Genomes for ALL Jeremy Edwards Molecular Genetics and Microbiology

1. Genomes for ALL Jeremy Edwards Molecular Genetics and Microbiology Cancer Research and Treatment Center Chemical and Nuclear Engineering jsedwards@salud.unm.edu

2. Sanger Sequencing

3. $1,000 Genome • Error rate must be significantly lower than the variation (~1 in 1kbp) • Error Rate goal of 1/100,000 • Raw read accuracy goal = 99.7% with 3x coverage • 3x coverage of the human diploid genome ~40 Billion bases • Requires 40 Million bases by $1 for the $1000 Genome • Is a 20 bp read long enough? • If the human genome sequence was random, one would expect that each 20bp read would be unique – 420>>3x109 • However, the human genome is not random. • Only 73% of the potential 20 bp reads could be uniquely placed in the genome. • Here, we collected 26 bp reads –extend to 34 bp. • The 6+7,7+6=26 bp read could be improved to 9+9,9+9=36 bp read using a different ligase • Mate Pairs – greatly helps the situation. • Should be able to increase this to 72 bp. • 60 bp is thought to be the minimum (Shendure, et al) • Which works for a 40 Mbp contig (34bp)

4. Polony History

5. Acrylamide gel + Thermal cycle + primers and DNA Polonies • Amplify template DNA in thin polyacrylamide gel • All normal PCR ingredients + polyacrylamide mixed and polymerized onto glass slide • Exponential DNA amplification obtained • DNA remains localized due to diffusion limitations Mitra and Church. (1999) Nucleic Acids Research. + PCR reagents

6. Polonies Mitra and Church. (1999) Nucleic Acids Research.

7. Polonies Mitra and Church. (1999) Nucleic Acids Research.

8. Polonies 75 ml PCR ~2 mm thick 15 ml PCR ~50 mm thick

9. Applying the technology Map Check

10. Mutants & function • 54 variants of the hG6PD gene were functionally tested • 50 mutants represent all possible amino acid substitutions in the 8 residue G6P binding site which can arise from single nucleotide substitutions • 3 each with reported protein activity, carried single nucleotide mutations located away from the G6P binding site • the last variant was the wild-type hG6PD.

11. 50% Mutant A 50% Mutant B Dilute DNA and Polymerize on a Glass Microscope Slide with PCR Components . . . . Parallel Competition Competitive Growth of Mutant Population Thermal Cycle to Amplify PCR Colonies (Polonies) Arising from Individual DNA Molecules . . . . Isolation of DNA from Population Merritt et al. (2003). Nucleic Acids Research Merritt et al. (2005). Biotech Bioeng

12. 0 hr 48 hr 96 hr Parallel Competition Growth competition used to determine relative activities of mutant proteins; Polonies and SBEs used to measure concentrations of each 0 hr 48 hr 96 hr Culture mutant pool Isolate plasmid DNA Polonies/SBE Green = Lys131Glu Red = WT (at this position)

13. Barcode design Molecular Barcode

14. Barcode Readout Tag2 Tag1 AATAAA AATAAA Tag2 = Tag1 = TCCCTT TCCCTT

15. Polony Sequencing (Old School) Mitra, et al. (2003) Anal Biochem

16. FISSEQ Mitra, et al. (2003) Anal Biochem b-mercaptoethanol treatment

17. FISSEQ – 34 bp reads Mitra, et al. (2003) Anal Biochem

18. Bead Based Higher Throughput Assays Map Check

19. Polonies or Beads?

20. BEAMing Dressman, D., et al. (2003) PNAS

21. ePCR

22. Polonies or Beads? Well, we will just call them both polonies if they are in a gel

23. Polony manipulations 5’ 5’ 5’ 5’ …and just about anything else ……To Be Continued

24. Strains that we sequenced

25. FBA

26. Acetate Data Edwards, et al. (2001) Nat Biotechnol

27. Predictive Capability Edwards, et al. (2001) Nat Biotechnol

28. Flux balance analysis:What if we are wrong? Map Check

29. We are wrong? Ibarra, et al. (2002) Nature

30. Adaptation to glycerol Ibarra, et al. (2002) Nature

31. Overview Genome ~1 kb fragments Sequence the 13 bp at the end of each ~1kb fragment

32. Overview Shendure, et al. (2005) Science

33. Fragmentation of Genome ~1kb Fragment Fragmented Genome – 10mg

34. Next few steps • A-Tailing • Circularization – Key step for improvement. (<0.1 % efficient) A A MmeI Sites T T T30 30 bp Contains MmeI sites Anchor Primer Hyb sites

35. RCA RCA MmeI

36. MmeI digested RCA product Gel Purified 66-68 bp band (Tag-T30-Tag) 1 kb

37. Ligate Primers to the end • Blunt end ligation produces three different products: • F –TAG-T30-TAG- R • F –TAG-T30-TAG- F • R –TAG-T30-TAG- R • Gel purify all the triplet of bands and then PCR amplify • Correct band is enriched, but other two are still present. • Gel purify the center (134 bp) band from the others (116 & 152 bp) Gel of Final Library

38. Overview

39. Ligation Sequencing +Direction 6 bp • Direction • 7 bp Inspired by Brenner, et al. (2000) Nat Biotechnol

40. Sequencing “Center” • Hybridize ‘anchor primer’ complementary to common library sequence • Ligate pool of fluorescently-labeled ‘query primers’ specific to one tag-position • Image to determine which primer pool ligated to each bead • Repeat

41. Pictures

42. Pictures

43. Pictures

44. Raw Data

45. “Reads” All data “Good Reads” Proximal -1 Proximal -7

46. Proximal +1

47. Proximal +2

48. Proximal +3

49. Proximal +4

50. Proximal +5