1. Targeted Sequencing of Human Genomes, Transcriptomes, and Methylomes Jin Billy Li George Church Lab Harvard Medical School jli@genetics.med.harvard.edu

2. Genetic Loci X Sample Size = Information # samples # genetic loci PCR seq Mass-spec Shotgun seq RNA-seq ChIP-seq SNP array

3. Target Capturing with Padlock Probes (aka MIPs) feature 1feature n pol lig … PCR (or RCA) … Porreca et al., Nat Methods 2007

4. Mass Production of Padlock Oligos 100 nt 150 nt 50 nt 55k features of up to 200nt

5. ~10,000-fold Improvement Since Nov 2007 1. longer hybridization time; 2. more probes; 3. right [dNTP] 123 * * 20-fold improvement already by better probe design and synthesis Li et al., in prepration

6. ~10,000-fold Improvement Since Nov 2007 1. longer hybridization time; 2. more probes; 3. right [dNTP] 123 * * 20-fold improvement already by better probe design and synthesis Li et al., in prepration

7. ~10,000-fold Improvement Since Nov 2007 1. longer hybridization time; 2. more probes; 3. right [dNTP] 123 * * 20-fold improvement already by better probe design and synthesis Li et al., in prepration

8. Improved Technology -> Better Performance 95% captured 85% within 100-fold range 55% within 10-fold range Sensitivity + Uniformity Correlation Nov 2007 Current Li et al., in prepration

9. Summary of Improvements Nov 2007Current Specificity~100% Sensitivity/Multiplexity (of 55k)18%95% Uniformity (in 100-fold range)16%85% Correlation of replicates (r)0.350.98 Accuracy (heterozygous calls)31%99%

10. Targeted Capturing of Genomes –Exome: PGP etc. –Contiguous regions or gene panels –SNPs –Hypermutable CpG dinucleotides Transcriptomes –Alleotyping –RNA editing sites Methylomes –CpG methylation

11. Targeted Capturing of Genomes –Exome: PGP etc. –Contiguous regions or gene panels –SNPs –Hypermutable CpG dinucleotides Transcriptomes –Alleotyping –RNA editing sites Methylomes –CpG methylation

12. Predicting Putative Editing Sites A in the genome G in some mRNAs or ESTs A -> I (G) RNA Editing Post-transcriptional A -> I I is read as G during translation Only 10 targets are known in human coding regions

13. 36,000 predicted editing sites gDNA + 7 tissue cDNAs from an individual Padlock + Solexa: 239 sites found to be edited Validation (PCR + Sanger): 18 of 20 random sites are obviously edited Discovery of 100’s of Novel Editing Sites with Erez Levanon, in preparation

14. Genomic DNA RNA - intestine RNA - kidney RNA - diencephalon RNA - frontal lobe RNA - corpus callosum RNA - cerebellum RNA - adrenal Example: VEZF1

15. Bisulfite Padlock Probes (BSP): CpG Methylation Bisulfite-treated genome “3-base” genome High specificity of padlock

16. Methylation Level Accurately Measured r = 0.979 BSP-BSP correlation BSP-Sanger correlation Methylation level measured by BSP sequencing Methylation level estimated by Sanger sequencing Methylation level, replicate 1 Methylation level, replicate 2 r = 0.966

17. Methylation Pattern around Genes Gene-Body Methylation with Madeleine Price Ball, in preparation (poster)

18. George Church Padlock technology Kun Zhang John Aach Abraham Rosenbaum Jay Shendure Greg Porreca Annika Ahlford RNA editing Erez Levanon Jung-Ki Yoon CpG methylation Madeleine Price Ball Church Lab Acknowledgements Agilent Emily Leproust Wilson Woo Sequencing Yuan Gao Bin Xie Bob Steen

19. Superior Quality of Padlock Oligos 100 nt 150 nt 50 nt PCR (2x) Solexa sequencing 55k features of up to 200nt Fraction of probes

20. U From Agilent Oligos to Padlock Probes amplification and selection T 18bp Agilent oligo, 136 bp 18bp PCR * p exonuclease USER + DpnII DpnII NN UAUA U Annealed with DpnII guide oligo Padlock probe * *

21. Heterozygous Genotypes Correctly Called Homozygous wild type Heterozygous variation Homozygous variation beforeafter

22. Methods in Comparison PadlockArray-based hyb Upfront probe cost (10-20% of exome) $12,000 per 55k 100mers$600 per 385k 70mers Probes amplifiable?YesNo Reaction phaseSolution, 10-20 μlSurface, 200 μl Enzymatic hyb?YesNo gDNA required~0.5-1 μg20 μg (WGA) Efficiency (->accuracy)1%N/A (<0.1%?) Uniformity100-fold range10-fold range Specificity~100% on target30-80% on or near target

23. Differential Clamping at Ligation Junction

24. % GC VS Capturing Efficiency

25. 99% Concordance Between Padlock and HapMap

26. The Editing “Calls” Are Well Correlated r = 0.964

27. Bisulfite-treated genome 10k CpG sites tiling the ENCODE regions –1 CpG site every 3kb region on average High specificity –79 of 80 Sanger reads match correct locations Bisulfite Padlock Probes (BSP): CpG Methylation

28. B strep B P P B B collected in a tube PCR λ exonuclease shearing, end polishing adapter ligation hybridization in closed-tube solution denaturing, PCR Li et al., unpublished

29. Methods in Comparison PadlockArray-based hybBiotin-coupled hyb Upfront probe cost (10-20% of exome) $12,000 per 55k 100mers $600 per 385k 70mers $500 per 244k 60mers Probes amplifiable?YesNoYes Reaction phaseSolution, 10-20 μlSurface, 200 μlSolution, 10-20 μl Enzymes in hyb?YesNo gDNA required~0.5-1 μg20 μg (WGA)~0.5-1 μg Efficiency (->accuracy)1%N/A (<0.1%?)~10%? Uniformity100-fold range10-fold range10-fold range? Specificity~100% on target 30-80% on or near target ~55% on or near target

30. Two Tech Replicates Are Well Correlated Ranked target sites Number of reads per site Counts, replicate 1 Counts, replicate 2 Uniformity Correlation of counts