "token operator", children: ">" }), " genome.fa\n"] }) }) }), "\n", _jsx(_components.p, { children: "构建index：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsx(_components.code, { className: "language-bash code-highlight", children: _jsx(_components.span, { className: "code-line", children: "seqkit faidx genome.fa\n" }) }) }), "\n", _jsx(_components.p, { children: "转化格式：\ngff -> gtf（更友好的格式）" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsx(_components.code, { className: "language-bash code-highlight", children: _jsxs(_components.span, { className: "code-line", children: ["gffread ", _jsx(_components.span, { className: "token parameter variable", children: "-T" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-o" }), " genes.gtf SWO.v3.0.gene.model.gff3\n"] }) }) }), "\n", _jsx(_components.p, { children: "可选： gtftk转化为ensembel格式：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 很难折腾" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["conda config ", _jsx(_components.span, { className: "token parameter variable", children: "--set" }), " channel_priority flexible\n"] }), _jsxs(_components.span, { className: "code-line", children: ["conda create ", _jsx(_components.span, { className: "token parameter variable", children: "-n" }), " pygtftk ", _jsx(_components.span, { className: "token assign-left variable", children: "python" }), _jsx(_components.span, { className: "token operator", children: "=" }), _jsx(_components.span, { className: "token number", children: "3.9" }), " pygtftk libgcc-ng", _jsx(_components.span, { className: "token operator", children: "=" }), _jsx(_components.span, { className: "token number", children: "12" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsx(_components.span, { className: "code-line", children: "gtftk convert_ensembl-i temp.gtf › genes.gtf\n" })] }) }), "\n", _jsx(_components.p, { children: "转录本研究？该物种研究到了基因水平还是转录本水平？" }), "\n", _jsxs(_components.p, { children: ["从 ", _jsx(_components.code, { className: "language-auto", children: "fasta" }), "和", _jsx(_components.code, { className: "language-auto", children: "gff3" }), "文件中提取最长转录本。\n", _jsx(_components.img, { src: "https://pic3.zhimg.com/v2-2d2fbca7a99a238d2b3976dc074210fe_1440w.jpg", alt: "", className: "internal" })] }), "\n", _jsxs(_components.p, { children: ["由于", _jsx(_components.a, { href: "https://zhida.zhihu.com/search?content_id=216752249&content_type=Article&match_order=1&q=%E5%8F%AF%E5%8F%98%E5%89%AA%E5%88%87&zhida_source=entity", target: "_blank", rel: "noopener noreferrer", children: "可变剪切" }), "的存在，通常一个基因可以转录为多个转录本。但是如果将多个转录本同时进行分析，那么分析会因此受到影响。所以，目前的解决办法是，选取一个最具代表性的转录本（最长转录本）来进行分析"] }), "\n", _jsx(_components.p, { children: "检查：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: ["gtftk count ", _jsx(_components.span, { className: "token parameter variable", children: "-i" }), " gene.gtf\n"] }), _jsxs(_components.span, { className: "code-line", children: ["gene\t", _jsx(_components.span, { className: "token number", children: "29875" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["transcript\t", _jsx(_components.span, { className: "token number", children: "49567" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["exon\t", _jsx(_components.span, { className: "token number", children: "307505" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["CDS\t", _jsx(_components.span, { className: "token number", children: "282437" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# gene < transcript，含有可变剪切" }), "\n"] })] }) }), "\n", _jsx(_components.p, { children: "筛选最长转录本+提取对应蛋白序列：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: ["gtftk short_long ", _jsx(_components.span, { className: "token parameter variable", children: "-l" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-i" }), " gene.gtf ", _jsx(_components.span, { className: "token operator", children: "|" }), " gtftk select_by_key ", _jsx(_components.span, { className: "token parameter variable", children: "-k" }), " feature ", _jsx(_components.span, { className: "token parameter variable", children: "-v" }), " transcript ", _jsx(_components.span, { className: "token operator", children: "|" }), " gtftk tabulate ", _jsx(_components.span, { className: "token parameter variable", children: "-k" }), " transcript_id,gene_id ", _jsx(_components.span, { className: "token operator", children: ">" }), "longest_mapid.txt\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "sed" }), " ", _jsx(_components.span, { className: "token string", children: "'1d'" }), " longest_mapid.txt ", _jsx(_components.span, { className: "token operator", children: "|" }), " ", _jsx(_components.span, { className: "token function", children: "cut" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-f" }), " ", _jsx(_components.span, { className: "token number", children: "1" }), " ", _jsx(_components.span, { className: "token operator", children: "|" }), " seqtk subseq SWO.v3.0.protein.fa -", _jsx(_components.span, { className: "token operator", children: ">" }), " longest_transcript.proteins.fa\n"] })] }) }), "\n", _jsx(_components.p, { children: "转化ID：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsx(_components.code, { className: "language-bash code-highlight", children: _jsxs(_components.span, { className: "code-line", children: ["seqkit replace ", _jsx(_components.span, { className: "token parameter variable", children: "-p" }), " ", _jsx(_components.span, { className: "token string", children: "'(.+)$'" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-r" }), " ", _jsx(_components.span, { className: "token string", children: "'{kv}'" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-k" }), " longest_mapid.txt longest_transcript.proteins.fa ", _jsx(_components.span, { className: "token operator", children: ">" }), "protein.fa\n"] }) }) }), "\n", _jsxs(_components.h2, { id: "几款软件详解", children: [_jsx(_components.a, { className: "heading-link", href: "#几款软件详解", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "几款软件详解"] }), "\n", _jsx(_components.p, { children: "seqtk：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# seq类" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["seqtk ", _jsx(_components.span, { className: "token function", children: "seq" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-l" }), " ", _jsx(_components.span, { className: "token number", children: "60" }), " atha.fasta ", _jsx(_components.span, { className: "token comment", children: "# 标准格式 " }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["seqtk ", _jsx(_components.span, { className: "token function", children: "seq" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-L" }), " ", _jsx(_components.span, { className: "token number", children: "100" }), " atha.fasta ", _jsx(_components.span, { className: "token comment", children: "# 删去短于100bp的序列" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["seqtk ", _jsx(_components.span, { className: "token function", children: "seq" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-U" }), " atha.fasta ", _jsx(_components.span, { className: "token comment", children: "# 转大写字母" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# sample类" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["seqtk sample ", _jsx(_components.span, { className: "token parameter variable", children: "-s" }), " ", _jsx(_components.span, { className: "token number", children: "1" }), " atha.fasta ", _jsx(_components.span, { className: "token number", children: "3" }), " ", _jsx(_components.span, { className: "token comment", children: "#伪随机抽样，-s制定种子，数字或百分数（eg 0.01）制定抽取数量" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# subseq类" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsx(_components.span, { className: "token string", children: "\"chr1\"" }), " ", _jsx(_components.span, { className: "token operator", children: ">" }), " sub.txt ", _jsx(_components.span, { className: "token comment", children: "# 其实是bed文件" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["seqtk subseq genome.fa sub.txt ", _jsx(_components.span, { className: "token operator", children: "|" }), " seqtk ", _jsx(_components.span, { className: "token function", children: "seq" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-l" }), " ", _jsx(_components.span, { className: "token number", children: "60" }), " ", _jsx(_components.span, { className: "token operator", children: "|" }), " le ", _jsx(_components.span, { className: "token comment", children: "# 通过基因名提取序列 " }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsx(_components.span, { className: "token string", children: "\"chr2 0 10\"" }), " ", _jsx(_components.span, { className: "token operator", children: ">" }), " sub.txt ", _jsx(_components.span, { className: "token comment", children: "# 可以选取一定片段" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["seqtk subseq genome.fa sub.txt ", _jsx(_components.span, { className: "token operator", children: "|" }), " seqtk ", _jsx(_components.span, { className: "token function", children: "seq" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-l" }), " ", _jsx(_components.span, { className: "token number", children: "60" }), " ", _jsx(_components.span, { className: "token operator", children: "|" }), " le\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token operator", children: ">" }), "chr2:1-10\n"] }), _jsx(_components.span, { className: "code-line", children: "ACCCTAAACC\n" })] }) }), "\n", _jsx(_components.p, { children: "seqkit，gffread基本只用于构建index和格式转化" }), "\n", _jsxs(_components.p, { children: [_jsx(_components.a, { href: "https://github.com/dputhier/pygtftk", target: "_blank", rel: "noopener noreferrer", children: "gtftk" }), "："] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 基础转化" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "gtftk convert_ensembl-i a.gtf › a_ensembl.gtf\n" }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 信息类" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["gtftk count ", _jsx(_components.span, { className: "token parameter variable", children: "-i" }), " a.gtf\n"] }), _jsx(_components.span, { className: "code-line", children: "gtftk nb_transcripts\n" }), _jsx(_components.span, { className: "code-line", children: "gtftk nb_exons\n" }), _jsx(_components.span, { className: "code-line", children: "gtftk feature_size\n" }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 筛查类" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: ["gtftk nb_transcripts ", _jsx(_components.span, { className: "token parameter variable", children: "-i" }), " a.gtf ", _jsx(_components.span, { className: "token operator", children: "|" }), " gtftk select_by_key ", _jsx(_components.span, { className: "token parameter variable", children: "-k" }), " feature ", _jsx(_components.span, { className: "token parameter variable", children: "-v" }), " gene ", _jsx(_components.span, { className: "token comment", children: "# select_by_key，选出基因" }), "\n"] })] }) }), "\n", _jsxs(_components.h1, { id: "测序数据", children: [_jsx(_components.a, { className: "heading-link", href: "#测序数据", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "测序数据"] }), "\n", _jsx(_components.p, { children: "NCBI GEO编号 -> SRA数据 -> Send to: Run Selector" }), "\n", _jsx(_components.p, { children: "GSE108930 -> SRR6451533" }), "\n", _jsxs(_components.h2, { id: "下载", children: [_jsx(_components.a, { className: "heading-link", href: "#下载", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "下载"] }), "\n", _jsxs(_components.h3, { id: "sra-tool下载", children: [_jsx(_components.a, { className: "heading-link", href: "#sra-tool下载", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "sra-tool下载"] }), "\n", _jsx(_components.pre, { children: _jsxs(_components.code, { className: "language-auto code-highlight", children: [_jsx(_components.span, { className: "code-line", children: "conda install -c bioconda sra-tools -y\n" }), _jsx(_components.span, { className: "code-line", children: "prefetch SRR6451531 -O ./\n" })] }) }), "\n", _jsxs(_components.h3, { id: "用sra-explorer生成", children: [_jsx(_components.a, { className: "heading-link", href: "#用sra-explorer生成", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "用", _jsx(_components.a, { href: "https://sra-explorer.info", target: "_blank", rel: "noopener noreferrer", children: "sra-explorer" }), "生成"] }), "\n", _jsxs(_components.ol, { children: ["\n", _jsx(_components.li, { children: "ftp：" }), "\n"] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsx(_components.code, { className: "language-bash code-highlight", children: _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "nohup" }), " ", _jsx(_components.span, { className: "token function", children: "wget" }), " ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR645/003/SRR6451533/SRR6451533_1.fastq.gz ", _jsx(_components.span, { className: "token operator", children: "&" }), " ", _jsx(_components.span, { className: "token comment", children: "# nohup &代表后台运行且不中止（服务器），一般配合 1>xx.log 2>&1 记录日志" }), "\n"] }) }) }), "\n", _jsxs(_components.ol, { start: "2", children: ["\n", _jsx(_components.li, { children: "ascp（安装麻烦）：" }), "\n"] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsx(_components.code, { className: "language-bash code-highlight", children: _jsxs(_components.span, { className: "code-line", children: ["ascp ", _jsx(_components.span, { className: "token parameter variable", children: "-QT" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-l" }), " 300m ", _jsx(_components.span, { className: "token parameter variable", children: "-P33001" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-i" }), " ", _jsx(_components.span, { className: "token environment constant", children: "$HOME" }), "/.aspera/connect/etc/asperaweb_id_dsa.openssh era-fasp@fasp.sra.ebi.ac.uk:vol1/fastq/SRR645/001/SRR6451531/SRR6451531_1.fastq.gz ", _jsx(_components.span, { className: "token builtin class-name", children: "." }), "\n"] }) }) }), "\n", _jsxs(_components.blockquote, { children: ["\n", _jsx(_components.p, { children: "下载前报错找不到ssh，需下载（或创建）openssh文件" }), "\n"] }), "\n", _jsxs(_components.h3, { id: "kingfisher下载", children: [_jsx(_components.a, { className: "heading-link", href: "#kingfisher下载", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "kingfisher下载"] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "sudo" }), " ", _jsx(_components.span, { className: "token function", children: "apt-get" }), " ", _jsx(_components.span, { className: "token function", children: "install" }), " aria2c\n"] }), _jsxs(_components.span, { className: "code-line", children: ["conda ", _jsx(_components.span, { className: "token function", children: "install" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-c" }), " bioconda kingfisher\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: ["kingfisher get ", _jsx(_components.span, { className: "token parameter variable", children: "-r" }), " SRR6451531 ", _jsx(_components.span, { className: "token parameter variable", children: "-m" }), " aws-http aws-cp ena-ftp prefetch\n"] })] }) }), "\n", _jsxs(_components.h2, { id: "质控fastp", children: [_jsx(_components.a, { className: "heading-link", href: "#质控fastp", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "质控（fastp）"] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: ["fastp ", _jsx(_components.span, { className: "token parameter variable", children: "-i" }), " ", _jsx(_components.span, { className: "token punctuation", children: ".." }), "/14.seq/SRR6451531_Cs1_1.fastq ", _jsx(_components.span, { className: "token parameter variable", children: "-I" }), " ", _jsx(_components.span, { className: "token punctuation", children: ".." }), "/14.seq/SRR6451531_Cs1_2.fastq ", _jsx(_components.span, { className: "token parameter variable", children: "-o" }), " Cs1_1-1.fastq ", _jsx(_components.span, { className: "token parameter variable", children: "-O" }), " Cs1_1-2.fastq ", _jsx(_components.span, { className: "token parameter variable", children: "-h" }), " Cs1_1.html ", _jsx(_components.span, { className: "token parameter variable", children: "-j" }), " Cs1_1.json \n"] }), _jsxs(_components.span, { className: "code-line", children: ["fastp v0.24.0, ", _jsx(_components.span, { className: "token function", children: "time" }), " used: ", _jsx(_components.span, { className: "token number", children: "148" }), " seconds\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token operator", children: ">" }), " ll\n"] }), _jsx(_components.span, { className: "code-line", children: "Permissions Size User Date Modified Name\n" }), _jsxs(_components.span, { className: "code-line", children: [".rw-rw-r-- ", _jsx(_components.span, { className: "token number", children: "9" }), ".5G parallels ", _jsx(_components.span, { className: "token number", children: "31" }), " Mar ", _jsx(_components.span, { className: "token number", children: "10" }), ":10 Cs1_1-1.fastq\n"] }), _jsxs(_components.span, { className: "code-line", children: [".rw-rw-r-- 487k parallels ", _jsx(_components.span, { className: "token number", children: "31" }), " Mar ", _jsx(_components.span, { className: "token number", children: "10" }), ":10 Cs1_1.html\n"] }), _jsxs(_components.span, { className: "code-line", children: [".rw-rw-r-- 136k parallels ", _jsx(_components.span, { className: "token number", children: "31" }), " Mar ", _jsx(_components.span, { className: "token number", children: "10" }), ":10 Cs1_1.json\n"] }), _jsxs(_components.span, { className: "code-line", children: [".rw-rw-r-- ", _jsx(_components.span, { className: "token number", children: "9" }), ".5G parallels ", _jsx(_components.span, { className: "token number", children: "31" }), " Mar ", _jsx(_components.span, { className: "token number", children: "10" }), ":10 Cs1_1-2.fastq\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" })] }) }), "\n", _jsxs(_components.h1, { id: "mappinghisat2", children: [_jsx(_components.a, { className: "heading-link", href: "#mappinghisat2", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "Mapping（hisat2）"] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: ["conda ", _jsx(_components.span, { className: "token function", children: "install" }), " hisat2\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# index构建，一般用" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["hisat2-build genome.fa genomeindex ", _jsx(_components.span, { className: "token comment", children: "# genomeindex为自定义输出名" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 还可以通过注释文件加入剪切位点与exon信息" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "hisat2_extract_splice_sites.py genes.gtf › genome.ss\n" }), _jsxs(_components.span, { className: "code-line", children: ["hisat2_extract_exons.py genes.gtf", _jsx(_components.span, { className: "token operator", children: ">" }), " genome.exon\n"] }), _jsxs(_components.span, { className: "code-line", children: ["hisat2-build ", _jsx(_components.span, { className: "token parameter variable", children: "--exon" }), " genome.exon ", _jsx(_components.span, { className: "token parameter variable", children: "--ss" }), " genome.ss genome.fa genomeindex\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: ["hisat2 --new-summary ", _jsx(_components.span, { className: "token parameter variable", children: "-p" }), " ", _jsx(_components.span, { className: "token number", children: "4" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-x" }), " genomeindex ", _jsx(_components.span, { className: "token parameter variable", children: "-1" }), " Cs1_1-1.fastq ", _jsx(_components.span, { className: "token parameter variable", children: "-2" }), " Cs1_1-2.fastq ", _jsx(_components.span, { className: "token parameter variable", children: "-S" }), " Cs1_1.sam --rna-strandness RF ", _jsx(_components.span, { className: "token comment", children: "# -p：线程 -x：index文件 --rna-strandness：链特异性" }), "\n"] })] }) }), "\n", _jsxs(_components.blockquote, { children: ["\n", _jsx(_components.p, { children: "其他：" }), "\n"] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "mkdir" }), " STARindex\n"] }), _jsxs(_components.span, { className: "code-line", children: ["STAR ", _jsx(_components.span, { className: "token parameter variable", children: "--runThreadN" }), " ", _jsx(_components.span, { className: "token number", children: "2" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "--runMode" }), " genomeGenerate ", _jsx(_components.span, { className: "token parameter variable", children: "--genomeDir" }), " ./STARindex/ ", _jsx(_components.span, { className: "token parameter variable", children: "--genomeFastaFiles" }), " genome.fa ", _jsx(_components.span, { className: "token parameter variable", children: "--sjdbGTFfile" }), " gene.gtf ", _jsx(_components.span, { className: "token parameter variable", children: "--sjdbOverhang" }), " ", _jsx(_components.span, { className: "token number", children: "149" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "--genomeSAindexNbases" }), " ", _jsx(_components.span, { className: "token number", children: "13" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "--limitGenomeGenerateRAM" }), " ", _jsx(_components.span, { className: "token number", children: "2000000000" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# --runThreadN：线程数" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# --runMode genomeGenerate：构建基因组索引" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# --genomeDir：索引目录（index_dir一定要是存在的文件夹，需提前建好）" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# --genomeFastaFiles：基因组文件" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# --sjdbGTFfile：基因组注释文件" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# --sjdbOverhang：reads长度减1" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# --limitGenomeGenerateRAM：限制使用内存" }), "\n"] })] }) }), "\n", _jsxs(_components.ul, { children: ["\n", _jsxs(_components.li, { children: ["sam(Sequence Alignment/Map)文件是最早由", _jsx(_components.a, { href: "http://bio-bwa.sourceforge.net/", target: "_blank", rel: "noopener noreferrer", children: "bwa" }), "的作者", _jsx(_components.a, { href: "http://www.liheng.org/", target: "_blank", rel: "noopener noreferrer", children: "Heng Li" }), "设计的一种记录reads mapping信息的文件格式，后来被广泛接受，得到了更普遍的应用。目前各种mapping的工具的结果基本上都是以bam/sam文件格式作为输出的。"] }), "\n", _jsx(_components.li, { children: "sam是一种纯文本文件，而bam文件是压缩过的，二进制版本的sam文件。在真实的生物信息分析中，为了节约存储，我们通常以bam文件的形式存储mapping的结果。" }), "\n"] }), "\n", _jsx(_components.p, { children: "安装samtools：" }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsx(_components.code, { className: "language-bash code-highlight", children: _jsxs(_components.span, { className: "code-line", children: ["conda ", _jsx(_components.span, { className: "token function", children: "install" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-y" }), " samtools\n"] }) }) }), "\n", _jsx(_components.p, { children: "生成sorted.bam文件和index：" }), "\n", _jsx(_components.pre, { className: "language-sh", children: _jsxs(_components.code, { className: "language-sh code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token shebang important", children: "#!/bin/bash" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# Convert SAM to BAM and sort the BAM files" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# sort是按默认的染色体位置进行排序,-@是线程数" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token keyword", children: "for" }), " ", _jsx(_components.span, { className: "token for-or-select variable", children: "sam" }), " ", _jsx(_components.span, { className: "token keyword", children: "in" }), " *.sam", _jsx(_components.span, { className: "token punctuation", children: ";" }), " ", _jsx(_components.span, { className: "token keyword", children: "do" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token assign-left variable", children: "sample" }), _jsx(_components.span, { className: "token operator", children: "=" }), _jsxs(_components.span, { className: "token variable", children: [_jsx(_components.span, { className: "token variable", children: "$(" }), _jsx(_components.span, { className: "token function", children: "basename" }), " $", _jsx(_components.span, { className: "token punctuation", children: "{" }), "sam", _jsx(_components.span, { className: "token punctuation", children: "}" }), " .sam", _jsx(_components.span, { className: "token variable", children: ")" })] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token assign-left variable", children: "bam" }), _jsx(_components.span, { className: "token operator", children: "=" }), _jsxs(_components.span, { className: "token string", children: ["\"", _jsx(_components.span, { className: "token variable", children: "${sample}" }), ".bam\""] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token assign-left variable", children: "sorted_bam" }), _jsx(_components.span, { className: "token operator", children: "=" }), _jsxs(_components.span, { className: "token string", children: ["\"", _jsx(_components.span, { className: "token variable", children: "${sample}" }), ".sorted.bam\""] }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsxs(_components.span, { className: "token string", children: ["\"Processing ", _jsx(_components.span, { className: "token variable", children: "$sam" }), " -> ", _jsx(_components.span, { className: "token variable", children: "$bam" }), " -> ", _jsx(_components.span, { className: "token variable", children: "$sorted_bam" }), "\""] }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token comment", children: "# Convert SAM to BAM" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" samtools view -@ ", _jsx(_components.span, { className: "token number", children: "2" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-bS" }), " ", _jsx(_components.span, { className: "token variable", children: "${sam}" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-o" }), " ", _jsx(_components.span, { className: "token variable", children: "${bam}" }), " ", _jsx(_components.span, { className: "token comment", children: "# sam转bam" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: " \n" }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token comment", children: "# Sort BAM file" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" samtools ", _jsx(_components.span, { className: "token function", children: "sort" }), " -@ ", _jsx(_components.span, { className: "token number", children: "6" }), " ", _jsx(_components.span, { className: "token variable", children: "${bam}" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-o" }), " ", _jsx(_components.span, { className: "token variable", children: "${sorted_bam}" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: " \n" }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token comment", children: "# Remove intermediate BAM file if sorting was successful" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token keyword", children: "if" }), " ", _jsx(_components.span, { className: "token punctuation", children: "[" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-f" }), " ", _jsxs(_components.span, { className: "token string", children: ["\"", _jsx(_components.span, { className: "token variable", children: "${sorted_bam}" }), "\""] }), " ", _jsx(_components.span, { className: "token punctuation", children: "]" }), _jsx(_components.span, { className: "token punctuation", children: ";" }), " ", _jsx(_components.span, { className: "token keyword", children: "then" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token function", children: "rm" }), " ", _jsx(_components.span, { className: "token variable", children: "${bam}" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token keyword", children: "else" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsxs(_components.span, { className: "token string", children: ["\"Error: Sorting failed for ", _jsx(_components.span, { className: "token variable", children: "${sam}" }), "\""] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token builtin class-name", children: "continue" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token keyword", children: "fi" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token comment", children: "# Index sorted BAM file" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsxs(_components.span, { className: "token string", children: ["\"Indexing ", _jsx(_components.span, { className: "token variable", children: "$sorted_bam" }), "\""] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" samtools index -@ ", _jsx(_components.span, { className: "token number", children: "4" }), " ", _jsx(_components.span, { className: "token variable", children: "${sorted_bam}" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsxs(_components.span, { className: "token string", children: ["\"Finished processing ", _jsx(_components.span, { className: "token variable", children: "$sam" }), "\""] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token keyword", children: "done" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsx(_components.span, { className: "token string", children: "\"All files have been processed.\"" }), "\n"] })] }) }), "\n", _jsx(_components.p, { children: "之后还能通过IGV可视化" }), "\n", _jsxs(_components.h1, { id: "定量表达stringtiefeaturecounts", children: [_jsx(_components.a, { className: "heading-link", href: "#定量表达stringtiefeaturecounts", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "定量表达（stringtie/featureCounts）"] }), "\n", _jsx(_components.p, { children: "RNA-Seq 方法旨在生成为基因组特征赋值的量化矩阵。在基于比对的 RNA-Seq 中，该过程需要将 BAM 比对文件与注释文件中列出的间隔相交。" }), "\n", _jsxs(_components.p, { children: ["交集过程通常称为“特征计数”，它会计算出与每个文件特征重叠的比对数量。例如，以下图中的 ", _jsx(_components.code, { className: "language-auto", children: "GENE A" }), " 为例。假设每片虚线都是一个比对。有多少特征与 ", _jsx(_components.code, { className: "language-auto", children: "GENE A" }), " 重叠？"] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsx(_components.span, { className: "code-line", children: "------- ------- -------\n" }), _jsx(_components.span, { className: "code-line", children: " ------ --------- ---------\n" }), _jsx(_components.span, { className: "code-line", children: " ------- ---------\n" }), _jsx(_components.span, { className: "code-line", children: " ------- --------- ---------\n" }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token operator", children: "|" }), _jsx(_components.span, { className: "token operator", children: "<" }), "--- GENE A ---", _jsx(_components.span, { className: "token operator", children: ">|" }), "\n"] })] }) }), "\n", _jsx(_components.p, { children: "计取决于“重叠”一词的含义。" }), "\n", _jsxs(_components.ul, { children: ["\n", _jsxs(_components.li, { children: ["任何重叠量——那么 ", _jsx(_components.code, { className: "language-auto", children: "11" }), " 对齐与该特征重叠。"] }), "\n", _jsxs(_components.li, { children: ["如果完全在特征内——那么计数只有 ", _jsx(_components.code, { className: "language-auto", children: "4" }), " 。"] }), "\n", _jsxs(_components.li, { children: ["至少 50% 的比对应该覆盖该特征吗——数量是 ", _jsx(_components.code, { className: "language-auto", children: "8" }), " 。"] }), "\n"] }), "\n", _jsx(_components.p, { children: "没有普遍正确的计数方法；根据情况，不同的策略可能更合适。默认设置将考虑任何重叠量来增加计数。" }), "\n", _jsxs(_components.h2, { id: "featurecounts", children: [_jsx(_components.a, { className: "heading-link", href: "#featurecounts", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "featureCounts"] }), "\n", _jsxs(_components.p, { children: ["OR: featurecounts ", _jsx(_components.a, { href: "http://git.genek.cn:3333/zhxd2/RunFeatureCounts", target: "_blank", rel: "noopener noreferrer", children: "R封装版" })] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 原版" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["conda ", _jsx(_components.span, { className: "token function", children: "install" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-y" }), " subread\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: ["featureCounts ", _jsx(_components.span, { className: "token parameter variable", children: "-T" }), " ", _jsx(_components.span, { className: "token number", children: "2" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-p" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-t" }), " exon ", _jsx(_components.span, { className: "token parameter variable", children: "-g" }), " gene_id ", _jsx(_components.span, { className: "token parameter variable", children: "-a" }), " ", _jsx(_components.span, { className: "token punctuation", children: ".." }), "/12.ref/genes.gtf ", _jsx(_components.span, { className: "token parameter variable", children: "-o" }), " ./Cs1_1_quant.txt ", _jsx(_components.span, { className: "token punctuation", children: ".." }), "/21.mapping/Cs1_1.bam\n"] })] }) }), "\n", _jsxs(_components.p, { children: ["产生表达矩阵", _jsx(_components.code, { className: "language-auto", children: "Cs1_1_quant.txt" }), "："] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# Program:featureCounts v2.0.8; Command:\"featureCounts\" \"-T\" \"2\" \"-p\" \"-t\" \"exon\" \"-g\" \"gene_id\" \"-a\" \"../12>" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["Geneid Chr Start End Strand Length ", _jsx(_components.span, { className: "token punctuation", children: ".." }), "/21.mapping/Cs1_1.bam\n"] }), _jsxs(_components.span, { className: "code-line", children: ["Cs_ont_1g000010 chr1", _jsx(_components.span, { className: "token punctuation", children: ";" }), "chr1", _jsx(_components.span, { className: "token punctuation", children: ";" }), "chr1 ", _jsx(_components.span, { className: "token number", children: "4607" }), _jsx(_components.span, { className: "token punctuation", children: ";" }), _jsx(_components.span, { className: "token number", children: "5477" }), _jsx(_components.span, { className: "token punctuation", children: ";" }), _jsx(_components.span, { className: "token number", children: "7112" }), " ", _jsx(_components.span, { className: "token number", children: "4819" }), _jsx(_components.span, { className: "token punctuation", children: ";" }), _jsx(_components.span, { className: "token number", children: "5572" }), _jsx(_components.span, { className: "token punctuation", children: ";" }), _jsx(_components.span, { className: "token number", children: "7117" }), " -", _jsx(_components.span, { className: "token punctuation", children: ";" }), "-", _jsx(_components.span, { className: "token punctuation", children: ";" }), "- ", _jsx(_components.span, { className: "token number", children: "315" }), " ", _jsx(_components.span, { className: "token number", children: "0" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 从左到右依次：" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# Geneid：基因的ensemble基因号；" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# Chr：多个外显子所在的染色体编号;" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# Start：多个外显子起始位点,与前面一一对应" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# End：多个外显子终止位点,与前面一一对应" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# Strand：正负链" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# Length：基因长度" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# sampleID：一列代表一个样本，数值表示比对到该基因上的read数目" }), "\n"] })] }) }), "\n", _jsx(_components.p, { children: "但是没有FPKM和TPM值，使用R计算" }), "\n", _jsx(_components.pre, { className: "language-r", children: _jsxs(_components.code, { className: "language-R code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: ["count_df ", _jsx(_components.span, { className: "token operator", children: "<-" }), " read.table", _jsx(_components.span, { className: "token punctuation", children: "(" }), _jsx(_components.span, { className: "token string", children: "\"Cs1_1_quant.txt\"" }), _jsx(_components.span, { className: "token punctuation", children: "," }), "header ", _jsx(_components.span, { className: "token operator", children: "=" }), " T", _jsx(_components.span, { className: "token punctuation", children: "," }), " row.names ", _jsx(_components.span, { className: "token operator", children: "=" }), " ", _jsx(_components.span, { className: "token number", children: "1" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["dim", _jsx(_components.span, { className: "token punctuation", children: "(" }), "count_df", _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["names", _jsx(_components.span, { className: "token punctuation", children: "(" }), "count_df", _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "##'@提取Count值" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["expr_df ", _jsx(_components.span, { className: "token operator", children: "<-" }), " count_df", _jsx(_components.span, { className: "token punctuation", children: "[" }), _jsx(_components.span, { className: "token punctuation", children: "," }), _jsx(_components.span, { className: "token number", children: "6" }), _jsx(_components.span, { className: "token operator", children: ":" }), "ncol", _jsx(_components.span, { className: "token punctuation", children: "(" }), "count_df", _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: "]" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "###'@基因表达量之和大于0" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["expr_df ", _jsx(_components.span, { className: "token operator", children: "<-" }), " expr_df", _jsx(_components.span, { className: "token punctuation", children: "[" }), "rowSums", _jsx(_components.span, { className: "token punctuation", children: "(" }), "expr_df", _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: ">" }), " ", _jsx(_components.span, { className: "token number", children: "0" }), _jsx(_components.span, { className: "token punctuation", children: "," }), _jsx(_components.span, { className: "token punctuation", children: "]" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["dim", _jsx(_components.span, { className: "token punctuation", children: "(" }), "expr_df", _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "##'@保存count值矩阵" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["write.csv", _jsx(_components.span, { className: "token punctuation", children: "(" }), "expr_df", _jsx(_components.span, { className: "token punctuation", children: "," }), _jsx(_components.span, { className: "token string", children: "\"count.filter.csv\"" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "#计算TPM" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "##'@提取基因长度，基因长度需要转化成kb" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["gene_length_kb ", _jsx(_components.span, { className: "token operator", children: "<-" }), " count_df", _jsx(_components.span, { className: "token operator", children: "$" }), "Length ", _jsx(_components.span, { className: "token operator", children: "/" }), " ", _jsx(_components.span, { className: "token number", children: "1000" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "### 每千碱基reads（per million scaling factor)长度标准化" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["data_rpk ", _jsx(_components.span, { className: "token operator", children: "<-" }), " expr_df ", _jsx(_components.span, { className: "token operator", children: "/" }), "gene_length_kb\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "##'@每百万" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["TPM ", _jsx(_components.span, { className: "token operator", children: "<-" }), " t", _jsx(_components.span, { className: "token punctuation", children: "(" }), "t", _jsx(_components.span, { className: "token punctuation", children: "(" }), "data_rpk", _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "/" }), " colSums", _jsx(_components.span, { className: "token punctuation", children: "(" }), "data_rpk", _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "*" }), " ", _jsx(_components.span, { className: "token number", children: "1000000" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## 求均值" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["avg_tmp ", _jsx(_components.span, { className: "token operator", children: "<-" }), " data.frame", _jsx(_components.span, { className: "token punctuation", children: "(" }), "avg_tmp ", _jsx(_components.span, { className: "token operator", children: "=" }), " rowMeans", _jsx(_components.span, { className: "token punctuation", children: "(" }), "TPM", _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "##'@保存数据" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["write.csv", _jsx(_components.span, { className: "token punctuation", children: "(" }), "TPM", _jsx(_components.span, { className: "token punctuation", children: "," }), _jsx(_components.span, { className: "token string", children: "\"TPM.csv\"" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "#计算FPKM值" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["FPKM ", _jsx(_components.span, { className: "token operator", children: "<-" }), " t", _jsx(_components.span, { className: "token punctuation", children: "(" }), "t", _jsx(_components.span, { className: "token punctuation", children: "(" }), "data_rpk", _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "/" }), " colSums", _jsx(_components.span, { className: "token punctuation", children: "(" }), "expr_df", _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "*" }), " ", _jsx(_components.span, { className: "token number", children: "10" }), _jsx(_components.span, { className: "token operator", children: "^" }), _jsx(_components.span, { className: "token number", children: "6" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "##'@保存数据" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["write.csv", _jsx(_components.span, { className: "token punctuation", children: "(" }), "FPKM", _jsx(_components.span, { className: "token punctuation", children: "," }), _jsx(_components.span, { className: "token string", children: "\"FPKM.csv\"" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] })] }) }), "\n", _jsxs(_components.h2, { id: "stringtie", children: [_jsx(_components.a, { className: "heading-link", href: "#stringtie", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "stringtie"] }), "\n", _jsx(_components.pre, { className: "language-sh", children: _jsxs(_components.code, { className: "language-sh code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "vim" }), " stringtie.sh\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "#!/bin/bash" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token keyword", children: "for" }), " ", _jsx(_components.span, { className: "token for-or-select variable", children: "sortbam" }), " ", _jsx(_components.span, { className: "token keyword", children: "in" }), " *.sorted.bam", _jsx(_components.span, { className: "token punctuation", children: ";" }), " ", _jsx(_components.span, { className: "token keyword", children: "do" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token assign-left variable", children: "sample" }), _jsx(_components.span, { className: "token operator", children: "=" }), _jsxs(_components.span, { className: "token variable", children: [_jsx(_components.span, { className: "token variable", children: "$(" }), _jsx(_components.span, { className: "token function", children: "basename" }), " $", _jsx(_components.span, { className: "token punctuation", children: "{" }), "sortbam", _jsx(_components.span, { className: "token punctuation", children: "}" }), " .sorted.bam", _jsx(_components.span, { className: "token variable", children: ")" })] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsxs(_components.span, { className: "token string", children: ["\"", _jsx(_components.span, { className: "token variable", children: "${sample}" }), "\""] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" stringtie ", _jsx(_components.span, { className: "token variable", children: "${sortbam}" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-o" }), " ", _jsx(_components.span, { className: "token variable", children: "${sample}" }), ".gtf ", _jsx(_components.span, { className: "token parameter variable", children: "-p" }), " ", _jsx(_components.span, { className: "token number", children: "4" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-G" }), " gene.gtf \n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token keyword", children: "done" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "#运行文档" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "bash" }), " stringtie.sh\n"] })] }) }), "\n", _jsx(_components.p, { children: "先组装好每个转录组表达矩阵，每个里面包含了TPM和FPKM数值。" }), "\n", _jsx(_components.p, { children: "将sample的gtf文件名整合进merge_list.txt文本，使用命令把参考物种的GTF与样本组装的进行合并，重新计算：" }), "\n", _jsx(_components.pre, { className: "language-text", children: _jsx(_components.code, { className: "language-text code-highlight", children: _jsx(_components.span, { className: "code-line", children: "stringtie --merge -p8 -G gene.gtf -o merge.gtf merge_list.txt\n" }) }) }), "\n", _jsx(_components.p, { children: "转录组定量：" }), "\n", _jsx(_components.pre, { className: "language-sh", children: _jsxs(_components.code, { className: "language-sh code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "vim" }), " merge_stringtie.sh\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "#!/bin/bash" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token keyword", children: "for" }), " ", _jsx(_components.span, { className: "token for-or-select variable", children: "sortbam" }), " ", _jsx(_components.span, { className: "token keyword", children: "in" }), " *.sorted.bam", _jsx(_components.span, { className: "token punctuation", children: ";" }), " ", _jsx(_components.span, { className: "token keyword", children: "do" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token assign-left variable", children: "sample" }), _jsx(_components.span, { className: "token operator", children: "=" }), _jsxs(_components.span, { className: "token variable", children: [_jsx(_components.span, { className: "token variable", children: "$(" }), _jsx(_components.span, { className: "token function", children: "basename" }), " $", _jsx(_components.span, { className: "token punctuation", children: "{" }), "sortbam", _jsx(_components.span, { className: "token punctuation", children: "}" }), " .sorted.bam", _jsx(_components.span, { className: "token variable", children: ")" })] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token builtin class-name", children: "echo" }), " ", _jsxs(_components.span, { className: "token string", children: ["\"", _jsx(_components.span, { className: "token variable", children: "${sample}" }), "\""] }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" stringtie ", _jsx(_components.span, { className: "token variable", children: "${sortbam}" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-p" }), " ", _jsx(_components.span, { className: "token number", children: "4" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-e" }), " ", _jsx(_components.span, { className: "token parameter variable", children: "-G" }), " merge.gtf ", _jsx(_components.span, { className: "token parameter variable", children: "-o" }), " ", _jsx(_components.span, { className: "token variable", children: "${sample}" }), ".gtf ", _jsx(_components.span, { className: "token parameter variable", children: "-A" }), " ", _jsx(_components.span, { className: "token variable", children: "${sample}" }), ".gene_abundance.txt\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token keyword", children: "done" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "#运行文档" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "bash" }), " merge_stringtie.txt\n"] })] }) }), "\n", _jsx(_components.p, { children: "计算得到的gene_abundance.txt文档将会含有新转录本的FPKM和TPM值。" }), "\n", _jsxs(_components.h1, { id: "下游分析", children: [_jsx(_components.a, { className: "heading-link", href: "#下游分析", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "下游分析"] }), "\n", _jsxs(_components.p, { children: ["表达数据定量要输出为一个矩阵，用于下游分析，需要用到一个", _jsx(_components.code, { className: "language-auto", children: "prepDE.py" }), "的脚本，这个脚本是stringtie的开发者提供好的，直接前往官网下载，在服务器上运行："] }), "\n", _jsx(_components.pre, { className: "language-bash", children: _jsxs(_components.code, { className: "language-bash code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "wget" }), " http://ccb.jhu.edu/software/stringtie/dl/prepDE.py3\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token function", children: "vim" }), " samplelist.tXTPuNc\n"] }), _jsxs(_components.span, { className: "code-line", children: ["python prepDE.py ", _jsx(_components.span, { className: "token parameter variable", children: "-i" }), " samplelist.txt ", _jsx(_components.span, { className: "token parameter variable", children: "-g" }), " gene_count_matrix.csv ", _jsx(_components.span, { className: "token parameter variable", children: "-t" }), " transcript_count_matrix.csv\n"] })] }) }), "\n", _jsx(_components.p, { children: "samplelist.txt内容应该如下：" }), "\n", _jsx(_components.pre, { className: "language-text", children: _jsxs(_components.code, { className: "language-text code-highlight", children: [_jsx(_components.span, { className: "code-line", children: "# 以样本名字（空格/n）含文件路径的样本gtf文件为一列\n" }), _jsx(_components.span, { className: "code-line", children: "HY1\t./HY1.gtf\n" }), _jsx(_components.span, { className: "code-line", children: "HY2\t./HY2.gtf\n" }), _jsx(_components.span, { className: "code-line", children: "HY3\t./HY3.gtf\n" }), _jsx(_components.span, { className: "code-line", children: "YJ1\t./YJ1.gtf\n" }), _jsx(_components.span, { className: "code-line", children: "YJ2\t./YJ2.gtf\n" }), _jsx(_components.span, { className: "code-line", children: "YJ3\t./YJ3.gtf\n" })] }) }), "\n", _jsxs(_components.h1, { id: "样本关系分析", children: [_jsx(_components.a, { className: "heading-link", href: "#样本关系分析", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "样本关系分析"] }), "\n", _jsxs(_components.h2, { id: "导入数据", children: [_jsx(_components.a, { className: "heading-link", href: "#导入数据", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "导入数据"] }), "\n", _jsx(_components.pre, { children: _jsxs(_components.code, { className: "language-{r code-highlight", children: [_jsx(_components.span, { className: "code-line", children: "library(tidyverse)\n" }), _jsx(_components.span, { className: "code-line", children: "library(pheatmap)\n" }), _jsx(_components.span, { className: "code-line", children: "library(RColorBrewer)\n" }), _jsx(_components.span, { className: "code-line", children: "library(scales)\n" }), _jsx(_components.span, { className: "code-line", children: "library(PCAtools)\n" }), _jsx(_components.span, { className: "code-line", children: "knitr::opts_chunk$set(echo = TRUE)\n" })] }) }), "\n", _jsxs(_components.ol, { children: ["\n", _jsx(_components.li, { children: "导入表达矩阵" }), "\n"] }), "\n", _jsx(_components.pre, { children: _jsx(_components.code, { className: "language-{r} code-highlight", children: _jsx(_components.span, { className: "code-line", children: "gene_exp = read.table(file = \"/Users/Yilong/Documents/Coding/R/23.Merge_result/genes.TMM.TPM.matrix\")\n" }) }) }), "\n", _jsxs(_components.ol, { start: "2", children: ["\n", _jsx(_components.li, { children: "导入样本信息表" }), "\n"] }), "\n", _jsx(_components.pre, { children: _jsxs(_components.code, { className: "language-{r} code-highlight", children: [_jsx(_components.span, { className: "code-line", children: "read.table(file = \"./23.Merge_result/sample_info.txt\") %>%\n" }), _jsx(_components.span, { className: "code-line", children: " rename(group = \"V1\", sample = \"V2\") %>%\n" }), _jsx(_components.span, { className: "code-line", children: " column_to_rownames(var = \"sample\") -> sample_info\n" })] }) }), "\n", _jsxs(_components.h2, { id: "样本相关性", children: [_jsx(_components.a, { className: "heading-link", href: "#样本相关性", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "样本相关性"] }), "\n", _jsxs(_components.ol, { children: ["\n", _jsx(_components.li, { children: "相关系数是什么" }), "\n"] }), "\n", _jsx(_components.span, { className: "katex-display", children: _jsxs(_components.span, { className: "katex", children: [_jsx(_components.span, { className: "katex-mathml", children: 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children: "=" }), " T", _jsx(_components.span, { className: "token punctuation", children: "," }), " encircleFill ", _jsx(_components.span, { className: "token operator", children: "=" }), " T", _jsx(_components.span, { className: "token punctuation", children: "," }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token comment", children: "# ellipse = T" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## Registered S3 methods overwritten by 'ggalt':" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## method from " }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## grid.draw.absoluteGrob ggplot2" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## grobHeight.absoluteGrob ggplot2" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## grobWidth.absoluteGrob ggplot2" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## grobX.absoluteGrob ggplot2" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "## grobY.absoluteGrob ggplot2" }), "\n"] })] }) }), "\n", _jsx(_components.img, { src: "https://github.com/Cafwell/picx-images-hosting/raw/master/RNAseq/RNAseq-pca1.jpg", alt: "pca1|600", className: "internal" }), "\n", _jsxs(_components.h2, { id: "prcomp--ggplot2-methods", children: [_jsx(_components.a, { className: "heading-link", href: "#prcomp--ggplot2-methods", children: _jsx(_components.svg, { xmlns: "http:www.w3.org/2000/svg", fill: "#ab2b65", viewBox: "0 0 20 20", className: "w-5 h-5", children: _jsx(_components.path, { fillRule: "evenodd", clipRule: "evenodd", d: "M9.493 2.853a.75.75 0 00-1.486-.205L7.545 6H4.198a.75.75 0 000 1.5h3.14l-.69 5H3.302a.75.75 0 000 1.5h3.14l-.435 3.148a.75.75 0 001.486.205L7.955 14h2.986l-.434 3.148a.75.75 0 001.486.205L12.456 14h3.346a.75.75 0 000-1.5h-3.14l.69-5h3.346a.75.75 0 000-1.5h-3.14l.435-3.147a.75.75 0 00-1.486-.205L12.045 6H9.059l.434-3.147zM8.852 7.5l-.69 5h2.986l.69-5H8.852z" }) }) }), "prcomp + ggplot2 methods"] }), "\n", _jsx(_components.pre, { className: "language-r", children: _jsxs(_components.code, { className: "language-R code-highlight", children: [_jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 转置矩阵（行为样本，列为基因）" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["p2_t ", _jsx(_components.span, { className: "token operator", children: "<-" }), " t", _jsx(_components.span, { className: "token punctuation", children: "(" }), "gene_exp", _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["variances ", _jsx(_components.span, { className: "token operator", children: "<-" }), " apply", _jsx(_components.span, { className: "token punctuation", children: "(" }), "p2_t", _jsx(_components.span, { className: "token punctuation", children: "," }), " ", _jsx(_components.span, { className: "token number", children: "2" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " var", _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 模拟removeVar = 0.3" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["p2_t_filtered ", _jsx(_components.span, { className: "token operator", children: "<-" }), " p2_t", _jsx(_components.span, { className: "token punctuation", children: "[" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " variances ", _jsx(_components.span, { className: "token operator", children: ">" }), " quantile", _jsx(_components.span, { className: "token punctuation", children: "(" }), "variances", _jsx(_components.span, { className: "token punctuation", children: "," }), " probs ", _jsx(_components.span, { className: "token operator", children: "=" }), " ", _jsx(_components.span, { className: "token number", children: "0.3" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: "]" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token comment", children: "# 执行PCA（若基因表达量纲差异大，建议scale=TRUE）" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: ["p2 ", _jsx(_components.span, { className: "token operator", children: "<-" }), " prcomp", _jsx(_components.span, { className: "token punctuation", children: "(" }), "p2_t_filtered", _jsx(_components.span, { className: "token punctuation", children: "," }), " center", _jsx(_components.span, { className: "token operator", children: "=" }), _jsx(_components.span, { className: "token boolean", children: "TRUE" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " scale", _jsx(_components.span, { className: "token operator", children: "=" }), _jsx(_components.span, { className: "token boolean", children: "FALSE" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: ["df_p2 ", _jsx(_components.span, { className: "token operator", children: "<-" }), " data.frame", _jsx(_components.span, { className: "token punctuation", children: "(" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" p2", _jsx(_components.span, { className: "token operator", children: "$" }), "x", _jsx(_components.span, { className: "token punctuation", children: "[" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " ", _jsx(_components.span, { className: "token number", children: "1" }), _jsx(_components.span, { className: "token operator", children: ":" }), _jsx(_components.span, { className: "token number", children: "2" }), _jsx(_components.span, { className: "token punctuation", children: "]" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " ", _jsx(_components.span, { className: "token comment", children: "# 取前两个主成分" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" Group ", _jsx(_components.span, { className: "token operator", children: "=" }), " sample_info", _jsx(_components.span, { className: "token operator", children: "$" }), "group\n"] }), _jsxs(_components.span, { className: "code-line", children: [_jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsxs(_components.span, { className: "code-line", children: ["ggplot", _jsx(_components.span, { className: "token punctuation", children: "(" }), "df_p2", _jsx(_components.span, { className: "token punctuation", children: "," }), " aes", _jsx(_components.span, { className: "token punctuation", children: "(" }), "x", _jsx(_components.span, { className: "token operator", children: "=" }), "PC1", _jsx(_components.span, { className: "token punctuation", children: "," }), " y", _jsx(_components.span, { className: "token operator", children: "=" }), "PC2", _jsx(_components.span, { className: "token punctuation", children: "," }), " color", _jsx(_components.span, { className: "token operator", children: "=" }), "Group", _jsx(_components.span, { className: "token punctuation", children: "," }), " group", _jsx(_components.span, { className: "token operator", children: "=" }), "Group", _jsx(_components.span, { className: "token punctuation", children: "," }), "label", _jsx(_components.span, { className: "token operator", children: "=" }), "rownames", _jsx(_components.span, { className: "token punctuation", children: "(" }), "df_p2", _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "+" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" geom_point", _jsx(_components.span, { className: "token punctuation", children: "(" }), "size", _jsx(_components.span, { className: "token operator", children: "=" }), _jsx(_components.span, { className: "token number", children: "3" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "+" }), " ", _jsx(_components.span, { className: "token comment", children: "# 显示样本名" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" ", _jsx(_components.span, { className: "token comment", children: "# stat_ellipse(level=0.95) + # 添加95%置信椭圆，但这儿每组数量不够多（一般> 5），不添加" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" labs", _jsx(_components.span, { className: "token punctuation", children: "(" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" x ", _jsx(_components.span, { className: "token operator", children: "=" }), " paste0", _jsx(_components.span, { className: "token punctuation", children: "(" }), _jsx(_components.span, { className: "token string", children: "\"PC1 (\"" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " round", _jsx(_components.span, { className: "token punctuation", children: "(" }), "summary", _jsx(_components.span, { className: "token punctuation", children: "(" }), "p2", _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token operator", children: "$" }), "importance", _jsx(_components.span, { className: "token punctuation", children: "[" }), _jsx(_components.span, { className: "token number", children: "2" }), _jsx(_components.span, { className: "token punctuation", children: "," }), _jsx(_components.span, { className: "token number", children: "1" }), _jsx(_components.span, { className: "token punctuation", children: "]" }), _jsx(_components.span, { className: "token operator", children: "*" }), _jsx(_components.span, { className: "token number", children: "100" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " ", _jsx(_components.span, { className: "token number", children: "1" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " ", _jsx(_components.span, { className: "token string", children: "\"%)\"" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: "," }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" y ", _jsx(_components.span, { className: "token operator", children: "=" }), " paste0", _jsx(_components.span, { className: "token punctuation", children: "(" }), _jsx(_components.span, { className: "token string", children: "\"PC2 (\"" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " round", _jsx(_components.span, { className: "token punctuation", children: "(" }), "summary", _jsx(_components.span, { className: "token punctuation", children: "(" }), "p2", _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token operator", children: "$" }), "importance", _jsx(_components.span, { className: "token punctuation", children: "[" }), _jsx(_components.span, { className: "token number", children: "2" }), _jsx(_components.span, { className: "token punctuation", children: "," }), _jsx(_components.span, { className: "token number", children: "2" }), _jsx(_components.span, { className: "token punctuation", children: "]" }), _jsx(_components.span, { className: "token operator", children: "*" }), _jsx(_components.span, { className: "token number", children: "100" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " ", _jsx(_components.span, { className: "token number", children: "1" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: "," }), " ", _jsx(_components.span, { className: "token string", children: "\"%)\"" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), _jsx(_components.span, { className: "token punctuation", children: "," }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" title ", _jsx(_components.span, { className: "token operator", children: "=" }), " ", _jsx(_components.span, { className: "token string", children: "\"PCA of RNA-seq Samples by Group\"" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "+" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" theme_minimal", _jsx(_components.span, { className: "token punctuation", children: "(" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), " ", _jsx(_components.span, { className: "token operator", children: "+" }), "\n"] }), _jsxs(_components.span, { className: "code-line", children: [" scale_color_brewer", _jsx(_components.span, { className: "token punctuation", children: "(" }), "palette ", _jsx(_components.span, { className: "token operator", children: "=" }), " ", _jsx(_components.span, { className: "token string", children: "\"Set1\"" }), _jsx(_components.span, { className: "token punctuation", children: ")" }), "\n"] })] }) }), "\n", _jsx(_components.img, { src: "https://github.com/Cafwell/picx-images-hosting/raw/master/RNAseq/RNAseq-pca2.jpg", alt: "pca1|600", className: "internal" }), "\n", _jsxs(_components.p, { children: ["DESeq2两两比对，", _jsx(_components.a, { href: "https://github.com/trinityrnaseq/trinityrnaseq", target: "_blank", rel: "noopener noreferrer", children: "trinity" }), "中的run_DE_analysis完成："] }), "\n", _jsx(_components.pre, { children: _jsxs(_components.code, { className: "language-zsh code-highlight", children: [_jsx(_components.span, { className: "code-line", children: "perl run_DE_analysis.pl \\\n" }), _jsx(_components.span, { className: "code-line", children: " --matrix genes.counts.matrix \\\n" }), _jsx(_components.span, { className: "code-line", children: " --method DESeq2 \\\n" }), _jsx(_components.span, { className: "code-line", children: " --samples_file sample_info.txt \\\n" }), _jsx(_components.span, { className: "code-line", children: " --contrasts contrasts.txt\n" })] }) }), "\n", _jsx(_components.p, { children: "其中：" }), "\n", _jsx(_components.pre, { className: "language-text", children: _jsxs(_components.code, { className: "language-text code-highlight", children: [_jsx(_components.span, { className: "code-line", children: "sample_info.txt:\n" }), _jsx(_components.span, { className: "code-line", children: "Cs1\tCs1_1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs1\tCs1_2\n" }), _jsx(_components.span, { className: "code-line", children: "Cs1\tCs1_3\n" }), _jsx(_components.span, { className: "code-line", children: "Cs2\tCs2_1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs2\tCs2_2\n" }), _jsx(_components.span, { className: "code-line", children: "Cs2\tCs2_3\n" }), _jsx(_components.span, { className: "code-line", children: "Cs3\tCs3_1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs3\tCs3_2\n" }), _jsx(_components.span, { className: "code-line", children: "Cs3\tCs3_3\n" }), _jsx(_components.span, { className: "code-line", children: "Cs4\tCs4_1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs4\tCs4_2\n" }), _jsx(_components.span, { className: "code-line", children: "Cs4\tCs4_3\n" }), _jsx(_components.span, { className: "code-line", children: "Cs5\tCs5_1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs5\tCs5_2\n" }), _jsx(_components.span, { className: "code-line", children: "Cs5\tCs5_3\n" }), _jsx(_components.span, { className: "code-line", children: "\n" }), _jsx(_components.span, { className: "code-line", children: "contrasts.txt\n" }), _jsx(_components.span, { className: "code-line", children: "Cs2 Cs1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs3 Cs1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs4 Cs1\n" }), _jsx(_components.span, { className: "code-line", children: "Cs5 Cs1\n" })] }) })] }); } function MDXContent(props = {}) { const {wrapper: MDXLayout} = { ..._provideComponents(), ...props.components }; return MDXLayout ? _jsx(MDXLayout, { ...props, children: _jsx(_createMdxContent, { ...props }) }) : _createMdxContent(props); } return { default: MDXContent };