Assistance Needed with Optimal ML Tree Selection and Sequence Alignment Errors in IQ-TREE2 Analysis

[azhengll]

I am currently conducting a phylogenetic analysis using MEGA11 and IQ-TREE2 and have encountered a couple of challenges.

Overview of My Workflow:

1. Multiple Sequence Alignment:
   o I retrieved 1,000 sequences from NCBI and performed a multiple sequence alignment using MEGA11.
   o After manually removing sequences with significant length discrepancies to ensure alignment quality, I saved the refined alignment file.
   o I then utilized trimAl to remove gaps, resulting in the final alignment file final.fas for phylogenetic tree construction.
2. Model Selection:
   o Using MEGA11's Best Model Finder, I determined that the most appropriate evolutionary model for my data is LG+G+I+F.
3. Bootstrap Analysis:
   o I conducted 1,000 bootstrap replicates by splitting the task into 20 parts, executing 50 bootstrap runs each with the following command:
   iqtree2 -s final.fas -m LG+G+I+F -T 4 -b 50 --prefix partX
   where X ranges from 1 to 20.

Consensus Tree Construction:
• After completing all bootstrap runs, I merged the resulting consensus tree files using:
cat part*.boottrees > alltrees

I then generated a consensus tree while excluding branches with bootstrap values below 70%:
iqtree2 -con -t alltrees -minsup 0.7
This produced the file alltrees.contree.

Challenges Encountered:

1. Selection of the Optimal ML Tree:
   o Given that I distributed the bootstrap analysis across 20 parts, I am uncertain about which ML tree should be designated as the optimal ML tree for subsequent analyses.
2. Sequence Alignment Errors When Mapping Branch Lengths:
   o I selected part1.treefile as my optimal ML tree and attempted to map branch lengths using the following command:
   iqtree2 -s final.fas -te alltrees.contree --prefix alltrees.contree
   o However, the program returned errors indicating that several taxa (e.g., 5XYO_A, AFX92969.1_endotype, etc.) do not appear in the alignment:
   ERROR: Tree taxon 5XYO_A does not appear in the alignment
   ERROR: Tree taxon AFX92969.1_endotype does not appear in the alignment
   ...
   ERROR: Tree taxa and alignment sequence do not match (see above)

Specific Questions:

1. Optimal ML Tree Selection:
   o Considering the bootstrap analysis was divided into 20 parts, which ML tree should I select as the optimal ML tree for mapping branch lengths and further analyses? Should I generate a single best ML tree from the entire dataset before splitting the bootstrap runs?
2. Resolving Sequence Alignment Errors:
   o What could be causing the discrepancy between the taxa in the consensus tree and those in the alignment file? How can I ensure that all taxa included in the tree are present in the alignment to avoid these errors?

Additional Context:
• All sequences used in the alignment are from the same dataset, and after manual curation, only sequences with consistent lengths were retained.
• The final.fas file has been successfully processed by trimAl without issues prior to running IQ-TREE2.
• I am using IQ-TREE2 version [please specify your version], and all software dependencies are properly installed.
Request for Guidance:
I would greatly appreciate your expert advice on:
• Best practices for selecting the optimal ML tree when bootstrap analyses are partitioned across multiple runs.
• Troubleshooting steps to resolve the mismatch between tree taxa and alignment sequences to successfully map branch lengths.