Hi Pariya Soltani Nezhad , I suggest Clustal Omega for the Alligment - https://www.ebi.ac.uk/Tools/msa/clustalo/

If you don't have experience with shell script, I suggest you split your fasta files using the FASTA Split tool in Galaxy.

Use a document reader to count the number of lines (I suggest Notepad++), and calculate how many files you will need to separate your large file into smaller files with 4000 lines (2000 sequences - fasta header + sequence). Go to https://usegalaxy.org/ , find the GENOMIC FILE MANIPULATION tab, click on FASTA/FASTQ, click on Split Fasta, upload the complete file, put Split mode : Split into a number of chunks, and select the number needed to have 4000 lines per file, Execute!

Hope it helps!

Tnx Wesley Elias Bhering Barrios ,

I did use Clustal Omega for the Alligment , but the file exceeds its maximum permitted size of 10485760 bytes.

Then, I did use Galaxy and with document reader counted the number of lines (more than 175000000 for multipel file and about 70000 for single sequence), and Go to https://usegalaxy.org/ , but couldn't Split into chunks, ....

could you help me more?

Pariya Soltani Nezhad ,

please check

https://molbiol-tools.ca/Genomics.htm

You Can try :

PGAweb

https://www.frontiersin.org/articles/10.3389/fmicb.2018.01910/full

Tnx...