I have a protein of interest which is ~300 amino acids in length. I also have around 40 short sequences (all 9 amino acids in length); these are all very different from each other. I would like to perform multiple pairwise alignments to see if these sequences match up with (or have high sequence identity similarity) any areas in the protein of interest.

Since the short 9-a.a. sequences are very heterogeneous, they will have similarities in different regions of the protein of interest. I would like to know if it is possible to perform the alignments for all 40 sequences in one step, rather than using COBALT 40 times (and checking each of the short sequences against the reference sequence individually).

Please let me know if the description of my problem is not clear enough, I would appreciate any help in identifying a way to do this.

  • $\begingroup$ Hi and welcome to Bio.SE! It sounds like you are reinventing BLAST. I'd be careful about aligning those short regions. I don't see a problem with running 40 pairwise alignments if you can run the pairwise alignment programmatically. $\endgroup$
    – James
    Nov 14, 2019 at 13:40
  • $\begingroup$ Possible duplicate of Do all multiple sequence alignments employ global alignment algorithms? $\endgroup$
    – David
    Nov 14, 2019 at 14:22
  • $\begingroup$ This appears to be a bioinformatics question rather than one about biology. —— Please take the time to take the tour and then go through the help pages starting with How to Ask questions effectively on this site. Thanks! 😊 $\endgroup$
    – tyersome
    Nov 14, 2019 at 19:23

3 Answers 3


What you want to perform is commonly called a multiple sequence alignment. As @Wayne_Yux said, the first step is to put all of your protein sequences in a single fasta file. You can then use one of several online tools to apply different alignment algorithms to your protein sequence set.

A popular sequence alignment algorithm is Clustal, which progressively builds a multiple sequence alignment from all of the pairwise alignments. genome.jp hosts a web-based alignment tool that allows you to choose from Clustal and other alignment types (1). For alignment of many small sequences to a single larger sequence, you should use the "SLOW/ACCURATE" option (2). After you upload your fasta (3) and execute multiple alignment (4), an aln file will be generated which you can download (5).

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You can then take this aln file and upload it to a different tool that allows you to visualize alignments. Personally, I like ESPript, which gives color-coded alignments in pdf or image formats. Because you want to compare many sequences, it will probably be necessary to change the "Alignments output layout" parameters, e.g. change "Gap between blocks" to a smaller number to fit all of your sequences in one frame.


In a multiple sequence alignment, the algorithms will attempt to align the sequences along their length (global alignment). What you need is a local alignment approach with a high penalty for base mismatch.

The E-INS-i algorithm in MAFFT might provide the desired functionality. Select the Advanced Settings and there:

  1. Strategy -> E-INS-i algorithm

  2. Align unrelated segments -> Leave gappy regions

Depending on how heterogeneous your sequences are, you might need to fine-tune other settings as well. Without the data, it is impossible to provide a more detailed guide.


You can store the 40 sequences in a fasta file and then use blastp to align them all at once to your reference sequence.

After that, you can inspect the alignment hits and see wether they fit your quality expectations


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