Any help with the error? I suspect this because the fasta file is too big (~1.75 gb).

import Bio
from Bio.Align.Applications import MuscleCommandline
in_file = "sequences.fasta"
muscle_exe = "/location/muscle3.8.31_i86win32.exe"
cmd_line = MuscleCommandline(muscle_exe, input = in_file, out = "sequences.aln", clw=" ")
print(cmd_line)
stdout, stderr = cmd_line()

/location/muscle3.8.31_i86win32.exe -in sequences.fasta -out sequences -clw

ApplicationError                          Traceback (most recent call last)
Cell In [4], line 8
      6 cmd_line = MuscleCommandline(muscle_exe, input = in_file, out = "sequences.aln", clw=" ")
      7 print(cmd_line)
----> 8 stdout, stderr = cmd_line()

File c:\Users\unico\AppData\Local\Programs\Python\Python311\Lib\site-packages\Bio\Application\__init__.py:574, in AbstractCommandline.__call__(self, stdin, stdout, stderr, cwd, env)
    571     stderr_arg.close()
    573 if return_code:
--> 574     raise ApplicationError(return_code, str(self), stdout_str, stderr_str)
    575 return stdout_str, stderr_str

ApplicationError: Non-zero return code 2 from '/location/muscle3.8.31_i86win32.exe -in sequences.fasta -out sequences.aln -clw', message 'MUSCLE v3.8.31 by Robert C. Edgar'