1. Download a viewer to open and look at your sequence data. To view your
data y
1. Download a viewer to open and look at your sequence data. To view your
data you will need to download one of three viewers for your computer. The three I
recommend looking at are Finch TV, 4 Peaks, or Chromas. If you use a Mac, then you
should use 4 Peaks. It’s the best viewer for this platform and it’s free. If you use a
PC, you can choose between Finch TV or Chromas (both of which are free). Finch TV
has versions for either PC or Mac, Chromas is just for PC’s. The links for all three are
on Canvas. Please download one and learn how to use it. There are not difficult to
learn, but they will require you to spend some time to appreciate all the features of
the software.
2. Compare forward and reverse sequences. Open both your forward and
reverse sequence files. These should be complements of each other. A fast way to
compare these two is to use the BLAST feature of NCBI (GenBank) to
BLAST2sequences. This will rapidly align these and show you where they do and do
not match. Where they do not match is something you should pay attention two.
The base callings routines are not infallible and if there is a discrepancy it is likely
because the base was called incorrectly by the software on one strand. You have to
step in and make a call as to which strand is correct. Once you have done this for all
mismatches the two strands should be perfectly complementary to each other. You
will notice that the ends of the two molecules are not matching – each will have 20
bases or so that are not in the other strand. You should be able to explain why that
is.
3. Determine your haplogroup by doing a BLAST search. Once you have opened
and viewed your DNA sequence, copy and paste your sequence and follow my
previous instructions on searching this sequence against all known genetic
sequence data on GenBank using the BLAST protocol. (BLAST stands for Basic Local
Alignment Search Tool). This will help you to determine your haplogroup. This is
an important piece of information and will give you quite a bit to write about. Once
you have your haplogroup determined you can research this on the web and find
out what it means. I have posted a few articles that discuss the concept of
haplogroups. I suggest starting with the Wikipedia entry – it gives a nice short
introduction to mitochondrial haplogroups and their distribution worldwide. Also
use the URL link I provided on Canvas for determining your haplogroup.
4. Compare your DNA sequence to others. You can open up any of your
colleagues data sets (using the same software you used to open and view your
sequence data) and compare the D-loop regions. What you will most likely find is
that your sequence is different from any other randomly picked sample. Over 400
bp of D-loop sequence you likely see at least a few SNP’s (single nucleotide
polymorphisms). This is what allows scientists to use this region to fingerprint
individuals using their DNA.
An easy way to align and compare D-loop sequences is to use a program called
Clustal. One of the easiest Clustal alignments can be done on the Dolan DNA
Learning Center’s site maintained by the Cold Spring Harbor Laboratories. You can
access this BioServer Site by clicking here. Use the Sequence Server (you can enter
as a guest or create a free login by registering). Click on Create Sequence and this
will allow you to cut and paste sequences into your workspace. You can enter up to
10 sequences to compare. After they are entered you just click on the boxes of the
sequences you want to align and then click on “Compare” (with the box reading
Align: Clustal W). You will then get a computer alignment of your sequences
showing where they are identical and where they are different (colored yellow).
This can be printed out and used in your report.
Another useful feature of this site is that you can download and use hundreds of
different D-loop sequences to compare to your own. These include ancient DNA
samples, modern groups of humans, non-human primates, and even Neanderthal
mtDNA D-loop sequences. To access these just click on “Manage Groups” and drop
down the window that reads classes. I have included the help file in a separate .pdf
file.
5. Phylogenetic Tree. Once your data has been entered and you perform a Clustal
Alignment you can easily create a tree diagram that will show visually how you are
related to others based on your D-loop sequence. This is called a phylogenetic tree
and can be created by clicking on the drop down box that reads Align: Clustal W
(next to compare). Select Phylogenetic Tree and hit “Compare.” A tree diagram will
be drawn to compare your selected sequences.
These are just some suggestions. Please feel free to search the web to get other
ideas for analysis and for discussion in your report.