DNA Sequencing: Techniques and Technologies for Obtaining Genetic Information

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: ~1/2,000 Other organisms have much higher polymorphism rates

5. Human population migrations • Out of Africa, Replacement • Single mother of all humans (Eve) ~150,000yr • Single father of all humans (Adam) ~70,000yr • Humans out of Africa ~40000 years ago replaced others (e.g., Neandertals) • Evidence: mtDNA • Multiregional Evolution • Fossil records show a continuous change of morphological features • Proponents of the theory doubt mtDNA and other genetic evidence

6. Why humans are so similar A small population that interbred reduced the genetic variation Out of Africa ~ 40,000 years ago Out of Africa

7. Migration of human variation http://info.med.yale.edu/genetics/kkidd/point.html

8. http://info.med.yale.edu/genetics/kkidd/point.html Migration of human variation

9. http://info.med.yale.edu/genetics/kkidd/point.html Migration of human variation

10. Human variation in Y chromosome

13. 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

14. 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 ~500 letters at a time

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

16. Different types of vectors

17. 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

18. Electrophoresis diagrams

19. Challenging to read answer

20. Challenging to read answer

21. Challenging to read answer

22. Reading an electropherogram • Filtering • Smoothening • Correction for length compressions • A method for calling the letters – PHRED PHRED – PHil’s Read EDitor (by Phil Green) Several better methods exist, but labs are reluctant to change

23. Output of PHRED: a read A read: 500-700 nucleotides 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

24. Method to sequence longer regions genomic segment cut many times at random (Shotgun) Get one or two reads from each segment ~500 bp ~500 bp

25. Reconstructing the Sequence (Fragment Assembly) reads Cover region with ~7-fold redundancy (7X) Overlap reads and extend to reconstruct the original genomic region

26. Definition of Coverage C Length of genomic segment: L Number of reads: n Length of each read: l Definition:Coverage C = n l / L How much coverage is enough? Lander-Waterman model: Assuming uniform distribution of reads, C=10 results in 1 gapped region /1,000,000 nucleotides

27. 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

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

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

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

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

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

33. AGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCTAGTAGCACAGACTACGACGAGACGATCGTGCGAGCGACGGCGTAGTGTGCTGTACTGTCGTGTGTGTGTACTCTCCT Sequencing and Fragment Assembly 3x109 nucleotides

34. 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

35. Hierarchical Sequencing

36. 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

37. Methods of physical mapping Goal: Make a map of the locations of each clone relative to one another Use the map to select a minimal set of clones to sequence Methods: • Hybridization • Digestion

38. 1. Hybridization Short words, the probes, attach to complementary words • Construct many probes • Treat each BAC with all probes • Record which ones attach to it • Same words attaching to BACS X, Y  overlap p1 pn

39. Hybridization – Computational Challenge p1p2 …………………….pm 0 0 1 …………………..1 Matrix: m probes  n clones (i, j): 1, if pi hybridizes to Cj 0, otherwise Definition: Consecutive ones matrix 1s are consecutive in each row & col Computational problem: Reorder the probes so that matrix is in consecutive-ones form Can be solved in O(m3) time (m > n) C1C2 ……………….Cn 1 1 0 …………………..0 1 0 1…………………...0 pi1pi2…………………….pim 1 1 1 0 0 0……………..0 0 1 1 1 1 1……………..0 0 0 1 1 1 0……………..0 Cj1Cj2 ……………….Cjn 0 0 0 0 0 0………1 1 1 0 0 0 0 0 0 0………0 1 1 1

40. Hybridization – Computational Challenge pi1pi2………………………………….pim pi1pi2…………………….pim If we put the matrix in consecutive-ones form, then we can deduce the order of the clones & which pairs of clones overlap 1 1 1 0 0 0……………..0 0 1 1 1 1 1……………..0 0 0 1 1 1 0……………..0 Cj1Cj2 ……………….Cjn Cj1Cj2 ……………….Cjn 0 0 0 0 0 0………1 1 1 0 0 0 0 0 0 0………0 1 1 1

41. Hybridization – Computational Challenge p1p2 …………………….pm 0 0 1 …………………..1 Additional challenge: A probe (short word) can hybridize in many places in the genome Computational Problem: Find the order of probes that implies the minimal probe repetition Equivalent: find the shortest string of probes such that each clone appears as a substring APX-hard Solutions: Greedy, probabilistic, lots of manual curation C1C2 ……………….Cn 1 1 0 …………………..0 1 0 1…………………...0

42. 2. Digestion Restriction enzymes cut DNA where specific words appear • Cut each clone separately with an enzyme • Run fragments on a gel and measure length • Clones Ca, Cb have fragments of length { li, lj, lk }  overlap Double digestion: Cut with enzyme A, enzyme B, then enzymes A + B

43. Online Clone-by-cloneThe Walking Method

44. The Walking Method • Build a very redundant library of BACs with sequenced clone-ends (cheap to build) • Sequence some “seed” clones • “Walk” from seeds using clone-ends to pick library clones that extend left & right

45. Walking: An Example

46. Walking off a Single Seed • Low redundant sequencing • Many sequential steps

47. Walking off a single clone is impractical • Cycle time to process one clone: 1-2 months • Grow clone • Prepare & Shear DNA • Prepare shotgun library & perform shotgun • Assemble in a computer • Close remaining gaps • A mammalian genome would need 15,000 walking steps !

48. Walking off several seeds in parallel • Few sequential steps • Additional redundant sequencing In general, can sequence a genome in ~5 walking steps, with <20% redundant sequencing Efficient Inefficient

49. Whole-Genome Shotgun Sequencing

50. cut many times at random Whole Genome Shotgun Sequencing genome plasmids (2 – 10 Kbp) forward-reverse paired reads known dist cosmids (40 Kbp) ~500 bp ~500 bp