3A Introduction to Phylogenetics - NU-CPGME/sl_workshop_2024 GitHub Wiki
July / August, 2024
Developed by:
Egon A. Ozer, MD PhD ([email protected])
Ramon Lorenzo Redondo, PhD ([email protected])
During this part of the workshop we will introduce basic concepts needed to perform phylogenetic analysis. We will run various steps to perform an exercise that will help us understand the general steps needed to complete phylogenetic and phylodynamics analyses.
First, we need to activate the phylogenetics environment
micromamba deactivate
micromamba activate phylogeneticsNavigate to the phylo_data directory in demo_data
cd ~/sl_workshop_2024/demo_data/phylo_dataImagine that you are studying the molecular evolution of SARS-CoV-2. After a huge surge of SARS-CoV-2 cases during the winter of 2021-2022 due to the appearance of the SARS-CoV-2 Omicron lineages, reports indicate that new lineages of this variant are spreading. These lineages, BA.4 and BA.5, seem to be spreading rapidly and you want to analyze their characteristics, population dynamics, and geographical origin. We will use full genome sequences from all viruses designated as BA.4 through April 30th 2022 to study this lineage.
After understanding the basic concepts of Multiple Sequence Alignment, we are going to generate an alignment as a first step of our analysis. We will use a subset of BA.4 SARS-CoV-2 full genome sequences that were publicly available by April 30th 2022 from GISAID. To speed up the alignment, we'll include the SARS-CoV-2 reference genome sequence:
mafft \
--auto \
--thread -1 \
--keeplength \
--addfragments SARSCoV2_BA4.fasta \
CoV2RefSeq.fasta \
> SARSCoV2_BA4.sequences_aligned.fasta
To compare well-resolved alignments with more difficult ones, we will also generate another alignment with a highly variable virus, HIV-1, using sequences dowloaded from the HIV sequence database https://www.hiv.lanl.gov/content/index. We will analyze the highly variable Envelope (receptor binding protein) gene.
High variability alignment
mafft \
--auto \
--thread -1 \
HIV1_CON_2021_env_DNA.fasta \
> HIV1_CON_2021_env_DNA_aligned.fasta
Visualize each alignment with Seaview. 
When viewing the SARSCoV2_BA4.sequences_aligned.fasta alignment in Seaview, remove the reference sequence "NC_045512.2" by selecting it, then using Edit --> Delete Sequence(s) in the menu bar to remove. Then File --> Save to save the edited sequence.
Now that we have covered the theoretical background behind phylogenetic tree inference, we are going to practice using the ML method.
Generate an ML tree using the alignment from Exercise 1. To save time, we will be providing the model rather than letting iqtree2 determine it for us and we will also not run bootstraps:
iqtree2 \
-s SARSCoV2_BA4.sequences_aligned.fasta \
-T AUTO \
-m TIM2+F+IClick here for the command to use if you want to use the ModelBuilder and bootstrap options instead (SLOWER!)
iqtree2 \
-s SARSCoV2_BA4.sequences_aligned.fasta \
-T AUTO \
-B 1000We will visualize the tree with FigTree 
figtree SARSCoV2_BA4.sequences_aligned.fasta.treefileOutput if it's still taking too long: SARSCoV2_BA4.sequences_aligned.fasta.treefile
(hCoV-19/Scotland/QEUH-3D9F5B6/2022:0.0000010000,(((((((((((((((((((hCoV-19/Germany/RP-RKI-I-740001/2022:0.0000334430,hCoV-19/SouthAfrica/NCV1151/2022:0.0000668889)24:0.0000010000,((hCoV-19/Denmark/DCGC-455749/2022:0.0000010000,hCoV-19/Denmark/DCGC-469344/2022:0.0000334430)50:0.0000010000,((hCoV-19/SouthAfrica/NICD-N39420/2022:0.0000334430,hCoV-19/SouthAfrica/NCV1121/2022:0.0001337897)33:0.0000010000,((hCoV-19/SouthAfrica/NICD-N38907/2022:0.0000334430,hCoV-19/SouthAfrica/NCV1108/2022:0.0000687791)100:0.0000010000,hCoV-19/Denmark/DCGC-493496/2022:0.0001003378)99:0.0000334430)18:0.0000010000)22:0.0000010000)64:0.0000010000,(hCoV-19/Denmark/DCGC-504706/2022:0.0000010000,hCoV-19/Denmark/DCGC-504119/2022:0.0000010000)100:0.0001003378)92:0.0000010000,hCoV-19/Denmark/DCGC-476838/2022:0.0000334430)99:0.0000668889,((((((((((((((((((((((((((((((((hCoV-19/SouthAfrica/SU-NHLS_4263/2022:0.0000010000,((hCoV-19/SouthAfrica/NCV1140/2022:0.0000334430,((((hCoV-19/SouthAfrica/NICD-N39107/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N39351/2022:0.0000668889)97:0.0000010000,(hCoV-19/SouthAfrica/NICD-N38270/2022:0.0000010000,(hCoV-19/SouthAfrica/NICD-N38535/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N38546/2022:0.0000668889)100:0.0001672446)61:0.0001337897)42:0.0000334430,(hCoV-19/Austria/LB-R00069-S356/2022:0.0000010000,hCoV-19/SouthAfrica/1068246/2022:0.0000010000)68:0.0000010000)41:0.0000010000,hCoV-19/SouthAfrica/NICD-N38527/2022:0.0000668889)5:0.0000010000)13:0.0000010000,hCoV-19/SouthAfrica/NICD-N38930/2022:0.0000334430)5:0.0000010000)4:0.0000010000,hCoV-19/SouthAfrica/NICD-N39414/2022:0.0000668889)5:0.0000010000,hCoV-19/SouthAfrica/NICD-N39146/2022:0.0000334430)4:0.0000010000,hCoV-19/SouthAfrica/NICD-N38872/2022:0.0000010000)8:0.0000010000,((((hCoV-19/SouthAfrica/NCV1208/2022:0.0000668889,(hCoV-19/SouthAfrica/NICD-N39172/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N38894/2022:0.0000334430)90:0.0000010000)100:0.0000334430,hCoV-19/SouthAfrica/NCV1201/2022:0.0000334430)52:0.0000010000,(hCoV-19/SouthAfrica/NICD-N38520/2022:0.0000668889,hCoV-19/SouthAfrica/NICD-N39302/2022:0.0000334430)35:0.0000010000)62:0.0000010000,(hCoV-19/Germany/BY-RKI-I-738206/2022:0.0000010000,hCoV-19/Germany/BY-RKI-I-749395/2022:0.0000334430)99:0.0000334430)97:0.0000334430)7:0.0000010000,((hCoV-19/SouthAfrica/NICD-N39203/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N38491/2022:0.0000010000)89:0.0000010000,(hCoV-19/SouthAfrica/NICD-N39242/2022:0.0000334430,((hCoV-19/SouthAfrica/NICD-N38557/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N39179/2022:0.0000317744)77:0.0000010000,hCoV-19/SouthAfrica/NICD-N38876/2022:0.0000334430)78:0.0000010000)98:0.0000010000)98:0.0000668889)11:0.0000010000,hCoV-19/England/PHEP-YYR3JSU/2022:0.0000010000)3:0.0000010000,(hCoV-19/SouthAfrica/CERI-KRISP-K040014/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N38014/2022:0.0000010000)99:0.0000668889)1:0.0000010000,hCoV-19/SouthAfrica/NICD-N38570/2022:0.0000010000)1:0.0000010000,hCoV-19/SouthAfrica/NICD-N39362/2022:0.0000010000)1:0.0000010000,hCoV-19/SouthAfrica/NCV1061/2022:0.0000334430)1:0.0000010000,hCoV-19/SouthAfrica/NICD-N39411/2022:0.0000668889)1:0.0000010000,hCoV-19/SouthAfrica/NCV1090/2022:0.0000668889)4:0.0000010000,hCoV-19/SouthAfrica/CERI-KRISP-K039713/2022:0.0000334430)0:0.0000010000,((hCoV-19/SouthAfrica/NICD-N38684/2022:0.0000334430,hCoV-19/SouthAfrica/NICD-N39067/2022:0.0000010000)86:0.0000010000,hCoV-19/SouthAfrica/CERI-KRISP-K039815/2022:0.0001745755)95:0.0000334430)0:0.0000010000,(hCoV-19/SouthAfrica/NICD-N39281/2022:0.0000334430,hCoV-19/SouthAfrica/NICD-N39354/2022:0.0000334430)96:0.0000334430)1:0.0000010000,hCoV-19/SouthAfrica/NICD-N37963/2022:0.0000334430)1:0.0000010000,(hCoV-19/SouthAfrica/NCV1156/2022:0.0000334430,hCoV-19/SouthAfrica/NCV1050/2022:0.0000010000)99:0.0000668889)2:0.0000010000,(hCoV-19/SouthAfrica/NICD-N39151/2022:0.0000334430,hCoV-19/SouthAfrica/NICD-N39400/2022:0.0000010000)100:0.0001003378)3:0.0000010000,hCoV-19/SouthAfrica/NICD-N38124/2022:0.0000334430)4:0.0000010000,hCoV-19/SouthAfrica/NHLS-UCT-GS-BH84/2022:0.0000010000)2:0.0000010000,(hCoV-19/SouthAfrica/NICD-N39252/2022:0.0000668889,hCoV-19/SouthAfrica/NICD-N37699/2022:0.0000334430)95:0.0000334430)4:0.0000010000,hCoV-19/SouthAfrica/NCV1187/2022:0.0000668889)8:0.0000010000,((((hCoV-19/SouthAfrica/NICD-N38008/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N38040/2022:0.0000010000)37:0.0000010000,hCoV-19/SouthAfrica/NICD-N37689/2022:0.0000010000)36:0.0000010000,hCoV-19/SouthAfrica/NICD-N39212/2022:0.0000334430)32:0.0000010000,hCoV-19/SouthAfrica/NICD-N38517/2022:0.0000010000)99:0.0000668889)3:0.0000010000,hCoV-19/SouthAfrica/NICD-N39405/2022:0.0001337897)5:0.0000039871,hCoV-19/SouthAfrica/NICD-N39438/2022:0.0000334430)8:0.0000010000,(hCoV-19/SouthAfrica/NCV1088/2022:0.0001017049,hCoV-19/SouthAfrica/CERI-KRISP-K040021/2022:0.0000668889)97:0.0000668889)2:0.0000010000,((((hCoV-19/USA/NJ-GBW-EWR000359/2022:0.0000668889,(hCoV-19/SouthAfrica/NICD-N39380/2022:0.0000668889,hCoV-19/SouthAfrica/CERI-KRISP-K039683/2022:0.0000347264)96:0.0000334430)3:0.0000010000,(((((hCoV-19/SouthAfrica/NCV1002/2022:0.0000010000,(hCoV-19/SouthAfrica/NICD-N38525/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N38672/2022:0.0000668889)95:0.0000334430)4:0.0000010000,((hCoV-19/SouthAfrica/NCV1193/2022:0.0000668889,hCoV-19/SouthAfrica/NICD-N39347/2022:0.0000668889)95:0.0000334430,hCoV-19/SouthAfrica/NICD-N39299/2022:0.0001003378)10:0.0000010000)2:0.0000010000,(((hCoV-19/SouthAfrica/NICD-N39202/2022:0.0000668889,hCoV-19/SouthAfrica/NICD-N39287/2022:0.0000668889)8:0.0000010000,hCoV-19/SouthAfrica/NCV1204/2022:0.0000334430)5:0.0000010000,(hCoV-19/SouthAfrica/NICD-N38648/2022:0.0000010000,(hCoV-19/SouthAfrica/NICD-N38576/2022:0.0000334430,hCoV-19/SouthAfrica/NCV1177/2022:0.0000334430)16:0.0000010000)2:0.0000010000)2:0.0000010000)1:0.0000010000,hCoV-19/SouthAfrica/NICD-N39301/2022:0.0000010000)4:0.0000010000,hCoV-19/Spain/IB-HUSE-05054/2022:0.0001031083)2:0.0000010000)2:0.0000010000,((hCoV-19/SouthAfrica/NCV1008/2022:0.0000334430,hCoV-19/SouthAfrica/CERI-KRISP-K038878/2022:0.0001672446)3:0.0000010000,hCoV-19/SouthAfrica/NICD-N38691/2022:0.0000010000)2:0.0000010000)2:0.0000010000,((hCoV-19/Netherlands/NH-AUMC-018612/2022:0.0000010000,hCoV-19/Austria/LB-R00071-S094/2022:0.0000010000)90:0.0000010000,hCoV-19/Austria/LB-R00072-S329/2022:0.0000334430)97:0.0000334430)2:0.0000010000)2:0.0000010000,((hCoV-19/Scotland/QEUH-3C0E7AE/2022:0.0001003378,hCoV-19/SouthAfrica/NICD-N38149/2022:0.0000334430)10:0.0000010000,(hCoV-19/SouthAfrica/CERI-KRISP-K040039/2022:0.0000321460,hCoV-19/SouthAfrica/CERI-KRISP-K039666/2022:0.0000694018)14:0.0000010000)2:0.0000010000)41:0.0000334430,((((((hCoV-19/SouthAfrica/NCV986/2022:0.0000668889,(hCoV-19/SouthAfrica/NICD-N39192/2022:0.0001013711,hCoV-19/SouthAfrica/NICD-N39236/2022:0.0000668889)99:0.0000334430)99:0.0000010000,(hCoV-19/SouthAfrica/CERI-KRISP-K039706/2022:0.0000334430,hCoV-19/SouthAfrica/NICD-N39514/2022:0.0001672446)99:0.0000010000)99:0.0000334430,hCoV-19/SouthAfrica/SU-NHLS_4729/2022:0.0001031801)18:0.0000010000,((((((hCoV-19/England/LSPA-3DD24AB/2022:0.0000000000,hCoV-19/England/LSPA-3DD0FD9/2022:0.0000000000):0.0000000000,hCoV-19/England/LSPA-3DD04BC/2022:0.0000000000):0.0000010000,hCoV-19/England/LSPA-3D312CE/2022:0.0000010000)90:0.0000010000,hCoV-19/England/LSPA-3D74E8F/2022:0.0000668889)90:0.0000010000,hCoV-19/England/LSPA-3D864F1/2022:0.0000334430)99:0.0000668889,((((hCoV-19/SouthAfrica/NICD-N38044/2022:0.0000010000,hCoV-19/SouthAfrica/NCV1045/2022:0.0000010000)99:0.0000010000,hCoV-19/SouthAfrica/NICD-N38098/2022:0.0000010000)100:0.0000668889,hCoV-19/SouthAfrica/NICD-N38888/2022:0.0000334430)6:0.0000010000,((hCoV-19/SouthAfrica/NCV1147/2022:0.0001337897,hCoV-19/SouthAfrica/NICD-N38618/2022:0.0000334430)64:0.0000010000,(hCoV-19/SouthAfrica/NICD-N39062/2022:0.0000010000,hCoV-19/SouthAfrica/NICD-N39113/2022:0.0001024015)33:0.0000010000)7:0.0000010000)10:0.0000010000)20:0.0000010000)31:0.0000010000,((hCoV-19/Denmark/DCGC-503974/2022:0.0000010000,hCoV-19/Denmark/DCGC-503775/2022:0.0000010000)100:0.0001003378,hCoV-19/SouthAfrica/CERI-KRISP-K039717/2022:0.0001373976)37:0.0000010000)46:0.0000010000,(hCoV-19/SouthAfrica/CERI-KRISP-K039919/2022:0.0001337897,hCoV-19/USA/NY-NYULH6630/2022:0.0000681695)98:0.0000668889)40:0.0000010000)28:0.0000334430,((hCoV-19/France/IDF-HMN-22042150668/2022:0.0000334430,((hCoV-19/Belgium/ULB-IBC_CV8142614476/2022:0.0001003378,hCoV-19/Belgium/MBLG-CTMAPF98541832/2022:0.0000334430)11:0.0000010000,hCoV-19/Belgium/MBLG-CTMAPF33795443/2022:0.0000334430)11:0.0000010000)99:0.0000668889,((((((((hCoV-19/Austria/LB-R00071-S177/2022:0.0000010000,hCoV-19/Austria/LB-R00071-S178/2022:0.0000010000)45:0.0000010000,((hCoV-19/Austria/LB-R00072-S316/2022:0.0000010000,hCoV-19/Austria/LB-R00072-S343/2022:0.0000010000)22:0.0000010000,hCoV-19/Austria/LB-R00071-S306/2022:0.0000334430)13:0.0000010000)13:0.0000010000,(((hCoV-19/Austria/LB-R00072-S340/2022:0.0000010000,hCoV-19/Austria/LB-R00071-S155/2022:0.0000010000)16:0.0000010000,hCoV-19/Austria/LB-R00072-S362/2022:0.0000010000)13:0.0000010000,hCoV-19/Austria/LB-R00072-S346/2022:0.0000010000)13:0.0000010000)31:0.0000316045,hCoV-19/Austria/LB-R00071-S303/2022:0.0000010000)14:0.0000010000,hCoV-19/Austria/LB-R00072-S361/2022:0.0000010000)6:0.0000010000,hCoV-19/Austria/LB-R00072-S337/2022:0.0000010000)16:0.0000010000,(hCoV-19/Austria/LB-R00072-S349/2022:0.0000010000,((hCoV-19/Austria/LB-R00072-S335/2022:0.0000334430,hCoV-19/Austria/LB-R00071-S292/2022:0.0000010000)23:0.0000010000,hCoV-19/Austria/LB-R00071-S158/2022:0.0000010000)21:0.0000010000)21:0.0000010000)99:0.0000668889,hCoV-19/SouthAfrica/CERI-KRISP-K039909/2022:0.0000010000)99:0.0000668889)38:0.0000010000)11:0.0000010000,(((((hCoV-19/SouthAfrica/NICD-N39515/2022:0.0000010000,(hCoV-19/SouthAfrica/NICD-N37688/2022:0.0000010000,((hCoV-19/SouthAfrica/NICD-N39169/2022:0.0000682434,hCoV-19/SouthAfrica/NICD-N39335/2022:0.0000010000)30:0.0000010000,hCoV-19/SouthAfrica/NICD-N39406/2022:0.0000010000)24:0.0000010000)8:0.0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We will apply simple concepts of coalescence and phylodynamics to analyze population dynamics and the origin of the BA.4 lineage of SARS-CoV-2. We will use the tree inferred in Exercise 2. We will use TreeTime to perform this analysis.
- Use the ML tree and metadata including dates to generate a temporal tree and coalescence analysis using ML approach.
treetime \
--confidence \
--relax 1.0 0.5 \
--aln SARSCoV2_BA4.sequences_aligned.fasta \
--tree SARSCoV2_BA4.sequences_aligned.fasta.treefile \
--dates SARSCoV2_BA4.metadata.tsv \
--coalescent skyline \
--clock-filter 4 \
--clock-rate 0.0008 \
--clock-std-dev 0.0004 \
--branch-length-mode marginal \
--outdir timetree_out- Perform ML ancestral state reconstruction to analyze phylogeography.
treetime mugration \
--tree timetree_out/timetree.nexus \
--states SARSCoV2_BA4.metadata.tsv \
--attribute countryWe will visualize the tree with FigTree
figtree With this final visualization we can estimate the most likely origin and expansion pattern of BA.4.
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