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reassortment-analysis

README.md

Reassortment analysis on H5N1 2.3.4.4b strains from the US, including the strains from the March-April 2024 outbreak in dairy cattle

This analysis uses TreeSort: https://github.com/flu-crew/TreeSort.

File structure

  • treesort-analysis.log: a TreeSort-generated reassortment report.
  • HA.annotated.treesort.d2.5.tre: HA phylogenetic tree with reassortment annotations on branches (the rea tag on the branches can be used to visualize reassortment events in FigTree).
  • requirements.txt: a list of Python packages required for this analysis. You can install all by running pip install -r requirements.txt.
  • EPI_ids.txt: lists the GISAID EPI identifiers for the seqeuences used in this analysis.
  • process-wgs.py: a Python script that can be used for processing a combined file of GISAID and cattle-outbreak sequences published in this work, to align, trim, and infer phylogenetic trees for each segment.
  • descriptor_H5N1_2344b_HA.csv: a TreeSort descriptor file that provides paths to trees and alignments.
  • run-iqtree.py: a script that runs IQ-Tree (iqtree should be installed and be callable as iqtree) and reverts the changes that IQ-Tree performs on taxa names (e.g., '|' -> '_').
  • treetime-root.py: a script that uses TreeTime to root a given tree based on the molecular clock constraints.

Step-by-step reassortment analysis:

  1. Combine the GISAID sequences (Epi IDs can be found in EPI_ids.txt) with the IRMA sequences from this analysis in a joint file called 'GISAID_NVSL_combined.fasta'

  2. Run python process-wgs.py. This will align, standardize, trim to cds, and build trees for each gene segment.

  3. Root the HA tree by running python treetime-root.py HA.iqtree.tre HA.final.aln

  4. mv HA.final.aln.rooted.tre HA.iqtree.rooted.tre

  5. Install TreeSort from https://github.com/flu-crew/TreeSort.

  6. Run treesort -i descriptor_H5N1_HA.csv -o HA.annotated.treesort.d2.5.tre --dev 2.5. TreeSort will identify reassortment events along the HA tree and will give a segment-by-segment report. The tree with reassortment annotations will be saved in HA.annotated.treesort.d2.5.tre. The TreeSort report and the annotated tree from this analysis are saved as treesort-analysis.log and HA.annotated.treesort.d2.5.tre respectively.