Genetic Research Using Bioinformatics: LESSON 9:

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

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’ 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

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.

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 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.

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

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

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 +1 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: