Q1
From this phrase, "The end goal is to identify every unique sequence", I am not 100% sure of what you mean by "every unique sequence". It sounds like maybe you want to group alignments of homologs and visualize them together?
If the proteins are similar enough, you might also benefit from trying BLAST to look at similar regions.
Otherwise MAFFT, Clustal, or MUSCLE are all appropriate tools to generate the alignments that you need. However I also do not know of any software aside from Geneious that has the option to align many proteins to a reference. If you were working with DNA I'm sure the stock suggestion would be to use a read aligner like bwa mem or minimap2 and to use a bam file.
Since I don't know of any software to do exactly what you would like, I wrote a small python script to run MAFFT many times for each sequence in a fasta after the first sequence, the reference.
Q2 - MAFFT/Python
In any case, here is a bit of python code that performs a MAFFT alignment of all of the sequences in a fasta file against the first sequence in the fasta file, which you have designated as the reference.
Here is the fasta file from Wikipedia. I called it seqs.fasta:
>reference
MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSTPVCPNGPGNCQVSLRDLFDRAVMVSHYIHDLSS
EMFNEFDKRYAQGKGFITMALNSCHTSSLPTPEDKEQAQQTHHEVLMSLILGLLRSWNDPLYHL
VTEVRGMKGAPDAILSRAIEIEEENKRLLEGMEMIFGQVIPGAKETEPYPVWSGLPSLQTKDED
ARYSAFYNLLHCLRRDSSKIDTYLKLLNCRIIYNNNC*
>MCHU - Calmodulin - Human, rabbit, bovine, rat, and chicken
ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTID
FPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA
DIDGDGQVNYEEFVQMMTAK*
>gi|5524211|gb|AAD44166.1| cytochrome b [Elephas maximus maximus]
LCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGTNLV
EWIWGGFSVDKATLNRFFAFHFILPFTMVALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLG
LLILILLLLLLALLSPDMLGDPDNHMPADPLNTPLHIKPEWYFLFAYAILRSVPNKLGGVLALFLSIVIL
GLMPFLHTSKHRSMMLRPLSQALFWTLTMDLLTLTWIGSQPVEYPYTIIGQMASILYFSIILAFLPIAGX
IENY
Here is a script, mafft_ref.py. Run it with python mafft_ref.py seqs.fasta:
from Bio import SeqIO
from Bio.Align.Applications import MafftCommandline
import tempfile
fasta = sys.argv[1]
reference = 0
count = 0
with tempfile.NamedTemporaryFile() as temp:
for record in SeqIO.parse(fasta, "fasta"):
if count == 0:
reference = record
else:
SeqIO.write([reference, record], temp.name, "fasta")
mafft_cline = MafftCommandline(input=temp.name)
stdout,stderr=mafft_cline()
print(stdout)
count += 1
Here is the output of python mafft_ref.py seqs.fasta printed to the terminal. Use python mafft.py prots.pep > output.txt to save the output to output.txt.
>reference
------------------MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSTPVCPNGPGNCQ
VSLRDLFDRA--------------VMVSHYIHDLSS-----EMFNEFDKRYAQGKGFITM
ALNSCHTSSLPTPEDKEQAQQTHHEVLMSLILGLLRSWNDPLYHLVTEVRGMKGAPDAIL
SRAIEIEEENKRLLEGMEMIFGQVIPGAKETEPYPVWSGLPSLQTKDEDARYSAFYNLLH
CLRRDSSKIDTYLKLLNCRIIYNNNC
>MCHU - Calmodulin - Human, rabbit, bovine, rat, and chicken
ADQLTEEQIAEFKEAFSLFDKDGDGTITTKEL------------GTVMRSL---GQNPTE
AELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDK---DGNGYISA
A---------------------------------------ELRHVMTN-----------L
GEKLTDEEVD-------EMIREADIDG-------------------DGQVNYEEFVQMM-
-----TAK------------------
>reference
------------MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSTPVCPNGPGNCQVS----
--LRDLF--------------------DRAVM----VSHYIHDLSSEMFNEFDKRYAQGK
GFITMALNSCHTSSL-------PTPEDKEQAQQTHHE-------------VLMSLILGLL
RS--WNDPLYHLVTEVRGMKGAPDAILSRAIEIEEE--------------NKRLLEGMEM
IFGQVI-------------------P-------------------GAKETE-PYPVWSGL
PSLQTKDEDARYSAFYNLLHCLRRDSSKIDTYLKLLNCRIIYNNNC
>gi|5524211|gb|AAD44166.1| cytochrome b [Elephas maximus maximus]
LCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGY----VLPWG----QMSFWGA
TVITNLFSAIPYIGTNLVEWIWGGFSVDKATLNRFFAFHFILP-----------------
-FTMVALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLGLLILILLLLLLALL
SPDMLGDPDNHM----------PADPLNTPLHIKPEWYFLFAYAILRSVPNK--LGGVLA
LFLSIVILGLMPFLHTSKHRSMMLRPLSQALFWTLTMDLLTLTWIGSQPVEYPYTIIGQM
ASI----------LYFSIILAFLPIAGXIENY--------------
References:
Some credit of mafft_ref.py to a biostars answer found here.
