Multipe Sequence Alignments:
ClustalW
Solution

1. clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln

2. clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap0 -gapopen=0
   clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap1 -gapopen=1
   clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap2 -gapopen=2
   clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap20 -gapopen=20
   clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap200 -gapopen=200
   clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap300 -gapopen=300
   clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap400 -gapopen=400
   clustalw -infile=1thm2_ref5.fasta -outfile=1thm2_ref5.aln.gap400 -gapopen=500
   

3. Gap penalties -gapopen=x
   Note that the alignments only change in a specific range. When the gap penalty is too high these alignments stop changing

4. Matrices The blosum matrix is especially well adapted to distantly related proteins, in which the blocks signal is weak.

5. Guide Tree : edit the original file and use the -usetree option

6. Phylogenic Tree: Do not use the guide tree, use the interactive option of clustal

7. Reference aln : Make sure the sequences are in the same order, use the option -outorder=input.