1. Sorry, some images removed for web posting, due to concern about copyrights.

2. ATGTTCTATCCCATTCATTTTGACGTTATTGTTGTTGGAGGAGGTCATGCTGGGACAGAAGCAGCTTTGGCTTCTGCTAGGATGCAATGTAATACGCTTTTGATTACTCATAATATTGATACTCTTG GACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACT TAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGA AGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGAT GAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCA AAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTTTCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCTTGTTATATTACCCATACTAATAACAA AACACATGAAATAGTTAGATCAAATTTATATCAGAGTCCTATGTATACAGGTTTAATAAAAGGAATAGCACCTCGGTATTGTCCATCTATAGAAGACAAAATAACTCGTTTTTCAGATCGTAATGCT CATCAAATTTTTTTAGAACCTGAAGGTTTGACAACACCTGAAGTATATCTTAATGGTATTTCTACTAGCTTACCTTTTTGTGTACAAATGCAAATGATTAAATCAATTCAGGGATTAGAGAATGCTTG TATAATTAGGCCAGGTTATGCTATTGAATACGATTTTTTTGATCCTCGTGATTTAAAATTAACATTAGAGAGTAAAATTATTTCTGGATTATTTTTTTCTGGTCAAATTAATGGTACTACTGGTTATGA AGAAGCAGCTGCCCAAGGATTATTGGCCGGAATTAATGCAGCTAGGTTTTCCAAAAATAAAGAAGGATGGTACCCTAGAAGAGATCAGGCATATTTAGGGGTGCTTGTAGATGATCTCTGTACA CATGGAACAGAAGAACCTTATCGTATGTTTACTTCGCGTGCTGAATATCGTCTGTCTTTACGTGAGGATAATGCTGATTTAAGATTAACTGAAATCGCGCGACAGTTAGGTTTGATAGATGAGTCA CGGTGGAAAGCTTTTTGTTGCAAAAAAGAAAACATTGAAAAAGAACGTCAGAGATTACGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAA CACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAAGTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAAT AAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTATAATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGAC AAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTATCACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTA ATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGGGTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAAT AATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTATGTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAAT AGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAAATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGT GTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGTGTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTC TATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACAACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTT GTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAATATTTTTTGAAAGGTGGTTTATGTCAGGAATCAAAGGCACTTCCCAAGAATATATTGGACATCATTTATATCA TCTGCAATTTGATTTAAGTACTTTTTCGTTAGTGAGCTCGGAGAATACTTCTTCATTCTGGGTATTAAATGTAGATTCGATGTTTTTTTCAATATTATTGGCTACTTTATTTTTATTAATTTTCGGTCG TTTGGCTACAGTGGCAACTTATGCGGTTCCCACAAAACTGCAAGTGTTTATTGAGTTAGTAATATTGTTTATTGATAGCAATGTAAAAGATATGTTTCATGGTAAAAATAAACTAATTGCGCCATTA TCTATGACGGTTTTTGTTTGGATTTTTTTAATGAATACCATGGATTTATTTCCCATAGATTTATTTCCTGCTATAGCTAAATTGTTAGGATTACCTGCTTTACGTGTTGTGCCGTCTGCCGATGTGAA TATCACTTCTTCGCTAGCTTTAAATGTATTTGTACTTGTTATGTACTACAATATTTACGTTAATGGCGTTCATGGTTTTATTAAAGGACTGATGTATCATCCATTTAATCATCCAACATGTATTCCTAT TAATTTTATTATTGAAGTTGTTAGTTTGTTATCTAAACCAGTATCACTTAGTCTTAGATTATTTGGCAATATGTATTCTGGTGAGTTGATTTTTATTTTGATATCTGGTTTATTACCATGGTGGGGACA ATGGGTTTTAAATTTACCATGGGCTATTTTTCATATTTTAGTCGTTACATTACAGGCTTTTATTTTTATGGTTTTAACGGTGATTTATTTATCTACAGCCCATGACTCCTGTTAAAATGAATCATGTAA TACAGAAGATTGCAGAGAGGTTATTATGGAACATTTAAATTTTGATATGTTATATATTGCTGCAGCAATAATGATGGGATTAGCAGCAATTGGAGCGGCTATCGGTATTGGCATCTTAGGTAGTAA ATTTTTAGAGGGGGCTGCACGTCAACCAGATCTCATTCCGATCCTTCGAACTCAGTTTTTTATTGTTATGGGATTGGTTGATGCAATACCAATGATTACTGTAGGTCTTGGTTTGTATGTGATGTTT TCTGCGGTTTAACAATGAAAATTAATTGCATGTATTTATTATATTGATTATTAAATATCAGGATTTGCGCTGTGAATCTTAATGCAACAATATTAGGTCAAACTATTTCATTTGTTTTGTTTGTTTGGT TTTGTATGAAGTATGTGTGGTATCCATTTATATCTATTATTGAGAAACGCCAAAAAGAAATTTCTGATAATTTAGTTTCTGCTACTCATGCCAAAACAGAATCTGAGCGTGTCAATGCTGAAGCTTT GCTTTGTTTGAGACAGGCTCGGGTCAAAGCTCAAGAAATTATAAAACAAGCAAATAAATGTAAAATGCAAATAATTAATGAAGCTAAACATGAAGCTGAAAAGGAGCAAAGTAGAATTTTATCTC AAGCGCGAGAACAGATTATTTATGAAAGAAAACGTGTTACTGACGAATTAAGAAAGCAAATTAGCGAACTTGTAATTGAGGGTACAGAGAAAGTTATAGAACATTCTATAAATGAAATGATTGAT ATAGATCTTTTAAATAATATTATTAATACGTTGTCATATAAGGATTAGATGTCTAGTATGCTTGTGGTTGCGCGTACGTATGCTCAAGCTATATTTGATATAGCTGTAGAACAGAAAAATATAAACA AGTGGAAATCAGTTCTTGATTTATTTTCTGAGATTAGTCTAAATAGACTAGTACAATCTTTATTTTTTAGATGTTTAGAACCAAAAAGATTGTCAGATATATTTATTGCTATTTGTGAAGATTACCAA AAGAAACAAGTTGATACCTTCAGTAAAAATATAATCTATATTATGGCGGAAAATAATCGTTTATTATTATTACCAATTGTATTTAAAGAGTTTACTTATTTATGTTCTATATATGTTCATACTGTAGAA ATAGAAATTATCTCTGCTTGGCCTTTGAAGTATAATCAGCTGAAAAAAATTACTGATATAATGGCTAAACGTTTATCTAAAACAGTGAATCCAGTACACAAAGTAGATAAAGATATATTGGCTGGT GTAATTATTCGTATTGGAGATACTGTGATTGATGGAAGTATACGTGGACGTATTTTTCGCTTAAATCACGTACTACAATCTTAATATTTTAACAGTAGTTAAAAGGTATAAACAAAAGATTATGCAA TTAAATTCAAATGAAATTTCAGATAATGGAGAAGTTATTATTAATGATTTAAAGTTATTTTATAATAAAGCTAGACAAACTAAAATTACAGAAGAACTTACAGAAATTGTTTCAGGAGCTTCTGTAAT ATAAACTTAAAAGATAGTTAGAGGTATAATTAATATGAGTTCTGGAAAAATTGTCCAGGTTATTGGAGCGGTGGTTGATGTTGCGTTCAATCAAGATGTGGTACCGACTGTATACCATGCACTTGA GGTGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAACACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAA GTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAATAAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTAT AATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGACAAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTAT CACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTAATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGG GTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAATAATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTAT GTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAATAGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAA ATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGTGTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGT GTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTCTATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACA ACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTTGTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAAT ATTTTTTGAAAGGTGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATT ATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGAAGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATG AAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGATGAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGA GCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCAAAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTT TCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCT DNA sequencing What, When, Why? How? Who and Where? DNA detective

3. ? ? DNA extraction PCR Gel electrophoresis Insect identification ACAGATGTCTTGTAATCCGGC CGTTGGTGGCATAGGGAAAG GACATTTAGTGAAAGAAATTG ATGCGATGGGTGGATCGATG GCTTATGCTATCGATCAATCA GGAATTCAATTTAGAGTACTT AATAGTAGCAAAGGAGCTGC TGTTAGAGCAACACGTGCTCA GGCAGATAAAATATTATATCG TCAAGCAATACGTAGTATTCT TGAATATCAAAAATTTTTGTTG GTTATTCA DNA sequencing ACAGATGTC TTGTAATCC GGCCGTTGG TGGCATAGG GAAAGGACA TTTAG Bioinformatics

4.  WHAT types of biological insights can DNA sequence data provide?  WHEN and WHY would we want to obtain this detailed information?  HOW is DNA sequencing actually performed?  WHO conducts DNA sequencing, and WHERE?

5. DNA sequencing DNA sequence of a particular gene ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAG GACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATG GCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTT AATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCA GGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCT TGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGA AGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTAC TCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTT GACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGAT GAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCAT CGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGA TTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCA AA What questions about this organism can DNA data help us to address? What good is it?

6. How is this organism related to other species? A F D E B H I J K ? ? G C

7. DNA sequences provide characters that are: Numerous, discrete characters (A, T, C, G) Directly comparable across species Unlikely to change due to culture conditions Can be sampled without ever culturing (important for endosymbionts!) ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAG GAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGACAGATGTCTTGTAATCC GGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGT GAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAA CACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTA GCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGG GAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAG CTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTCAATACGCGTGCTCAGGCAGATAAAATATTA Molecular phylogenetics: Inference of evolutionary relationships based on molecular data

8. Secondary structure of 16S rRNA in E. coli Molecular Phylogenetics Step 1. Select a DNA region that is homologous, or similar across species due to common ancestry. Ribosomal RNA (rRNA) Ideal gene for phylogenetic studies because it : is an essential gene that is present in all organisms. is a common target for sequencing studies; large database for comparisons. contains sites that are relatively conserved (stems) and sites that are more free to vary (loops).

9. 2. Amplify and Sequence this region across isolates…. ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG CGATGGGTGGATCGATGGCTTATGCTATCGATC AATCAGGAATTCAATTTAGAGTACTTAATAGTA GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT CAGGCAGATAAAATATTATATCGTCAAGCAATA CGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG GTFTGGGTGGATCGATGGCTTATGCTATCGATC AATCAGGAATTCAATTTAGAGTACTTAATAGTA GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT CAGGCAGATAAAATATTATATCGTCAAGCAATA CGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG CGATGGGTGGATCGATGGCTTATGCTATCGATC AATTTAGAATTCAATTTAGAGTACTTAATAGTAG CAAAGGAGCTGCTGTTAGAGCAACACGTGCTC AGGCAGATAAAATATTATATCGTCAAGCAATAC GT Sequence the PCR product PCR

10. 3. Sequence alignment is crucial for inferring how DNA sites have changed. Poor alignment Implies that species “I” is divergent from the others, but this is not the case. Good alignment. Species “I” has probably experienced a deletion event at position #6 or #7.

11. 4. Estimate relationships based on extent of DNA similarity. ATGTTGGCAGTCCGATGTAAGC ATGTTGGCAGTCCGATGTAACC ACGGTAGCAGTCTGATGTATCC CTGCTGGTAGTCGTTTGTAACC CTGCTGGCAGTCGGTTGTAACC ATGCTGGCAGTCGGGTGTAACC ATGGTGGCAGTCGGGTGTCACC Colored letters = different from top sequence (taxon G) Molecular phylogeny of taxa A-I. At variable DNA positions, related groups will tend to share the same nucleotide. The sheer number of characters is helpful to distinguish the ‘phylogenetic signal’ from noise.

12. Example: Molecular phylogenies have revealed unexpected features of bacterial evolution. For instance, an endosymbiotic lifestyle has evolved several times independently. Moran and Wernegreen (2000)

13. How does this organism fit into the world of available sequence data? ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG CGATGGGTGGATCGATGGCTTATGCTATCGATC AATCAGGAATTCAATTTAGAGTACTTAATAGTA GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT CAGGCAGATAAAATATTATATCGTCAAGCAATA CGT PCR Sequence the PCR product “Blast” sequence to Genbank GENBANK = NIH genetic database with all publicly available DNA sequences. As of 2004: > 44 billion bp, and > 40 million sequences Blast output: Lists sequences that are most similar to yours

14. ? ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT AGGGAAAGGACATTTAGTGAAAGAAATTGATG CGATGGGTGGATCGATGGCTTATGCTATCGATC AATCAGGAATTCAATTTAGAGTACTTAATAGTA GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT CAGGCAGATAAAATATTATATCGTCAAGCAATA CGT Is the bacterium inside this insect really Wolbachia? PCR and sequence a gene of interest (e.g., 16S rDNA) Blast results: Wolbachia sp. 1 Wolbachia sp. 2 Wolbachia sp. 3 …. Blast results: Wolbachia sp. 1 Wolbachia sp. 2 Wolbachia sp. 3 …. YES!! “Blast” sequence to Genbank

15.  WHAT types of biological insights can DNA sequence data can provide?  WHEN and WHY would we want to obtain this detailed information?  HOW is DNA sequencing actually performed?  WHO conducts DNA sequencing, and WHERE?

16. The elegant idea behind DNA sequencing Technology changes quickly, but for many years we’ve on Sanger’s cool trick. Fred Sanger In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators. This method is still in use today. What is the basis of Sanger’s method? Shared with Walter Gilbert and Paul Berg

17. Deoxynucleotides (dNTPs) are the building blocks of DNA. dNTPs are held together by phosophodiester bonds, which comprise the backbone of the DNA chain. Figures in this and subsequent slides from Hartl and Jones, Essential Genetics, 1999 [removed here]

18. DNA is replicated 5’ to 3’. The addition of a new deoxynucleotide in DNA synthesis depends on the presence of that 3’-OH.

19. Dideoxynucleotides (ddNTPs) are missing that critical 3’-OH end. When incorporated into DNA chain, they terminate chain elongation because a phosphodiester bond with the next deoxynucleotide cannot be formed. = chain termination

20. Fascinating. Now how do we actually sequence ??

21. ? ? DNA extraction PCR Gel electrophoresis Insect identification PCR products

22. DNA Sequencing: Step 1: Purify template (the DNA to be sequenced) Amplified DNA + used Taq + extra primer and dNTPs + salts from PCR buffer, etc…. On to sequencing…. clean PCR product + water PCR clean-up

23. Step 2: Set up cycle sequencing reaction NUCLEOTIDES DIDEOXYNUCLEOTIDES TEMPLATE DNA (e.g., YOUR PCR PRODUCT) DNA PRIMER

24. Today, nearly all sequencing is Fluorescence-based, and uses chain terminators that are labeled with different dyes. The dyes are “spectrally distinct,” and each has a different emission wavelength.

25. denaturation Primer annealing Product extension Reaction steps seems a lot like PCR, but not quite… Step 3: Perform cycle sequencing in PCR machine

26. Go to: http://www.dnalc.org/ddnalc/resources/animations.html >> Cycle sequencing

27. Use of capillaries filled with a matrix for higher through-put of sequencing reactions. e.g. ABI 3730

28. 1. Purify PCR product 2. Set up sequencing reaction 3. Perform cycle sequencing 4. Resolve sequence fragments 5. Read order of terminators (DNA sequence) In sum: From PCR to sequence data

29. Quality of sequence data may vary, depending on: Purity and concentration of template DNA Presence of extra PCR bands (artifacts) Quality of dye-terminators, electrophoresis matrix, and other reagents Ideally, look at chromatograms and convince yourself that base calls are robust.

30. DNA sequence assembly : Combining sequence reads to build the entire sequence of the template DNA PCR PRODUCT of 1,200 bp Sequence read #1 Sequence read #2 Sequence read #3 Sequence “reads” typically range from 500-800 bp. Often the total DNA fragment is much longer. Regions of overlap among sequence reads: Let us piece together the linear “puzzle” of the template DNA sequence. Often confirm base calls and improve overall add data quality.

31. The “next generation” is here.

32. Steps in “next-generation” sequencing This generates millions of clonally amplified (identical) DNA fragments on each bead Isolate DNA-containing beads Link DNA fragments to beads (one fragment per bead) Attach adaptors (A and B) to ends of DNA fragments A B Create “Water-in-oil” emulsion + PCR Reagents + Emulsion Oil Perform emulsion PCR

33. 44 um PicoTiterPlate (a fancy microtiter plate with >400,000 wells) ~400 wells per sq. millimeter Load Enzyme Beads Load DNA beads One bead per well

34. dNTP’s are added one at a time to the reaction. Incorporation of one (or more) nucleotide(s) that are complementary to the template results in a chemiluminescent signal, which is recorded by a camera and reflected as a peak in a ‘pyrogram.’ Pyrosequencing - Sequence by synthesis, not electrophoresis. ~500 MB on one plate, in just a few hours. = pyrophosphate Peaks in pyrogram reflect nucleotide sequence

35.  WHAT types of biological insights can DNA sequence data can provide?  WHEN and WHY would we want to get this detailed information?  HOW is DNA sequencing actually performed?  WHO conducts DNA sequencing and has access to the data? WHERE is sequencing performed? How might these things change in the future?

36. Major sequencing centers have facilities to sequence multiple full genomes each year Due to genomic research over the past decade, DNA sequencing has become: Increasingly automated Higher-throughput Less $$ per reaction And (good news) more accessible to researchers and educators

37. Many public universities to have DNA sequencing facilities in house. Such facilities are usually able to run small numbers of reactions for a cost of ~$10-15 per reaction, but often much less for “internal users” with ties to the university. Ideal situation: Team up with faculty at your local university to act as facilitators. Possible avenues for DNA sequencing: Even if DNA sequencing not an option for classroom labs, important to discuss: how DNA sequencing is performed, how data is evaluated, ethical issues surrounding its interpretation and use.

38. Ethical issues involving DNA sequencing Truro: A tranquil Cape community shaken by the murder of a young woman in January 2002

39. Should police have requested DNA of every man in Truro??

40. Thylacine pup preserved in alcohol in 1866. Its cells could be used for cloning. By chance this Thylacine was stored in a jar of alcohol rather than formalin, which would have destroyed the DNA. Should scientists attempt to clone the now-extinct Thylacine to revive this species?? (The technology will be there eventually.)

41.  WHAT, WHEN and WHY? DNA data provides new insights into evolutionary relationships, including closest matches in vast public databases. Take-home messages  WHO and WHERE? Increasingly accessible, DNA sequencing is already woven into the fabric of health and legal systems. Evaluating DNA evidence is key in debates of ethics and policies surrounding medical care, individual privacy, and conservation of biodiversity.  HOW? Though technology changes rapidly, chain termination, developed in the 1970’s, remains central to much DNA sequencing. However, much faster and cheaper approaches are gradually replacing these methods.