分子鐘假說

基因序列中密碼子隨著時(shí)間的推移以幾乎恒定的比例相互替換，即具有恒定的演化速率，兩個(gè)物種之間的遺傳距離將與物種的分化時(shí)間成正比。我們通常采用單拷貝基因家族中的四重簡(jiǎn)并位點(diǎn)來估算分子鐘（替換速率）以及物種間的分化時(shí)間，密碼子中的四重簡(jiǎn)并位點(diǎn)由于第三堿基不改變所編碼的氨基酸屬于中性進(jìn)化，其中中性替換速率一般用每個(gè)位點(diǎn)每年的變異數(shù)來衡量。

1. 分子鐘應(yīng)當(dāng)被看作是氨基酸或核苷酸突變的隨機(jī)性所導(dǎo)致的隨機(jī)鐘
2. 分子鐘假說允許不同蛋白質(zhì)間進(jìn)化速率不同，以不同的速率跳動(dòng)
3. 速率恒定性未必對(duì)所有物種適用

全局分子鐘模型：序列間的期望距離隨分歧時(shí)間線性增加。
局部分子鐘模型：對(duì)分支賦予不同的進(jìn)化速率

常用軟件：

image.png

分析實(shí)操

使用軟件：PAML 軟件包中的mcmctree 工具
準(zhǔn)備文件：

1. 多序列比對(duì)文件，Phylip 格式
2. 有化石時(shí)間標(biāo)定的進(jìn)化樹
3. mcmctree 配置文件

使用氨基酸序列進(jìn)行估計(jì)，使用orthofinder 的進(jìn)化樹及單拷貝基因數(shù)據(jù)做輸入。

#將orthofinder的進(jìn)化樹SpeciesTree_rooted.txt和多序列比對(duì)結(jié)果SpeciesTreeAlignment.fa拷貝過來


#重命名 
mv SpeciesTree_rooted.txt  input.tree
#fasta格式轉(zhuǎn)phylip格式
trimal -in SpeciesTreeAlignment.fa -out  Sequence.phy -phylip_paml

step1 估算位點(diǎn)替換率

mcmctree mcmctree.ctl

• mcmctree.ctl 配置文件

          seed = -1
       seqfile = ./Sequence.phy
      treefile = ./input.tree
       outfile = out

         ndata = 1
       seqtype = 2  * 0: nucleotides; 1:codons; 2:AAs
       usedata = 3    * 0: no data; 1:seq like; 2:use in.BV; 3: out.BV
         clock = 3    * 1: global clock; 2: independent rates; 3: correlated rates
       RootAge = <1.0  * safe constraint on root age, used if no fossil for root.

         model = 0    * 0:JC69, 1:K80, 2:F81, 3:F84, 4:HKY85
         alpha = 0    * alpha for gamma rates at sites
         ncatG = 5    * No. categories in discrete gamma

     cleandata = 0    * remove sites with ambiguity data (1:yes, 0:no)?

       BDparas = 1 1 0    * birth, death, sampling
   kappa_gamma = 6 2      * gamma prior for kappa
   alpha_gamma = 1 1      * gamma prior for alpha

   rgene_gamma = 2 2   * gamma prior for overall rates for genes
  sigma2_gamma = 1 10    * gamma prior for sigma^2     (for clock=2 or 3)

      finetune = 1: 0.1  0.1  0.1  0.01 .5  * auto (0 or 1) : times, musigma2, rates, mixing, paras, FossilErr

         print = 1
        burnin = 2000
      sampfreq = 2
       nsample = 20000

將生成的tmp0001.trees文件進(jìn)行修改，定根（添加一對(duì)括號(hào)即可），添加化石時(shí)間
修改生成的tmp0001.ctl文件為

seqfile = tmp0001.txt
treefile = tmp0001.trees
outfile = tmp0001.out
noisy = 3
seqtype = 2
model = 0
Small_Diff = 0.1e-6
getSE = 0
method = 1
clock = 1

再次運(yùn)行

codeml  tmp0001.ctl

step2 使用近似似然法計(jì)算分化時(shí)間

#調(diào)整mcmctree.ctl 中的rgene_gamma 第二個(gè)參數(shù)b, 使得a/b 約等于s上一步tmp0001.out文件得到的替換率， 將usedata 設(shè)置為3 再運(yùn)行

mcmctree  mcmctree.ctl

# 將生成的out.BV 文件重命名成 in.BV
mv out.BV in.BV

# 將mcmctree.ctl配置文件usedata 設(shè)置為2 ，運(yùn)行mcmctree
mcmctree  mcmctree.ctl

• 結(jié)果文件FigTree.tre