# 使用JCVI进行MCScan共线性分析与可视化 > > > ## 安装 推荐使用`conda`安装`jcvi`，并安装依赖软件`BEDTools`、`EMBOSS`和`LAST` ```bash conda create --name jcvi python=3.9 jcvi bedtools emboss last ``` 在此之前，建议按照[官网教程](https://tug.org/texlive/quickinstall.html)安装`texlive`,而不要使用`conda`中的`texlive-core` ```bash wget https://mirror.ctan.org/systems/texlive/tlnet/install-tl-unx.tar.gz tar -xzf install-tl-unx.tar.gz cd install-tl-* perl install-tl # 若无管理员权限需要安装在家目录 # 在交互界面按"D"，再按"1" # 更改"TEXDIR"至合适位置 # 再按提示完成安装（约需1.5h) ``` ## 准备数据 `jcvi`支持直接从[phytozome](https://phytozome-next.jgi.doe.gov/)下载数据（需要先注册） 查看可下载的物种，常见的植物物种都可以直接下载 ```bash python -m jcvi.apps.fetch phytozome ``` 下载与整理数据 ```bash # download sequences and coordinates of grape and peach python -m jcvi.apps.fetch phytozome Vvinifera,Ppersica # convert the GFF to BED file python -m jcvi.formats.gff bed --type=mRNA --key=ID --primary_only Vvinifera_145_Genoscope.12X.gene.gff3.gz -o grape.bed python -m jcvi.formats.gff bed --type=mRNA --key=ID --primary_only Ppersica_298_v2.1.gene.gff3.gz -o peach.bed # clean headers to remove description fields from Phytozome FASTA files python -m jcvi.formats.fasta format Vvinifera_145_Genoscope.12X.cds.fa.gz grape.cds python -m jcvi.formats.fasta format Ppersica_298_v2.1.cds.fa.gz peach.cds ``` ## 共线性分析 ```bash python -m jcvi.compara.catalog ortholog --no_strip_names grape peach ``` 程序默认按照可用线程，调用全部进行`LAST`比对 ## 可视化 ### 点阵图 在共线性分析后，程序会自动绘制点阵图。手动运行方式如下 ```bash python -m jcvi.graphics.dotplot grape.peach.anchors ``` 在两者的点阵图中，不论水平方向或者竖直方向查看，都可以发现不超过3次的共线性区域。这说明，🍇和🍑之间发生了基因组三倍体化的事件，使得出现了这样3:3的模式。 如果仔细观察，还可以发现3个共线性区域中常常有一个信号更强，对应着两个基因组之间的直系同源区域。如果我们只想要得到这些1:1直系同源的区域呢？我们只需要重复之前的比对，同时加上选项`--cscore=.99`即可。`C-score`是由`LAST`比对区域到`BLAST`比对区域的比值确定。(C-score is defined by the ratio of LAST hit to the best BLAST hits to either the query and hit)。`0.99`的`C-score`阈值有效地过滤`LAST`比对结果，从而得到最佳相互比对结果(reciprocal best hit, RBH)。 ```bash # remove old file rm grape.peach.last.filtered grape.peach.anchors python -m jcvi.compara.catalog ortholog grape peach --cscore=.99 ``` ![Grape-peach-synteny-.99](/files/QGk2iANq2xvXcWSnJb0u) 还可以手动查看synteny depth分布 ```bash python -m jcvi.compara.synteny depth --histogram grape.peach.anchors ``` ![Grape-peach-depth](/files/YNqbCCHzefj3QZw0V83c) ### 共线性图 首先先从`.anchors`文件中产生更简要的`.simple`文件 ```bash python -m jcvi.compara.synteny screen --minspan=30 --simple grape.peach.anchors grape.peach.anchors.simple ``` 此外，除了原始BED文件和`.simple`文件文件，还需要手动设置两个文件用于绘图（注意，文件末尾不能存在多余的空行） 1. `seqids`：设定绘制哪些染色体，一般会去掉较小和未定位的scaffolds，下方设定了19个🍇的染色体与8个🍑的染色体 ```vim chr1,chr2,chr3,chr4,chr5,chr6,chr7,chr8,chr9,chr10,chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19 Pp1,Pp2,Pp3,Pp4,Pp5,Pp6,Pp7,Pp8 ``` 2. `layout`：设定画板上如何绘制。整体上`canvas`绘图的`x`,`y`轴的定位在`0-1`之间，前三行设定了染色体轨道的位置，循环，颜色，标签，垂直排列以及对应的BED文件。下方的`track0`就是🍇，`track1`就是🍑。下一节就是设定在哪些tracks之间绘制共线性关系。`e,0,1`代表着绘制`track0`与`track1`之间的共线性，使用`grape.peach.anchors.simple`文件中的详细信息。 ```vim # y, xstart, xend, rotation, color, label, va, bed .6, .1, .8, 0, , Grape, top, grape.bed .4, .1, .8, 0, , Peach, top, peach.bed # edges e, 0, 1, grape.peach.anchors.simple ``` 绘图 ```bash python -m jcvi.graphics.karyotype seqids layout ``` ![Grape-peach-karyotype](/files/psxkjizsrEXCExZrdwDu) ### 局部可视化 首先从基因水平提取匹配的区块 ```bash python -m jcvi.compara.synteny mcscan grape.bed grape.peach.lifted.anchors --iter=1 -o grape.peach.i1.blocks ``` 参数`--iter=1`表明从每个🍇区域中提取1个最佳匹配区域。若将`--iter`设置为2，那么每个🍇区域就会有2个对应的🍑区域。这一点对于基因组重复区域的绘制很有帮助。 目前`grape.peach.i1.blocks`中包含有很多局部区域，我们可以手动选择需要绘制的部分。 ```bash head -50 grape.peach.i1.blocks > blocks.select ``` 最终，我们仍然需要一个`layout`文件进行绘图，`blocks.layout`文件如下 ```vim # x, y, rot, ha, va, color, ratio, label 0.5, 0.6, 0, left, center, m, 1, grape Chr1 0.5, 0.4, 0, left, center, #fc8d62, 1, peach Pp1 # edges e, 0, 1 ``` 然后，我们就可以进行绘制了 ```bash cat grape.bed peach.bed > grape_peach.bed python -m jcvi.graphics.synteny blocks.select grape_peach.bed blocks.layout ``` ![Grape-peach-blocks](/files/tKUvrLQrHXtKy7ArCIvt) ### 调整与美化 #### 设置颜色 **species1.species2.anchors.simple** ```bash g*GSVIVT01012028001 GSVIVT01000604001 ppa011886m ppa008534m 392 + GSVIVT01010441001 GSVIVT01000970001 ppa022891m ppa001358m 115 - ... # g: green; r: red; etc. ``` #### 多物种 **layout** ```bash # y, xstart, xend, rotation, color, label, va, bed .7, .2, .4, 45, , Grape, top, grape.bed .5, .2, .8, 0, , Peach, top, peach.bed .7, .4, .8, -45, , Cacao, bottom, cacao.bed # edges e, 0, 1, grape.peach.anchors.simple e, 1, 2, peach.cacao.anchors.simple ``` **seqids** ```bash chr1,chr2,chr3,chr4,chr5,chr6,chr7,chr8,chr9,chr10,chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19 scaffold_1,scaffold_2,scaffold_3,scaffold_4,scaffold_5,scaffold_6,scaffold_7,scaffold_8 scaffold_1,scaffold_2,scaffold_3,scaffold_4,scaffold_5,scaffold_6,scaffold_7,scaffold_8,scaffold_9,scaffold_10 ``` **三角形layout** ```bash # y, xstart, xend, rotation, color, label, va, bed .5, .025, .625, 60, , Grape, top, grape.bed .2, .2, .8, 0, , Peach, top, peach.bed .5, .375, .975, -60, , Cacao, top, cacao.bed # edges e, 0, 1, grape.peach.anchors.simple e, 1, 2, peach.cacao.anchors.simple e, 0, 2, grape.cacao.anchors.simple ``` --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. 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