1. Genetic Research Using Bioinformatics: LESSON 9:
Analyzing DNA Sequences and DNA Barcoding
PowerPoint slides to accompany
Using Bioinformatics:
Genetic Research
Chowning, J., Kovarik, D., Porter, S., Grisworld, J., Spitze, J., Farris, C., K. Petersen, and T. Caraballo.  Using Bioinformatics: Genetic Research.  Published Online October 2012. figshare.

2. 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

3. 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

4. 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

5. 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’
3’- T A C T G C C T A G T C G – 5’
Sequence #2:
Bottom (“R”)
Bioinformatics tools like BLAST can be used to compare the sequences from the two strands.
Image Source: Wikimedia Commons

6. Analyzing DNA Sequences
Day One:
1. Obtain two chromatograms for each sample.
Sequence #1:
Top Strand
Sequence #2:
Bottom Strand
A T G A C G G A T C A G C
T A C T G C C T A G T C G
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.
Day Three:
6. Translate the sequence to check for stop codons.
ATGCCGTAA
M P STOP
7. Use BLAST to identify origin of sequence.
8. Use BOLD to confirm identity and make phylogenetic tree.
Image Source: NCBI, FinchTV, BOLD.

7. Viewing DNA Sequences with FinchTV
Image Source: FinchTV

8. DNA Peaks Can Vary in Height and Width
Image Source: FinchTV

9. 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.

10. 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

11. Background “Noise” May Be Present
Image Source: FinchTV

12. The Beginning and Ends of Sequences Are Likely To Be Poor Quality
Image Source: FinchTV

13. 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

14. Analyzing DNA Sequences
Day One:
1. Obtain two chromatograms for each sample.
Sequence #1:
Top Strand
Sequence #2:
Bottom Strand
A T G A C G G A T C A G C
T A C T G C C T A G T C G
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.
Day Three:
6. Translate the sequence to check for stop codons.
ATGCCGTAA
M P STOP
7. Use BLAST to identify origin of sequence.
8. Use BOLD to confirm identity and make phylogenetic tree.
Image Source: NCBI, FinchTV, BOLD.

15. Transcription and Translation Begin at the Start Codon
5’- A T G A C G G A T G A G C – 3’
3’- T A C T G C C T A C T C G – 5’
Sequence #1:
Sequence #2:
Reading Frame M T D Q

16. There Are Six Potential Reading Frames in DNA
5’- A T G A C G G A T G A G C – 3’
3’- T A C T G C C T A C T C G – 5’
Sequence #1:
Sequence #2:
Reading Frame M T D Q
Reading Frame +2
Reading Frame +3
Reading Frame -2
Reading Frame -1
Reading Frame -3

17. Frame-Shifts, Amino Acid Changes, and Stop Codons
M D G STOP
Reading Frame +2
5’- A T G G A C G G A T G A G – 3’
Accidental insertion of an extra “G” when editing
Reading Frame M T G E
5’- A T G A C G G A T G A G C – 3’
3’- T A C T G C C T A C T C G – 5’
Sequence #1:
Sequence #2: