利用核酸序列估算分歧時間

前期準備

使用phylosuite對葉綠體基因提取，比對，串聯(lián)和建樹

期間發(fā)現(xiàn)的問題：

• 部分物種注釋缺失

Species accD atpA atpB atpE atpF atpH atpI cemA chlB chlL chlN clpP ilvH matK ndhK petA petB petD petG psbA psbA_copy2 psbD psbE psbF psbI psbJ psbK psbL psbM psbN psbT psbZ rpl14 rpl16 rpl2 rpl20 rpl22 rpl23 rpl32 rpl33 rpl36 rpoA rpoB rpoC1 rpoC2 rps11 rps12 rps12_copy2 rps12_copy3 rps14 rps15 rps16 rps18 rps19 rps2 rps3 rps4 rps7 rps7_copy2 rps8 ycf1 ycf2 ycf3 ycf4

加黑基因去除，去除后共計57種基因

1 tre文件處理

modeltest中發(fā)現(xiàn)使用modelfinder查找模型速度很慢，可能是數(shù)據(jù)問題，把之前的數(shù)據(jù)去除重復序列后重新mafft，再串聯(lián)和modelfinder，速度上升很多
分析過程發(fā)現(xiàn)重復序列，于是刪除 重復序列
外類群選擇：根據(jù)實驗選擇

• 去除枝長信息

  cat species.tre |sed -r 's/:[0-9\.]+//g' |sed -r 's/\)[0-9\.]+/)/g'>species_formated.tre
  

• 查找分化時間-http://timetree.org/

• 添加分化時間，單位是100Mya

  可以使用去掉枝長等信息的tre文件，用figtree打開，給分化枝加顏色信息，再打開去除掉figtree添加的枝長信息，即可對照生成合適的tre文件，注意應在第一行寫入物種數(shù)和樹數(shù)，空格分開

  81 1
  (Ectocarpus_siliculosus_FP102343,(((((Conocephalum_salebrosum_MT023022,Wiesnerella_denudata_NC_050394)'>1.203<1.626',(Dumortiera_hirsuta_NC_039590)),Reboulia_hemisphaerica_MK477551),Marchantia_emarginata_subsp__cuneiloba_LC749600)'<2.152>1.190',(((Andreaea_regularis_NC_070057,(Eosphagnum_rigescens_KU725456,((Flatbergium_novo_caledoniae_KU725454,Flatbergium_sericeum_KU725458),(((((((((((((GZ1_GZ1__GZ1,HBD_2_HBD_2__HBD_2_r_ds_from),(MT2_MT2__MT2,TC2_2__Contigs__TC2_2_)),Sphagnum_centrale_KU725466),Sphagnum_palustre_KU726621),Sphagnum_magellanicum_KU725440),MT1_MT1__MT1),Sphagnum_papillosum_KU725474),HNZW_HNZW__HNZW),(HND_2_HND_2__HND_2_r_ds_from,(Sphagnum_austinii_KU725462,Sphagnum_portoricense_KU725469))),Sphagnum_australe_KU725452),((((((((GZ3_GZ3__GZ3_3_r_ds_from,GZ4_GZ4__GZ4_3_r_ds_from),Sphagnum_capillifolium_KU725443),(HNXS_HNXS__HNXS,Sphagnum_fuscum_KU725465)),(Sphagnum_bartlettianum_KU725444,Sphagnum_rubellum_KU725473)),(HNJY_2_HNJY_2__HNJY_2__ads_fro,XJX_XJX__XJX)),((JX1_JX1__JX1,tc3_tc3__tc3),Sphagnum_subnitens_KU725472)),(Sphagnum_aongstroemii_KU725451,(Sphagnum_girgensohnii_KU725439,(Sphagnum_rubiginosum_KU725449,sc2_sc2__sc2)))),((((HNCY_2_HNCY_2__HNCY_2__ads_fro,cbcy_cbcy__CBCY),(Sphagnum_squarrosum_KU725464,sc11_sc11__sc11)),Sphagnum_teres_KU725438),Sphagnum_wulfianum_KU725459))),(((((((GZ2_GZ2__GZ2,GZ5_GZ5__GZ5),Sphagnum_cuspidatum_KU725471),Sphagnum_majus_KU725470),(((Sphagnum_angustifolium_KU725442,Sphagnum_fallax_KU725463),Sphagnum_balticum_KU725468),(Sphagnum_obtusum_KU725445,Sphagnum_recurvum_KU725467))),Sphagnum_riparium_KU725448),Sphagnum_contortum_KU725460),((((((GZ6_GZ6__GZ6,TC1_Contigs_1_TC1_5),((YN5__4_YN5__4__Contigs__YN5__4,yn2_yn2__yn2),yn1_yn1__yn1)),HNHY_HNHY__HNHY),(Sphagnum_lenense_KU725446,Sphagnum_orientale_KU725447)),((Sphagnum_falcatulum_KU725441,Sphagnum_tumidulum_KU725455),Sphagnum_lescurii_KU725457)),Sphagnum_pylaesii_KU725461))),(Sphagnum_compactum_KU725453,Sphagnum_strictum_KU725450))))'<3.804>3.550'),((((Ditrichum_rhynchostegium_LC716918,Trematodon_longicollis_LC761299),Fissidens_protonematicola_LC761303),Timmiella_anomala_LC761301),((Entosthodon_attenuatus_NC_058543,Physcomitrium_patens_NC_005087),Funaria_hygrometrica_NC_058544))'<2.248>1.217'),(Anthoceros_angustus_AB086179,(Azolla_filiculoides_MF177094,Azolla_rubra_MF177091)'<0.172>0.022'))))；
  

2 fas序列保存為phylip_paml格式

trimal -in 86.mafft.fas -out 86.trimal.fas -automated1 -phylip_paml

3 ctl文件處理

• ctl文件處理

  seed = -1
         seqfile = input.phy
        treefile = input.tree
         outfile = out.txt
  
           ndata = 1
         seqtype = 0  * 0: nucleotides; 1:codons; 2:AAs
         usedata = 3    * 0: no data; 1:seq like; 2:use in.BV; 3: out.BV
           clock = 2    * 1: global clock; 2: independent rates; 3: correlated rates
         RootAge =   * safe constraint on root age, used if no fossil for root.
  
           model = 7    * 0:JC69, 1:K80, 2:F81, 3:F84, 4:HKY85
           alpha = 0.5    * 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: .1  .1  .1  .1 .01 .5  * auto (0 or 1) : times, musigma2, rates, mixing, paras, FossilErr
  
           print = 1
          burnin = 10000
        sampfreq = 5
         nsample = 30000
  
  *** Note: Make your window wider (100 columns) before running the program.
  

報錯：

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

問題原因：tree文件或序列文件有問題（大概是tre文件有問題，重新跑樹）
問題解決：原因為在跑完樹后對樹修剪，導致部分node自帶括號。重新跑樹加時間后解決

代碼片段_通過PAML中的CODEML模塊計算dnds的過程以及踩坑

(((((Conocephalum_salebrosum_MT023022,Wiesnerella_denudata_NC_050394),Dumortiera_hirsuta_NC_039590),Reboulia_hemisphaerica_MK477551),Marchantia_emarginata_subsp__cuneiloba_LC749600),((((((Ditrichum_rhynchostegium_LC716918,Trematodon_longicollis_LC761299),Fissidens_protonematicola_LC761303),Timmiella_anomala_LC761301),((Entosthodon_attenuatus_NC_058543,Physcomitrium_patens_NC_005087),Funaria_hygrometrica_NC_058544)),(Andreaea_regularis_NC_070057,(Eosphagnum_rigescens_KU725456,((Flatbergium_novo_caledoniae_KU725454,Flatbergium_sericeum_KU725458),(((((((((((((GZ1_GZ1__GZ1,HBD_2_HBD_2__HBD_2_r_ds_from),(MT2_MT2__MT2,TC2_2__Contigs__TC2_2_)),Sphagnum_centrale_KU725466),Sphagnum_palustre_KU726621),Sphagnum_magellanicum_KU725440),MT1_MT1__MT1),Sphagnum_papillosum_KU725474),HNZW_HNZW__HNZW),(HND_2_HND_2__HND_2_r_ds_from,(Sphagnum_austinii_KU725462,Sphagnum_portoricense_KU725469))),Sphagnum_australe_KU725452),((((((((GZ3_GZ3__GZ3_3_r_ds_from,GZ4_GZ4__GZ4_3_r_ds_from),Sphagnum_capillifolium_KU725443),(HNXS_HNXS__HNXS,Sphagnum_fuscum_KU725465)),(Sphagnum_bartlettianum_KU725444,Sphagnum_rubellum_KU725473)),(HNJY_2_HNJY_2__HNJY_2__ads_fro,XJX_XJX__XJX)),((JX1_JX1__JX1,tc3_tc3__tc3),Sphagnum_subnitens_KU725472)),(Sphagnum_aongstroemii_KU725451,(Sphagnum_girgensohnii_KU725439,(Sphagnum_rubiginosum_KU725449,sc2_sc2__sc2)))),((((HNCY_2_HNCY_2__HNCY_2__ads_fro,cbcy_cbcy__CBCY),(Sphagnum_squarrosum_KU725464,sc11_sc11__sc11)),Sphagnum_teres_KU725438),Sphagnum_wulfianum_KU725459))),(((((((GZ2_GZ2__GZ2,GZ5_GZ5__GZ5),Sphagnum_cuspidatum_KU725471),Sphagnum_majus_KU725470),(((Sphagnum_angustifolium_KU725442,Sphagnum_fallax_KU725463),Sphagnum_balticum_KU725468),(Sphagnum_obtusum_KU725445,Sphagnum_recurvum_KU725467))),Sphagnum_riparium_KU725448),Sphagnum_contortum_KU725460),((((((GZ6_GZ6__GZ6,TC1_Contigs_1_TC1_5),((YN5__4_YN5__4__Contigs__YN5__4,yn2_yn2__yn2),yn1_yn1__yn1)),HNHY_HNHY__HNHY),(Sphagnum_lenense_KU725446,Sphagnum_orientale_KU725447)),((Sphagnum_falcatulum_KU725441,Sphagnum_tumidulum_KU725455),Sphagnum_lescurii_KU725457)),Sphagnum_pylaesii_KU725461))),(Sphagnum_compactum_KU725453,Sphagnum_strictum_KU725450))))‘>3.550<3.804’)),Anthoceros_angustus_AB086179));