NCBI BLAST (Part B): Comparing Sequences

Open NCBI Nucleotide

in another browser window to work through this tutorial side by side.

Comparing Sequences with BLAST

1 of 8You have now used BLAST to identify an unknown sequence of nucleotides.

Now let's compare sequences.

For this example we will again look at human mitochondrial DNA, but this time we will compare three different homo sapiens:

  • "Modern" human
  • Neanderthal
  • Denisova

We will use the reference sequences for the mitochondria for these three organisms and compare them using BLAST.

In the Nucleotide database, search for:

mitochondrion[ti] AND human[orgn]

mitochondrion[ti] AND human[orgn]

Then, because we are looking for the best quality sequences we can find, use the Source databases limit on the left of your screen to limit to RefSeq (curated) records.

refseq limit

Please use the Sort by Default order to follow along with the exercise.

Comparing Sequences with BLAST

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The results are 3 RefSeq records for human mitochondria sequences: One from modern humans, one from Neanderthal, and one from Denisova ("Homo sp. Altai").

You can click on each record to learn more about each, but, when you are ready, return to the summary display of all three sequences, then use the link under Analyze these sequences in the right panel to Run BLAST.


This link feature from Nucleotide copies the record numbers into the BLAST query box.

Comparing Sequences with BLAST

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You should now be on the Standard Nucleotide BLAST page and your three accession numbers should populate the Enter Query Sequence box.

Because we want to align these sequences, click the box, "Align two or more sequences" and move the accession numbers "NC_011137.1" "NC_013993.1" to the new box.

BLAST align 3 sequences

This will compare the sequence in the first box (the modern human - NC_012920.1) with the other two (Neanderthal - NC_011137.1 and Denisova "Homo sp. Altai" - NC_013993.1).

Leave the other options at their default settings and click BLAST.

Comparing Sequences with BLAST

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Hopefully you won't be surprised that the results page shows two alignments against the modern human sequence: one for "Homo sapiens neanderthalensis mitochondrion, complete genome" and one for "Homo sp. Altai mitochondrion, complete genome."

What is interesting is in the Alignments view, which you can see by clicking on the Alignments tab.

alignments tab

If you scroll down or use the "Next" and "Previous" navigation, you will see that there are two tables:

  • The first comparing modern human with Neanderthal, and 
  • the second comparing modern human to Denisova ("Homo sp. Altai").
Before going further, please check that you have the Alignment view options at the top set to Pairwise with dots for entities, and that the CDS feature is selected:

alignment view options

With these display options, where there is a difference between the two sequences, the different base(s) are shown in the Subject line. Where there is no difference, a dot appears in the Subject line.

highlighting where alignments don't match

Comparing Sequences with BLAST

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The CDS display option means that, directly from this screen, you can see the sequence changes that may result in amino acid changes.

Scroll down and find a section with CDS data:

amino acid change

The CDS data appears as two lines: One above, showing the amino acid translation for the Query (modern human) sequence; and one below, showing the amino acid translation for the Subject (ancient human) sequence.

Comparing Sequences with BLAST

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Note the section starting around base 3301 in the Query (modern human) sequence as compared to Neanderthal. this section shows CDS data.

Comparing the modern human mitochondrial sequence to the Neanderthal sequence, answer these questions:

A base difference around 3800 in the modern human sequence (an "A" versus a "G") results in a difference in amino acids. In modern humans, it is a T (threonine). In Neanderthal, it was a(n):

There is a base difference around 3308 in the Query (modern human) sequence that has an effect. What seems to be the effect?

Comparing Sequences with BLAST

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Scroll down to the second table to view this same section comparing modern human to the Denisova ("Homo sp. Altai") sequence.

Looking at this same region around 3308 in the human (query) sequence, do modern humans differ from Denisova here, like the difference we saw with Neanderthal?

Comparing Sequences with BLAST

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You have reached the end of this tutorial on BLAST.

You might have guessed from all of the options on the screens, that there's a lot more to learn about BLAST. This exercise gave you a taste of a couple of things BLAST can do. To learn more about BLAST, check out the NCBI playlist on the NLM YouTube channel.

Want more examples? See Teaching NCBI Resources Through Case Studies "Five Examples for NCBI BLAST."

Close both windows to end the Guide.

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