通过PAML中的CODEML模块计算dnds的过程以及踩坑

最近帮女朋友做毕业设计的时候用到了 PAML这个软件的codeml功能，发现网上相关的资料很少，于是把自己踩的一些坑分享一下，希望能帮到其他有相同困难的人

一、下载与安装

PAML软件下载地址

http://abacus.gene.ucl.ac.uk/software/paml4.9j.tgz

DAMBE软件下载地址

http://dambe.bio.uottawa.ca/DAMBE/dambe_install_win.aspx

二、使用方法

首先准备好你的fas文件

我们需要将fas文件转换一下格式，方法很多，我这边说两种方法，这两种方法最后得到的文件内容完全相同，只是后缀名不同

方法一：

使用python脚本转换

将你的*.fas文件与脚本放在同一目录下，执行脚本，会生成一个.phy文件

import re
with open('seven.fas', 'r') as fin:
    sequences = [(m.group(1), ''.join(m.group(2).split()))
                 for m in re.finditer(r'(?m)^>([^ 
]+)[^
]*([^>]*)', fin.read())]
with open('seven.phy', 'w') as fout:
    fout.write('%d %d
' % (len(sequences), len(sequences[0][1])))
    for item in sequences:
        fout.write('%-20s %s
' % item)

方法二：

使用DAMBE软件转换格式

1.打开DAMBE，选择 File -> Open standard sequence file -> 文件类型选择为包含 fas 类型 -> 选择你的fas文件

 2.点击 go

 3.点击 File -> save or convert sequence format  -> 选择 paml 格式

 4.手动修改 *.pml 的后缀名为 *.nuc

通过以上两个方法会得到一份 *.phy 或者 *.nuc 文件

接下来需要去除序列中的终止密码子 

 TAG,TAA,TGA

你可以全选文件内容查找替换 将  TAG/TAA/TGA 替换为 ---

也可以使用下面这个python脚本

import re

with open(r'seven.phy', 'r') as f:
    content = f.read()
    content = content.replace("TAG","---")
    content = content.replace("TAA", "---")
    content = content.replace("TGA", "---")
    # print(content)

with open('sevenend.phy', 'w') as f:
    f.write(content)

会生成一个去除过终止密码子的文件

现在将这个处理过后的序列文件*.phy与树文件、配置文件codeml.ctl三个放在  paml4.9jin 目录下

配置文件codeml.ctl内容如下可参考 一般修改前面三行即可 按顺序为序列文件名 树文件名 输出文件名

      seqfile = seven.nuc * sequence data filename
     treefile = Newick      * tree structure file name
      outfile = test.txt           * main result file name

        noisy = 0  * 0,1,2,3,9: how much rubbish on the screen
      verbose = 0  * 0: concise; 1: detailed, 2: too much
      runmode = -2  * 0: user tree;  1: semi-automatic;  2: automatic
                   * 3: StepwiseAddition; (4,5):PerturbationNNI; -2: pairwise

      seqtype = 1  * 1:codons; 2:AAs; 3:codons-->AAs
    CodonFreq = 2  * 0:1/61 each, 1:F1X4, 2:F3X4, 3:codon table

*        ndata = 5504
        clock = 0  * 0:no clock, 1:clock; 2:local clock; 3:CombinedAnalysis
       aaDist = 0  * 0:equal, +:geometric; -:linear, 1-6:G1974,Miyata,c,p,v,a
   aaRatefile = dat/jones.dat  * only used for aa seqs with model=empirical(_F)
                   * dayhoff.dat, jones.dat, wag.dat, mtmam.dat, or your own

        model = 0
                   * models for codons:
                       * 0:one, 1:b, 2:2 or more dN/dS ratios for branches
                   * models for AAs or codon-translated AAs:
                       * 0:poisson, 1:proportional, 2:Empirical, 3:Empirical+F
                       * 6:FromCodon, 7:AAClasses, 8:REVaa_0, 9:REVaa(nr=189)

      NSsites = 0  * 0:one w;1:neutral;2:selection; 3:discrete;4:freqs;
                   * 5:gamma;6:2gamma;7:beta;8:beta&w;9:betaγ
                   * 10:beta&gamma+1; 11:beta&normal>1; 12:0&2normal>1;
                   * 13:3normal>0

        icode = 0  * 0:universal code; 1:mammalian mt; 2-10:see below
        Mgene = 0
                   * codon: 0:rates, 1:separate; 2:diff pi, 3:diff kapa, 4:all diff
                   * AA: 0:rates, 1:separate

    fix_kappa = 0  * 1: kappa fixed, 0: kappa to be estimated
        kappa = 2  * initial or fixed kappa
    fix_omega = 0  * 1: omega or omega_1 fixed, 0: estimate 
        omega = .4 * initial or fixed omega, for codons or codon-based AAs

    fix_alpha = 1  * 0: estimate gamma shape parameter; 1: fix it at alpha
        alpha = 0. * initial or fixed alpha, 0:infinity (constant rate)
       Malpha = 0  * different alphas for genes
        ncatG = 8  * # of categories in dG of NSsites models

        getSE = 0  * 0: don't want them, 1: want S.E.s of estimates
 RateAncestor = 1  * (0,1,2): rates (alpha>0) or ancestral states (1 or 2)

   Small_Diff = .5e-6
    cleandata = 1  * remove sites with ambiguity data (1:yes, 0:no)?
*  fix_blength = 1  * 0: ignore, -1: random, 1: initial, 2: fixed, 3: proportional
       method = 0  * Optimization method 0: simultaneous; 1: one branch a time

* Genetic codes: 0:universal, 1:mammalian mt., 2:yeast mt., 3:mold mt.,
* 4: invertebrate mt., 5: ciliate nuclear, 6: echinoderm mt., 
* 7: euplotid mt., 8: alternative yeast nu. 9: ascidian mt., 
* 10: blepharisma nu.
* These codes correspond to transl_table 1 to 11 of GENEBANK.

在此目录下打开命令行

输入一下命令即可

codeml

 当前目录下就会出现结果文件 test.txt  以及其他文件了

在过程中我遇到过许多报错提供给大家参考一下

67 columns are converted into ??? because of stop codons

这个报错是因为没有去除文件中的终止密码子，可以参考上面的步骤去除

Error: Error in sequence data file: . in 1st seq.?.

Error: check #seqs and tree: perhaps too many '('?.

Make sure to separate the sequence from its name by 2 or more spaces.

以上报错均为你的序列文件内容/格式有问题，麻烦按照上面的步骤重新生成序列文件或者参考其他人的文件格式