I'm having trouble aligning 3 sequences together - the total gene (~2500bp) and the forward and reverse sequences that are ~1000bp in length. Now I'm using Clustal X2, but the problem is that the program does complete alignment, which is useless for me right now. Could anyone recommend or suggest any parameters or methods on how to align more that 2 sequences together, but that each sequence is aligned to the first one (the total gene)? Thanks!


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  • $\begingroup$ Which kind of result do you want to have? One alignment with three sequences, or two pair-wise alignments? If the former, what would you do if one of the 1000bp sequences causes a gap in the reference sequence? $\endgroup$ – Michael Kuhn Nov 20 '12 at 9:57
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    $\begingroup$ Could you please clarify your question? The reverse strand of a sequence is not alignable to the forward strand. Also, i don't understand what you mean by complete gene, forward and reverse sequences. Do you mean the genomic sequence, the transcribed transcript and the reverse complement of that transcript? $\endgroup$ – terdon Nov 20 '12 at 10:59
  • $\begingroup$ Like the others, not sure that I understand, but if these 1000 bp sequences are perfect matches to the 2500 bp sequence then can't you generate what you want just by editing a file which shows the dsDNA sequence? Shouldn't take too long. $\endgroup$ – Alan Boyd Nov 20 '12 at 15:20

Your question is not very clear, but I think what you are trying to do is align sequencing results to a reference gene sequence.

Your total gene sequence spans ~2500 bp. It sounds like you have a forward sequence at the start of gene, reading only about 1000 bp and the reverse strand reading in 1000 bp from the end (if this is Sanger sequencing, you likely only have 800-900 bp of accurate base reads by the way). Therefore, you have a gap of about 500 bp in the middle of your gene of interest.

There are (at least) two ways to do the alignment, and I would suggest the manual method if you are trying to verify gene sequencing.

Manual Method:

  1. In a word processing document, paste in your gene-of-interest (or the recombinant gene you are trying to make), as well as the base reads from the forward strand, and the reverse complementof your reverse strand. Make sure you remove any whitespace like line breaks and spaces as this will disrupt the rest of the process.

  2. Manually select a small portion of your forward read (20-30 bp for example) and search for the same "phrase" in your gene-of-interest. If you find it, highlight it in both the sequence it came from and the gene-of-interest so that you know you have already matched them.

  3. Carry on with adjacent bases for the forward strand. Repeat for the reverse complement of the reverse strand.

Semi-Automated Method:

  1. Go to NCBI Blast, and under the heading Basic Blast, click "nucleotide blast".

  2. Check "Align 2 or more sequences."

  3. Enter your Query Sequence as your 1000 bp sequences, entered in FASTA format. FASTA format will allow you to enter multiple sequences, but if you just enter one at a time, that will work perfectly well. Results will only display one at a time anyway.

For example:

> sequence name here
  1. Enter your gene of interest as your Subject Sequence. Format does not matter here, but for consistency, I use FASTA format.

  2. Keep "Highly similar sequences (megablast)" selected. You shouldn't need to play with these parameters, they just change how the alignment method penalizes alignments for gaps and mismatches.

  3. Hit BLAST.

  4. Interpret your results. I've included an example of BLAST results below.

BLAST example output aligning two sequences.

At the top of this window is a schematic, showing exactly how much of the the query sequence aligns with the subject sequence. The longer the line, the more bases align. Similarly, the closer the colour of the line is to right of the scale (e.g., red), then the more continuous the segment is indicating a longer, continuous alignment.

Below the graphic is a table of results. Briefly, the table highlights all possible alignments that the BLAST algorithm has found, including how much coverage there was, confidence of the alignment and length of alignment coverage.

Below the results table, are the detailed results which were summarized in the table. This detailed report shows you exactly which bases align or mismatch, or any gaps. The top line is the subject sequence; the bottom is the query sequence. The other thing to keep in mind here is the Strand line. In this case, it says "Plus/Plus, meaning that the aligned sequences were the forward sense, or plus strand, of each of subject and query sequence, respectively. If you entered a reverse complement sequence instead (such as the reverse sequencing results) then this would change to "Plus/Minus".

From here, you will still need to manually line these results up to your gene of interest to determine exactly which portion of the gene is missing sequence coverage, as well as any mutations that may have cropped up in the cloning process.


Multiple sequence alignment is a common bioinformatics problem and there are many tools available to solve it.
This paper provides a good comparison of the following tools:

Thompson, et al. A comprehensive benchmark study of multiple sequence alignment methods: a current challenge and future perspectives

The paper addresses the following methods:

  • ClustalW
  • ProbCons
  • $\begingroup$ this answer may be a bit sparse, but these are the most typical solutions to the question $\endgroup$ – shigeta Aug 30 '13 at 13:06

If you want to align sequencing results over the reference sequence and check the sequences for presence of spontaneous mutations, the easiest way would be to use Sequlator.

Sequlator is a convenient because it has a pairwise alignment, reverse complement, editing and navigation functions but lacks irrelevant functions. With Sequlator one can manipulate any way existing alignments, or manually assemble sequencing reads into an alignment with the reference sequence. After elimination of poorly sequenced fragments, discrepancies with the reference sequence are revealed in the consensus of the resulting alignment. And it is free and Java-based meaning that it works on Windows, Linux and Mac computers


In Mafft, the command to align new sequences to an existing reference alignment is:

mafft --add new_sequences.fta existing_alignment.fta > output.fta

See this part of the documentation for more details. It is a nice program to use for alignment, in any case...


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