LESSON 9:
Analyzing DNA Sequences and DNA Barcoding
PowerPoint slides to accompany
Using Bioinformatics:
Genetic Research
How DNA Sequence Data is Obtained
for Genetic Research
Obtain Samples:
Blood , Saliva, Hair
Follicles, Feathers, Scales
Genetic Data
Compare
DNA
Sequences to
One Another
Extract DNA from Cells
Sequence DNA
…TTCACCAACAGGCCCACA…
TTCAACAACAGGCCCAC
TTCACCAACAGGCCCAC
TTCATCAACAGGCCCAC
GOALS:
• Identify the organism from which the DNA was obtained.
• Compare DNA sequences to each other.
Image Source: Wikimedia Commons
Overview of DNA Sequencing
Mix with primers
Perform sequencing reaction
DNA
Sample
…T T C A C C A A C T G G C C C A C A…
DNA Sequence
Chromatogram
Sequence Both Strands of DNA
Sequence #1: Top Strand
Sequence #2: Bottom Strand
A T G A C G G A T C A G C
Sequence #1:
Top Strand
T A C T G C C T A G T C G
Sequence #2:
Bottom Strand
Image Source: Wikimedia Commons
Compare the Two Sequences
Sequence #1: Top Strand
Sequence #2: Bottom Strand
Sequence #1:
Top (“F”)
5’-
A T G A C G G A T C A G C
– 3’
Sequence #2:
Bottom (“R”)
3’-
T A C T G C C T A G T C G
– 5’
Bioinformatics tools like BLAST can be used to
compare the sequences from the two strands.
Image Source: Wikimedia Commons
Analyzing DNA Sequences
Sequence #1: A T G A C G G A T C A G C
Top Strand
T A C T G C C T A G T C G
Sequence #2:
Bottom Strand
Day One:
1. Obtain two chromatograms for each sample.
2. Align the sequences with BLAST.
Day Two:
3. Visualize the chromatograms using FinchTV.
Compare BLAST alignments against base calls in
chromatogram.
Sequence #1
Sequence #2
4. Review any differences and determine which
base is most likely correct.
5. Edit and trim the DNA sequence using quality data.
ATGCCGTAA
M P STOP
Day Three:
6. Translate the sequence to check for stop codons.
7. Use BLAST to identify origin of sequence.
8. Use BOLD to confirm identity and make
phylogenetic tree.
Image Source: NCBI, FinchTV, BOLD.
Viewing DNA Sequences with FinchTV
Image Source: FinchTV
DNA Peaks Can Vary in
Height and Width
Image Source: FinchTV
Quality Values Represent
the Accuracy of Each Base Call
Quality values represent the ability of the DNA sequencing software to identify the
base at a given position.
Quality Value (Q) = log10 of the error probability * -10.
Q10 means the base has a one in ten chance (probability) of being misidentified.
Q20 = probability of 1 in 100 of being misidentified.
Q30 = probability of 1 in 1,000 of being misidentified.
Q40 = probability of 1 in 10,000 of being misidentified.
Quality Values Are Used When
Comparing Sequences
Quality values represent the ability of the DNA sequencing software to identify the
base at a given position.
Image Source: FinchTV
Background “Noise” May Be Present
Image Source: FinchTV
The Beginning and Ends of Sequences
Are Likely To Be Poor Quality
Image Source: FinchTV
Examples of Chromatogram Data
#1
#2
#3
Circle #1: Example of a series of the same nucleotide (many T’s in a row). Notice the highest peaks are
visible at each position.
Circle #2: Example of an ambiguous base call. Notice the T (Red) at position 57 (highlighted in blue) is just
below a green peak (A) at the same position. Look at the poor quality score on bottom left of screen (Q12).
An A may be the actual nucleotide at this position.
Circle #3: Example of two A’s together. The peaks look different, but are the highest peaks at these positions.
Image Source: FinchTV
Analyzing DNA Sequences
Sequence #1: A T G A C G G A T C A G C
Top Strand
T A C T G C C T A G T C G
Sequence #2:
Bottom Strand
Day One:
1. Obtain two chromatograms for each sample.
2. Align the sequences with BLAST.
Day Two:
3. Visualize the chromatograms using FinchTV.
Compare BLAST alignments against base calls in
chromatogram.
Sequence #1
Sequence #2
4. Review any differences and determine which
base is most likely correct.
5. Edit and trim the DNA sequence using quality data.
ATGCCGTAA
M P STOP
Day Three:
6. Translate the sequence to check for stop codons.
7. Use BLAST to identify origin of sequence.
8. Use BOLD to confirm identity and make
phylogenetic tree.
Image Source: NCBI, FinchTV, BOLD.
Transcription and Translation Begin
at the Start Codon
Reading Frame +1
Sequence #1:
5’-
Sequence #2:
3’-
M
T
D
Q
A T G A C G G A T G A G C
T A C T G C C T A C T C G
– 3’
– 5’
There Are Six Potential Reading Frames
in DNA
Reading Frame +3
Reading Frame +2
Reading Frame +1
Sequence #1:
5’-
M
T
D
Q
A T G A C G G A T G A G C
T A C T G C C T A C T C G
Sequence #2:
3’Reading Frame -1
Reading Frame -2
Reading Frame -3
– 3’
– 5’
Frame-Shifts, Amino Acid Changes,
and Stop Codons
Reading Frame +2
5’-
M
D
G
STOP
A T G G A C G G A T G A G
– 3’
Accidental insertion of an extra “G” when editing
Reading Frame +1
Sequence #1:
5’-
Sequence #2:
3’-
M
T
G
E
A T G A C G G A T G A G C
T A C T G C C T A C T C G
– 3’
– 5’