Copy and paste the entire string of nucleotide symbols, below, into the box under Enter Query Sequence.
Uncheck this box labeled "Align two more sequences:"
then scroll down and click the BLAST button:
Once your results are displayed, you will see a header followed by the results of your search. The results can be displayed in several different views, including a list of sequence "Descriptions," via a "Graphic Summary," and via a more detailed "Alignments" view.
Select the Graphic Summary by clicking on this tab:
to see a graphic summary of the top 100 results.
Click on the Descriptions tab to learn more about each of the sequences that aligned with yours.
Click on the description of the sequence to see the alignment.
For this exercise, select one of the sequences labeled, "Homo sapiens isolate PNG## haplogroup...mitochondrion, complete genome."
Clicking on a sequence will bring you to the Alignments view.
To go to the subject sequence in the Nucleotide database, there are several links from the alignment.
The first two: one in the header next to Download labeled GenBank, and another link from the Sequence ID, take you to the record for the full sequence as it was submitted (or created). Remember that our match started around base 7585. The third link, adjacent to the range (also labeled GenBank), takes you to a record displaying just the range of interest (around 7585 to 8268).
Either record might be useful, but let's look at the record for the entire sequence that was submitted, and look at our query sequence in that context.
Follow the link to the GenBank record in the Nucleotide database from your Sequence ID (MN849867.1 in this example):
[If this page insists on opening in a new browser tab, use this link instead to go to MN849867.1]
A GenBank Record2 of 3
An interesting part of a Nucleotide record is the section labeled "FEATURES." Called the "feature table," this is the part that reflects scientists' annotations -- notes on what biological features of interest are known about a sequence.
Scroll down the feature table of this mitochondrial DNA record. Definitions of some of the feature labels can be found in the GenBank Sample Record.
Two features of major interest include:
CDS = a coding sequence, or region of nucleotides that corresponds with amino acids in a protein.
gene = a region identified as a gene. A gene may include multiple sections of coding sequences, so the same nucleotide sequence (shown in a number range) may be labeled as CDS and gene.
Comparing Sequences with BLAST1 of 10
You 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
We will use the reference sequences for the mitochondria for these three organisms and compare them using BLAST.
Comparing Sequences with BLAST2 of 10
In the Nucleotide database, search for:
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.
Please use the Sort by Default order to follow along with the exercise.
Comparing Sequences with BLAST3 of 10
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.
Comparing Sequences with BLAST4 of 10
This link feature from Nucleotide copies the record numbers into the BLAST query 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.
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 BLAST5 of 10
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. There are two tables: The first comparing modern human with Neanderthal, and the second comparing modern human to Denisova ("Homo sp. Altai").
Comparing Sequences with BLAST6 of 10
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:
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.
Comparing Sequences with BLAST7 of 10
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:
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 BLAST9 of 10
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?
BONUS!: To explore what differences in amino acids might make, biologically, go to the Amino Acid Explorer and use the "Compare" feature on the left side of the screen.
For example, compare isoleucine to valine.