Custom scripts and files for Han et al. (2021)

Citation

Han, A. X., Felix Garza, Z. C., Welkers, M. R., Vigeveno, R. M., Tran, N. D., Le, T. Q. M., Pham Quang, T., Dang, D. T., Tran, T. N. A., Ha, M. T., Nguyen, T. H., Le, Q. T., Le, T. H., Hoang, T. B. N., Chokephaibulkit, K., Puthavathana, P., Nguyen, V. V. C., Nghiem, M. N., Nguyen, V. K., … Russell, C. A. (2021). Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children. ELife, 10. https://doi.org/10.7554/eLife.68917.

For any questions/queries with regards to the scripts/notebooks, please email Alvin Han.

Sequencing data

All raw sequence data have been deposited at NCBI sequence read archive under BioProject Accession number PRJNA722099.

Installation

You will need Python 3 and several dependencies to run the Jupyter notebooks/scripts and reproduce our analyses. We have only ran our scripts/notebooks on MacOS. Linux should work just as well. We recommend using conda to download and install all dependencies:

1. Clone this repository.
2. cd Within_Host_H3vH1
3. conda env create -f environment.yml
4. Prior to running the notebooks/scripts: conda activate h3vh1

Running the analyses

1. Separate folders were created for the different subtypes (H3N2 and H1N1pdm09). Move the raw sequencing data files thtat you have downloaded into the respective <subtype>/data/raw subfolder.
2. Read mapping and variant calling were performed by running the SEA_<subtype>_01_NGS_Mapping.ipynb jupyter notebook.
3. For H3N2, we were able to analyse the precision of our variant calls (SEA_H3N2_02a_Variant_Precision.ipynb).
4. Phylogenetic analyses of the concatenated sequences were performed to identify samples with potential mixed infections and/or were contaminated (SEA_<subtype>_02b*_Phylogenetic_Analyses.ipynb). You may use R script, available in H3N2/scripts/ggtree001-2.R to create the Supplementary figures S9 and S10. You will need to change the working directory and output filename accordingly as stipulated in the script.
5. Within-host genetic diversity and evolutionary dynamics analyses were performed using SEA_<subtype>_03_Genetic_Diversity_Analyses.ipynb.
6. Haplotype reconstruction was performed by running H3N2/scripts/reconstruct_haplotype_wrapper.py using the following commands (for H3N2 as an example):

cd H3N2  
python ./scripts/reconstruct_haplotype_wrapper.py -v ./results/variant_call_MinCoV100_MinProp0.02_MinFreq0_ErrTol0.01.csv -f ./results/metadata_wo_mixed_infections.csv -m ./results/mapped --DMNFuncs_fpath ../python_lib/DMNFuncs.py  

8. For H1N1pdm09, transmmission bottleneck size is estimated by running SEA_H1N1pdm09_04_Transmission_Analyses.ipynb.
9. Within-host simulations was performed by running simulations/scripts/WHDEL.py. A bash script is enclosed to run multiple simulations in parallel:

cd simulations
bash WHDEL_wrapper.sh