<p>Hi, Im a begginer in MEGA software. I have 89 protein sequence for which I need to construct a <strong>phylogenetic tree</strong> using <strong>bootstrap method with 1000 replication</strong> with data set parameter with <strong>complete deletion</strong>. But I am not able to construct a tree because of <strong>3 </strong>sequence whose protein length is <strong>very less</strong> when compared to other 86 sequence. Even I tried by <strong>deleting non conserved regions </strong>in all protein sequence but still I am not able to get a tree because the size of the smaller proteins become smaller and smaller. Kindly help me out in solving this problem.</p>

Regardless of the approach or program you are using, the input for any phylogenetic estimation approach is an alignment, i.e., an inference of homology. Therefore, by necessity, your sequences **must have a shared ancestry** to even begin to infer a phylogeny. If the sequences are shorter but homologous, a multiple sequence alignment (of nucleotides or amino acids or both via a translation alignment for protein-coding sequences) ought to resolve the sequences by introducing gaps - insertions or deletions. It sounds like you're not doing this; when you say

> The 3 short protein sequence are upregulated in abiotic stresses. Is it ok if I omit the sequence because they have role in abiotic stresses?

It suggests that your dataset may consist of multiple proteins, not the same protein across samples, which is a **completely inappropriate** input for phylogenetic techniques.

In other words, your workflow would be:

1. Construct a dataset of the same locus across all samples
2. Align the amino acids or nucleotides
3. Model selection for ML analysis or NJ distance corrections/uncorrected NJ/UPGMA/etc.
4. [If you decide to use a model: With an appropriate model, any likelihood (maximum likelihood or Bayesian) approach.]
5. Bootstrapping etc. for support.

If you do have sequences with a shared history, I would follow Istvan Albert's recommendation and remove the short sequences if they are truly unalignable.