DNA Sequencing

1. DNA Sequencing

2. DNA sequencing How we obtain the sequence of nucleotides of a species …ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…

3. Which representative of the species? Which human? Answer one: Answer two: it doesn’t matter Polymorphism rate: number of letter changes between two different members of a species Humans: ~1/1,000 Other organisms have much higher polymorphism rates • Population size!

4. Why humans are so similar Out of Africa N A small population that interbred reduced the genetic variation Out of Africa ~ 40,000 years ago Heterozygosity: H H = 4Nu/(1 + 4Nu) u ~ 10-8, N ~ 104  H ~ 410-4

5. Human population migrations • Out of Africa, Replacement • “Grandma” of all humans (Eve) ~150,000yr • Ancestor of all mtDNA • “Grandpa” of all humans (Adam) ~100,000yr • Ancestor of all Y-chromosomes • Multiregional Evolution • Fossil records show a continuous change of morphological features • Proponents of the theory doubt mtDNA and other genetic evidence • New fossil records bury “multirigionalists” • Nice article in Economist on that http://www.economist.com/science/displaystory.cfm?story_id=9507453

6. DNA Sequencing – Overview 1975 • Gel electrophoresis • Predominant, old technology by F. Sanger • Whole genome strategies • Physical mapping • Walking • Shotgun sequencing • Computational fragment assembly • The future—new sequencing technologies • Pyrosequencing, single molecule methods, … • Assembly techniques • Future variants of sequencing • Resequencing of humans • Microbial and environmental sequencing • Cancer genome sequencing 2015

7. DNA Sequencing Goal: Find the complete sequence of A, C, G, T’s in DNA Challenge: There is no machine that takes long DNA as an input, and gives the complete sequence as output Can only sequence ~900 letters at a time

8. DNA Sequencing – vectors DNA Shake DNA fragments Known location (restriction site) Vector Circular genome (bacterium, plasmid) + =

9. Different types of vectors

10. DNA Sequencing – gel electrophoresis • Start at primer (restriction site) • Grow DNA chain • Include dideoxynucleoside (modified a, c, g, t) • Stops reaction at all possible points • Separate products with length, using gel electrophoresis

11. Electrophoresis diagrams

12. Reading an electropherogram • Filtering • Smoothening • Correction for length compressions • A method for calling the letters – PHRED PHRED – PHil’s Read EDitor (by Phil Green) Almost 15 years old method Newer methods may be better, but labs are reluctant to change

13. Output of PHRED: a read A Sanger read: 500-1000 bp A C G A A T C A G …A 16 18 21 23 25 15 28 30 32 …21 Quality scores: -10log10Prob(Error) Reads can be obtained from leftmost, rightmost ends of the insert Double-barreled sequencing: (1990) Both leftmost & rightmost ends are sequenced, reads are paired

14. Method to sequence longer regions genomic segment cut many times at random (Shotgun) Get one or two reads from each segment ~900 bp ~900 bp

15. Reconstructing the Sequence (Fragment Assembly) reads Cover region with high redundancy Overlap & extend reads to reconstruct the original genomic region

16. Definition of Coverage C Length of genomic segment: G Number of reads: N Length of each read: L Definition:Coverage C = N L / G How much coverage is enough? Lander-Waterman model: Prob[ not covered bp ] = e-C Assuming uniform distribution of reads, C=10 results in 1 gapped region /1,000,000 nucleotides

17. Repeats Bacterial genomes: 5% Mammals: 50% Repeat types: • Low-Complexity DNA (e.g. ATATATATACATA…) • Microsatellite repeats (a1…ak)N where k ~ 3-6 (e.g. CAGCAGTAGCAGCACCAG) • Transposons • SINE(Short Interspersed Nuclear Elements) e.g., ALU: ~300-long, 106 copies • LINE(Long Interspersed Nuclear Elements) ~4000-long, 200,000 copies • LTRretroposons(Long Terminal Repeats (~700 bp) at each end) cousins of HIV • Gene Families genes duplicate & then diverge (paralogs) • Recent duplications ~100,000-long, very similar copies

18. AGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCTAGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCT Sequencing and Fragment Assembly 3x109 nucleotides 50% of human DNA is composed of repeats Error! Glued together two distant regions

19. What can we do about repeats? Two main approaches: • Cluster the reads • Link the reads

20. What can we do about repeats? Two main approaches: • Cluster the reads • Link the reads

21. What can we do about repeats? Two main approaches: • Cluster the reads • Link the reads

22. AGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCTAGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCT A R B D R C Sequencing and Fragment Assembly 3x109 nucleotides ARB, CRD or ARD, CRB ?

23. AGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCTAGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCT Sequencing and Fragment Assembly 3x109 nucleotides

24. Strategies for whole-genome sequencing • Hierarchical – Clone-by-clone • Break genome into many long pieces • Map each long piece onto the genome • Sequence each piece with shotgun Example: Yeast, Worm, Human, Rat • Online version of (1) – Walking • Break genome into many long pieces • Start sequencing each piece with shotgun • Construct map as you go Example: Rice genome • Whole genome shotgun One large shotgun pass on the whole genome Example: Drosophila, Human (Celera), Neurospora, Mouse, Rat, Dog

25. Hierarchical Sequencing

26. a BAC clone map Hierarchical Sequencing Strategy • Obtain a large collection of BAC clones • Map them onto the genome (Physical Mapping) • Select a minimum tiling path • Sequence each clone in the path with shotgun • Assemble • Put everything together genome