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I'm trying to align sequences using MUSCLE in my own computer. To compute Percent Identity Matrix I'm trying to use Clustal2.1 like it's done in https://www.ebi.ac.uk/Tools/msa/muscle/. Even though I'm doing the same thing, I get rounded up values in my computer.

For example, this is the webtool output:

#
#
#  Percent Identity  Matrix - created by Clustal2.1 
#
#

     1: JX310755.1  100.00   92.79   65.29   63.98
     2: M97264.1     92.79  100.00   65.66   63.92
     3: AJ292230.1   65.29   65.66  100.00   66.88
     4: KM379144.1   63.98   63.92   66.88  100.00

And this is the output in my computer.

#
#
#  Percent Identity  Matrix - created by Clustal2.1 
#
#

     1: JX310755.1     100      93      65      64
     2: M97264.1        93     100      66      64
     3: AJ292230.1      65      66     100      67
     4: KM379144.1      64      64      67     100

I have both installed clustalw using sudo apt install clustalw and by downloading the tarballs from http://www.clustal.org/download/current/clustalw-2.1.tar.gz, with exact results.

The commmand I'm using is clustalw2 alignment -tree -pim.

Any thoughts on why this could be? Thanks in advance.

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Try clustal omega, instead of clustalw because it is really old. It should report to 2 dp. Variations do occur depending on how indels are treated, but this is just rounding.

You could try

alignment -tree -pim -score

The idea is to convert to absolute rather than percentage and might overcome the rounding error

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So, after a year I came out with the solution - as M__ kindly suggested, using Clustal Omega.

First, align usign muscle:

muscle -in fasta -quiet -out alignment

Then, generate percent distance matrix using clustal omega:

clustalo --infile alignment --distmat-out output --percent-id --full

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