1. 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. http://dx.doi.org/10.6084/m9.figshare.936568 http://dx.doi.org/10.6084/m9.figshare.936568

2. Image Source: Wikimedia Commons How DNA Sequence Data is Obtained for Genetic Research Genetic Data …TTCACCAACAGGCCCACA… Extract DNA from Cells Sequence DNA Compare DNA Sequences to One Another Obtain Samples: Blood, Saliva, Hair Follicles, Feathers, Scales TTCAACAACAGGCCCAC TTCACCAACAGGCCCAC TTCATCAACAGGCCCAC GOALS: Identify the organism from which the DNA was obtained. Compare DNA sequences to each other.

3. Overview of DNA Sequencing DNA Sample Mix with primers Perform sequencing reaction …T T C A C C A A C T G G C C C A C A… DNA Sequence Chromatogram

4. Image Source: Wikimedia Commons Sequence Both Strands of DNA Sequence #1: Top Strand 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 Sequence #2: Bottom Strand

5. Compare the Two Sequences 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 #1: Top (“F”) Sequence #2: Bottom (“R”) Bioinformatics tools like BLAST can be used to compare the sequences from the two strands. Sequence #1: Top Strand Sequence #2: Bottom Strand Image Source: Wikimedia Commons

6. Viewing DNA Sequences with FinchTV Image Source: FinchTV

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

8. 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) = log 10 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.

9. 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

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

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

12. Examples of Chromatogram Data 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. #1 #2#3 Image Source: FinchTV

13. 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 +1 M T D Q

14. 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 +1 M T D Q Reading Frame +2 Reading Frame +3 Reading Frame -2 Reading Frame -1 Reading Frame -3

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