ETE for phylogenetic analysis

http://www.manongjc.com/detail/50-qamdejytlfikukk.html
http://blog.genesino.com/2016/07/ete/
http://www.360doc.com/content/18/1124/08/52645714_796869344.shtml

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ETE能做什么

A Python framework for the construction, analysis and visualization of trees.

一、安装和使用

conda安装

# Install Minconda  (you can ignore this step if you already have Anaconda/Miniconda)
wget http://repo.continuum.io/miniconda/Miniconda-latest-Linux-x86_64.sh -O Miniconda-latest-Linux-x86_64.sh
bash Miniconda-latest-Linux-x86_64.sh -b -p ~/anaconda/
export PATH=~/anaconda/bin:$PATH;

# Install ETE
conda install -c etetoolkit ete3 ete3_external_apps

# Check installation
ete3 version
ete3 build check

github源码安装

wget https://github.com/etetoolkit/ete/archive/master.zip -O ete3.20160719.zip
unzip ete3.20160719.zip
python setup.py install
yum install python-six.noarch
ete3 upgrade-external-tools

二、ETE运行

1. 输入序列

输入序列为标准的fasta格式文件，名字可以为任意形式
如果需要在序列名字中区分物种信息，fasta序列名需满足 SpeciesCode_SequenceName例如HUMAN_p53 = HUMAN, p53。可以通过参数--spname-delimiter指定使用其它字符作为分隔符。

2. ete预先定义了多个流程用以完成从原始fasta序列到后续进化树生成的各个步骤。

运行以下命令可以列出系统自带的流程及其解释

ete3 build workflows genetree

3. 使用既定流程最简单运行

-w指定所用的流程；
-a指定输入序列；
--tools-dir指定安装的外部程序的路径；

ete3 build \
  -w standard_fasttree \
  -a diTPS.prot.fa \
  -o standard_fasttree \
  --tools-dir /root/.etetoolkit/ext_apps-latest/

4. 自己定制流程

获取可以定制的各部分命令

ete3 build apps

选择预定义好的模块，如tree builders: phyml_default_bootstrap, aligners: mafft_einsi, model testers: pmodeltest_full_slow, alg cleaners: trimal_gappyout.
流程定制模板: 顺序为aligner-trimmer-model_tester-builder
totally 4 parts included as stated above, multiple sequence alignment, trimming MSA results, select best model, use appropriate softwares to build tree.
- represents command separator
none represents skipping related operations
基于我们的选择定制的流程mafft_einsi-trimal_gappyout-pmodeltest_full_slow-phyml_default_bootstrap

ete3 build \
  -w mafft_einsi-trimal_gappyout-pmodeltest_full_slow-phyml_default_bootstrap \
  -a diTPS.prot.fa -o custom_phymltree

-w可以接受多个流程(空格分开)，进而得到不同的比对工具、处理方式和建树工具输出的多个结果，可以通过ete3 compare比较这些结果的吻合度, 比如 Robinson-Foulds距离等。

ete3 compare \
  -r newtree1.nwq \
  -t "tree2.nw tree3.nw tree4.nw" \
  --unrooted

# Tree file can be got using find
find custom_phymltree -name *.nw

定制不同的分析模块
查看已有分析模块的定义

ete3 build show phyml_default

[phyml_default]
   _desc = Phyml tree using +G+I+F, 4 classes and aLRT branch supports. Default models JTT/GTR
   _app = phyml
   _aa_model = JTT
   -nt_model = GTR
      --pinv = e
     --alpha = e
  --nclasses = 4
          -o = tlr
          -f = m
 --bootstrap = -2

修改部分定义获得新的模块

ete3 build show phyml_default >customized.config

## 修改后的customized.config
            
[phyml_bootstrap_100]
        _desc = Phyml tree using +G+I+F, 4 classes and aLRT branch supports. Default models JTT/GTR
         _app = phyml
    _aa_model = JTT
    -nt_model = GTR
       --pinv = e
      --alpha = e
   --nclasses = 4
           -o = tlr
           -f = m
  --bootstrap = 100
 [trimal_auto]
          _desc = trimal alignment cleaning using auto algorithm
          _app  = trimal
  -automated1 = 

## 使用新定义的模块
ete3 build -a diTPS.prot.fa --clearall -o phyml_bootstrap_100 -w
mafft_einsi-trimal_auto-none-phyml_bootstrap_100 -c customized.cfg --cpu 5

5. 氨基酸比对指导核苷酸比对的进化树构建 (要求氨基酸序列与核苷酸序列名字一一对应，核苷酸序列可以含有终止密码子，最终获得的核苷酸比对序列存储在`*.used_alg.fa`文件中。)

ete3 build -a diTPS.prot.fa -n diTPS.nucl.fa -o aa2nt 
   -w standard_fasttree --clearall --nt-switch-threshold 0.9
   -C 20

使用预先比对好的序列, 使用none代替aligner

ete3 build -a diTPS.prot.aln.fa -w none-none-none-fasttree
   -o manual_alg --clearall

设置树的根节点

from ete3 import Tree

tree = Tree('tree.nw')

root = 'one_node_name'
tree.set_outgroup(root)

#use mid-point as root
mid = tree.get_midpoint_outgroup()
tree.set_outgroup(mid)

tree.write('tree.rooted.nw')
tree.render('tree.rooted.pdf')

问题解决

ETE: cannot connect to X server 如果程序运行出现错误ETE: cannot connect to X server则安装Xvfb，并运行 xvfb-run ete3取代ete3, 后面的代码不变。
```
yum install xorg-x11-server-Xvfb.x86_64
xvfb-run ete3 build -w standard_fasttree -a diTPS.prot.fa -o standard_fasttree 
```

ETE: cannot connect to X server (solve in python script or jupyter ref)

Install xvfbwrapper using pip install xvfbwrapper

# Add the following 4 lines at the beginning of python code 
# or the first cell in Jupyter
from xvfbwrapper import Xvfb

vdisplay = Xvfb()
vdisplay.start()

# launch stuff inside virtual display here
# other python codes here

# Add this line at the end of python code
# or the last cell in Jupyter
vdisplay.stop()

External applications directory are not found 指定ETE使用的工具的安装路径；一般发生在普通用户使用根用户编译的ETE时。
```
--tools-dir /root/.etetoolkit/ext_apps-latest/
```

Tree annotation

# A virtual X-server XVFB is used in case you do not have X-server
from xvfbwrapper import Xvfb

vdisplay = Xvfb()
vdisplay.start()

# launch stuff inside virtual display here

#vdisplay.stop()

from ete3 import Tree, faces, TreeStyle, NodeStyle
from ete3 import ClusterTree, RectFace, AttrFace, ProfileFace, TextFace
from ete3.treeview.faces import add_face_to_node
import pandas as pd
import numpy as np
import colorsys

The most simple way of showing a tree.

t = Tree()
t.populate(7,names_library=['A','B','C','D','E','F','G'])
## %%liline is used for showing plots in ipythonnotebook.
## t.render(file_name="tree.pdf") # will save tree into pdf file
t.render(file_name="%%inline")

Get the randomly generated tree in newick format and save to a string variable which can be read using Tree() function.

t_str = t.write(outfile=None, format=0)
t_str

'(((B:1,A:1)1:1,(G:1,F:1)1:1)1:1,(E:1,(D:1,C:1)1:1)1:1);'

t = Tree(t_str)
ts = TreeStyle()
ts.show_leaf_name = True
ts.show_branch_length = True
ts.show_branch_support = True
t.render(file_name="%%inline", tree_style=ts)

Get the randomly generated tree in newick format and save to file which can also be read using Tree() function.

t.write(outfile="tree.nw", format=0)
t = Tree("tree.nw")
ts.mode = "c"
ts.arc_start = -180 # 0 degrees = 3 o'clock
ts.arc_span = 180
t.render(file_name="%%inline", w=500, tree_style=ts)

设置根节点、叶节点和中间节点的属性

ts = TreeStyle()
ts.show_leaf_name = True
ts.show_branch_length = True
ts.show_branch_support = True

# Draws nodes as small red spheres of diameter equal to 10 pixels
for n in t.traverse():  # Traverse each node and set attribute for each type of nodes
    if n.is_leaf(): # Decide if leaf node
        nstyle = NodeStyle()
        nstyle["shape"] = "sphere"
        nstyle["size"] = 10
        nstyle["fgcolor"] = "darkred"
        n.set_style(nstyle)
    else:
        nstyle = NodeStyle()
        nstyle["shape"] = "square"
        nstyle["size"] = 15
        nstyle["fgcolor"] = "orange"
        n.set_style(nstyle)        

t.img_style["size"] = 30
t.img_style["fgcolor"] = "blue"

t.render(file_name="%%inline", w=500, tree_style=ts)

修改节点的名字

t = Tree(t_str)

nameMap = {'A': 'American', 'B': 'Britain', 'C':'China', 
           'D':'Dutch', 'E':'Egypt','F':'France','G':'German'}

ts = TreeStyle()
ts.show_leaf_name = False
ts.show_branch_length = True
ts.show_branch_support = True

# Creates my own layout function. I will use all previously created
# faces and will set different node styles depending on the type of
# node.
def mylayout(node):   
    # If node is a leaf, add the nodes name and a its scientific
    # name
    if node.is_leaf():
        # We can also create faces on the fly
        newName = nameMap.get(node.name, node.name)
        newNameFace = faces.TextFace(newName)
        faces.add_face_to_node(newNameFace, node, column=1, aligned=True)

        # Sets the style of leaf nodes
        node.img_style["size"] = 12
        node.img_style["shape"] = "sphere"
        node.img_style["fgcolor"] = "blue"
    #If node is an internal node
    else:
        # Sets the style of internal nodes
        node.img_style["size"] = 1
        node.img_style["shape"] = "circle"
        node.img_style["fgcolor"] = "darkred"

ts.layout_fn = mylayout

t.img_style["size"] = 30
t.img_style["fgcolor"] = "black"

t.render(file_name="%%inline", w=600, tree_style=ts)

不同的节点设置不同的背景色，树枝的颜色

t = Tree(t_str)

nameMap = {'A': 'American', 'B': 'Britain', 'C':'China', 
           'D':'Dutch', 'E':'Egypt','F':'France','G':'German'}

colorMap = { 'American': '#ACFFFF',
             'Britain': '#ACACFF',
             'China': '#ACACAC',
             'Dutch': '#59ACAC',
             'Egypt': '#5959AC',
             'France': '#595959',
             'German': '#065959'}

ts = TreeStyle()
ts.show_leaf_name = False
ts.show_branch_length = True
ts.show_branch_support = True

# Creates my own layout function. I will use all previously created
# faces and will set different node styles depending on the type of
# node.
def mylayout(node):   
    #Change branch color
    node.img_style["hz_line_color"] = 'orange'  # change horizontal branch color 
    node.img_style["vt_line_color"] = 'red' # Change vertical branch color

    # If node is a leaf, add the nodes name and a its scientific
    # name
    if node.is_leaf():
        # We can also create faces on the fly
        newName = nameMap.get(node.name, node.name)
        newNameFace = faces.TextFace(newName)
        faces.add_face_to_node(newNameFace, node, column=1, aligned=True)

        # Sets the style of leaf nodes
        node.img_style["size"] = 12
        node.img_style["shape"] = "sphere"
        node.img_style["fgcolor"] = "blue"
        node.img_style["bgcolor"] = colorMap[newName]

        node.img_style["hz_line_color"] = 'blue'  # change branch color 
    #If node is an internal node
    else:
        # Sets the style of internal nodes
        node.img_style["size"] = 1
        node.img_style["shape"] = "circle"
        node.img_style["fgcolor"] = "darkred"

ts.layout_fn = mylayout

t.img_style["size"] = 30
t.img_style["fgcolor"] = "black"

t.render(file_name="%%inline",tree_style=ts)

树+热图（自定义颜色+列名字）

自定义热图函数，同时该函数也支持替换或新增节点的名字。

nameFace = AttrFace("name", fsize=12) #Set leaf node attribute

def setup_heatmap(tree, tree_style, header, center_value=0.0, nameMap ={}, nameLabel = '',
                  color_up=0.7, color_down=0.2, color_center="white"):
    DEFAULT_COLOR_SATURATION = 0.5
    BASE_LIGHTNESS = 0.7
    def gradient_color(value, max_value, saturation=0.5, hue=0.1):    
        def rgb2hex(rgb):
            return '#%02x%02x%02x' % rgb
        def hls2hex(h, l, s):
            return rgb2hex( tuple(map(lambda x: int(x*255), 
                          colorsys.hls_to_rgb(h, l, s))))

        lightness = 1 - (value * BASE_LIGHTNESS) / max_value
        return hls2hex(hue, lightness, DEFAULT_COLOR_SATURATION)

    # Calculate max gradient value from the ClusterTree matrix
    maxv = abs(center_value - tree.arraytable._matrix_max)
    minv = abs(center_value - tree.arraytable._matrix_min)
    if center_value <= tree.arraytable._matrix_min:
        MAX_VALUE = minv + maxv
    else:
        MAX_VALUE = max(maxv, minv)

    # Add heatmap colors to tree
    cols_add_before_heat = 0
    if nameMap:
        cols_add_before_heat = 1
    for lf in tree:
        if nameMap:
            longNameFace = faces.TextFace(nameMap.get(lf.name, lf.name))
            lf.add_face(longNameFace, column=0, position="aligned")

        for i, value in enumerate(getattr(lf, "profile", [])):
            if value > center_value:
                color = gradient_color(abs(center_value - value), MAX_VALUE, 
                                       hue=color_up)
            elif value < center_value:
                color = gradient_color(abs(center_value - value), MAX_VALUE, 
                                       hue=color_down)
            else:
                color = color_center
            lf.add_face(RectFace(20, 20, color, color), position="aligned", 
                        column=i+cols_add_before_heat)
            # Uncomment to add numeric values to the matrix
            #lf.add_face(TextFace("%0.2f "%value, fsize=5), position="aligned", column=i)
        lf.add_face(nameFace, column=i+cols_add_before_heat+1, position="aligned")

    if nameMap and nameLabel:
        nameF = TextFace(nameLabel, fsize=7)
        #nameF.rotation = -90
        tree_style.aligned_header.add_face(nameF, column=0)
    # Add header 
    for i, name in enumerate(header):
        nameF = TextFace(name, fsize=7)
        nameF.rotation = -90
        tree_style.aligned_header.add_face(nameF, column=i+cols_add_before_heat)
 #-------------END setup_heatmap----------------------------------------------

读入矩阵（可把文后的测试矩阵存储到文件中读入）

矩阵需满足三个条件：

矩阵为TAB键分割，第一行是每列的名字
矩阵每一行第一列为行名字，与树的节点对应
矩阵可以存储与一个文件中，也可以是如下的字符串

data = pd.read_table("matrix", header=0, index_col=0)
data.index.name = "#Names"  #修改第一行的名字使其符合ETE的要求
data_mat = data.to_csv(None, sep="\t", float_format="%.2f")
header = list(data.columns.values)  #获取列的名字用于标记

data

	col1	col2	col3	col4	col5	col6	col7
#Names
---	---	---	---	---	---	---	---
A	-1.23	-0.81	1.79	0.78	-0.42	-0.69	0.58
B	-1.76	-0.94	1.16	0.36	0.41	-0.35	1.12
C	-2.19	0.13	0.65	-0.51	0.52	1.04	0.36
D	-1.22	-0.98	0.79	-0.76	-0.29	1.54	0.93
E	-1.47	-0.83	0.85	0.07	-0.81	1.53	0.65
F	-1.04	-1.11	0.87	-0.14	-0.80	1.74	0.48
G	-1.57	-1.17	1.29	0.23	-0.20	1.17	0.26

data_mat

'#Names\tcol1\tcol2\tcol3\tcol4\tcol5\tcol6\tcol7\nA\t-1.23\t-0.81\t1.79\t0.78\t-0.42\t-0.69\t0.58\nB\t-1.76\t-0.94\t1.16\t0.36\t0.41\t-0.35\t1.12\nC\t-2.19\t0.13\t0.65\t-0.51\t0.52\t1.04\t0.36\nD\t-1.22\t-0.98\t0.79\t-0.76\t-0.29\t1.54\t0.93\nE\t-1.47\t-0.83\t0.85\t0.07\t-0.81\t1.53\t0.65\nF\t-1.04\t-1.11\t0.87\t-0.14\t-0.80\t1.74\t0.48\nG\t-1.57\t-1.17\t1.29\t0.23\t-0.20\t1.17\t0.26\n'

header

['col1', 'col2', 'col3', 'col4', 'col5', 'col6', 'col7']

调用函数绘制热图

t = ClusterTree(t_str, data_mat)

ts = TreeStyle()
ts.show_leaf_name = False
ts.show_branch_length = True
ts.show_branch_support = True

setup_heatmap(t, ts, header, center_value=0, color_up=0.9, color_down=0.3, color_center="white")

t.render(file_name="%%inline", tree_style=ts)

绘制热图时修改Layout

def mylayout_only(node):   
    #Change branch color
    node.img_style["hz_line_color"] = 'orange'  # change horizontal branch color 
    node.img_style["vt_line_color"] = 'red' # Change vertical branch color

    # If node is a leaf, add the nodes name and a its scientific
    # name
    if node.is_leaf():
        newName = nameMap.get(node.name)
        node.img_style["size"] = 12
        node.img_style["shape"] = "sphere"
        node.img_style["fgcolor"] = "blue"
        node.img_style["bgcolor"] = colorMap[newName]        
        node.img_style["hz_line_color"] = 'blue'  # change branch color 
    #If node is an internal node
    else:
        # Sets the style of internal nodes
        node.img_style["size"] = 1
        node.img_style["shape"] = "circle"
        node.img_style["fgcolor"] = "darkred"

t = ClusterTree(t_str, data_mat)

ts = TreeStyle()
ts.show_leaf_name = False
ts.show_branch_length = True
ts.show_branch_support = True
ts.layout_fn = mylayout_only

setup_heatmap(t, ts, header, center_value=0, color_up=0.9, color_down=0.3, 
              color_center="white", nameMap=nameMap, nameLabel="Full")

t.render(file_name="%%inline", tree_style=ts)

测试矩阵

## 矩阵为TAB键分割
## 矩阵每一行第一列为行名字，与树的节点对应
## 矩阵可以存储于一个文件中，也可以是如下的字符串
matrix = """
#Names\tcol1\tcol2\tcol3\tcol4\tcol5\tcol6\tcol7
A\t-1.23\t-0.81\t1.79\t0.78\t-0.42\t-0.69\t0.58
B\t-1.76\t-0.94\t1.16\t0.36\t0.41\t-0.35\t1.12
C\t-2.19\t0.13\t0.65\t-0.51\t0.52\t1.04\t0.36
D\t-1.22\t-0.98\t0.79\t-0.76\t-0.29\t1.54\t0.93
E\t-1.47\t-0.83\t0.85\t0.07\t-0.81\t1.53\t0.65
F\t-1.04\t-1.11\t0.87\t-0.14\t-0.80\t1.74\t0.48
G\t-1.57\t-1.17\t1.29\t0.23\t-0.20\t1.17\t0.26
"""

产生颜色的辅助函数，给定一个列表，这个函数会自动返回一个字典包含每个字段对应的颜色。

def hex2rgb(hexcolor):
    return [(hexcolor>>16) & 0xff, (hexcolor>>8) & 0xff, hexcolor & 0xff]

def rgb2hex(rgbcolor):
    r, g, b = rgbcolor
    rgb = hex((r << 16) + (g << 8) +b)[2:].upper()
    zero = '0'* (6-len(rgb))
    return '#'+zero+rgb
#----------------------------------
def generateColor(labelL):
    labelL = list(set(labelL))
    labelL.sort()
    colorD = {}

    r = 255
    g = 255
    b = 255
    len_label = int(len(labelL) / 3 + 1)
    step = int(250 / len_label)

    cnt = 1
    for labels in labelL:
        if cnt % 3 == 1:
            r = r - step 
        elif cnt % 3 == 2:
            g = g -step
        else:
            b = b - step
        cnt += 1
        color = rgb2hex([r, g, b])
        colorD[labels] = color
    return colorD

clan_colorD = generateColor(nameMap.values())
clan_colorD

{'American': '#ACFFFF',
 'Britain': '#ACACFF',
 'China': '#ACACAC',
 'Dutch': '#59ACAC',
 'Egypt': '#5959AC',
 'France': '#595959',
 'German': '#065959'}

带有Support value的Newick树，供测试不同的属性使用

nw = """
(((Dre:0.008339,Dme:0.300613)1.000000:0.596401,
(Cfa:0.640858,Hsa:0.753230)1.000000:0.182035)1.000000:0.106234,
((Dre:0.271621,Cfa:0.046042)1.000000:0.953250,
(Hsa:0.061813,Mms:0.110769)1.000000:0.204419)1.000000:0.973467);

关闭Virtual X-server

vdisplay.stop()