Object: Starting with two or more sequences, compare them and find the differences.
Example: In the NCBI database Nucleotide, enter the following search:
human[organism] AND mitochondrion[title]
This will search for nucleic acid sequences from humans with the word "mitochondrion" in the title. Mitochondrial DNA is often used in evolutionary comparisons because it is inherited only through the maternal lineage and has a low rate of recombination.
Limit the results to NCBI Reference Sequences by selecting the RefSeq limit under Source databases in the left-hand Filter menu. These are high-quality sequences that have been curated and annotated by NCBI staff.
There are three Reference Sequences for the mitochondrial genome in humans: one for modern humans (hom*o sapiens), one for Neanderthals (hom*o sapiens neanderthalensis), and one for Denisovans (hom*o sp. Altai).
In the right-hand discovery menu under Analyze these sequences click Run BLAST.
This will openBLASTn, Nucleotide BLAST, and automatically add the accession numbers of these Reference Sequences into the Query Sequence box.
To compare sequences, check the box next to Align two or more sequences under the Query Sequence box. To BLAST the modern human mitochondrial genome sequence (NC_012920.1) against the subject sequences of Neanderthal (NC_011137.1) and Denisovan (NC_013993.1), move the latter two accession numbers from the Query Sequence box into the Subject Sequence box using copy and paste.
Enter a job title and click BLAST, leaving the other settings at their default options.
You should see two results, in which the query sequence (modern human) is compared to one of the subject sequences, Neanderthal or Denisovan. Note that the query sequence is 99% similar to the Neanderthal sequence, and 98% similar to the Denisovan sequence.
To see how the sequences differ and what the biological significance might be:
- Go to the Alignments tab and in the Alignment view drop-down menu select Pairwise with dots for identities.
- Click the checkbox next to CDS feature.
Click on the name of the first result (hom*o sapiens neanderthalis). You should see a base-by-base comparison of the two sequences in two lines. The top line is the query sequence (modern human). In the second line, representing the subject sequence (ancient human), bases where the subject sequence is identical to the query sequence are replaced by dots, and bases where the subject sequence differs from the query sequence appear in red.
Scroll down to the first coding sequence (CDS). The CDS regions are displayed in four lines: the first line shows the amino acid translation for the query sequence (modern human) on the second line. The third line is the subject sequence (ancient human), and the one below shows the amino acid translation for the subject sequence.
Note that there are two additional amino acids, M (methionine) and P (proline), at the beginning of the protein sequence in modern humans compared to Neanderthal. This is due to the substitution of T (thymine) at position 3308 in the modern human sequence for C (cytosine) in the analogous position in the Neanderthal sequence.
Note as well that the substitution of A (adenine) at position 3334 in the modern human sequence for G (guanine) in the Neanderthal sequence results in an amino acid difference in the protein sequences. In the modern human protein sequence an I (isoleucine) replaces a V (valine) present in the Neanderthal protein sequence.
Go to "Amino acid properties" and "Amino acid properties and consequences of substitution: Valine" to investigate the biological significance of this change. Would the substitution of I for V have a large effect on protein structure or function? Does this seem to be a conservative mutation (that is, one that results in little or no change in protein structure or function) or a non-conservative mutation (that is, one that results in a significant change in protein structure or function)?
Now scroll down to the Denisovan result and look at positions 3308 and 3334 in the query sequence. Are there any differences in the Denisovan sequence at these positions?
To see how the species are related in evolutionary terms:
- Go to the Description tab and click on the Distance tree of results link.
- When the rectangle cladogram displays, go to the menu Tools > Layout and select Slanted Cladogram.
To which species, Denisovans or Neanderthals, are modern humans more closely related?
