Targeted Sequencing of Human Genomes, Transcriptomes, and Methylomes

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 PCR seq Mass-spec SNP array # samples Shotgun seq RNA-seq ChIP-seq # genetic loci

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

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

5. ~10,000-fold Improvement Since Nov 20071. longer hybridization time; 2. more probes; 3. right [dNTP] 1 2 3 * Li et al., in prepration *20-fold improvement already by better probe design and synthesis

6. ~10,000-fold Improvement Since Nov 20071. longer hybridization time; 2. more probes; 3. right [dNTP] 1 2 3 * Li et al., in prepration *20-fold improvement already by better probe design and synthesis

7. ~10,000-fold Improvement Since Nov 20071. longer hybridization time; 2. more probes; 3. right [dNTP] 1 2 3 * Li et al., in prepration *20-fold improvement already by better probe design and synthesis

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

9. Summary of Improvements

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 • HypermutableCpGdinucleotides • Transcriptomes • Alleotyping • RNA editing sites • Methylomes • CpGmethylation

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

13. Discovery of 100’s of Novel Editing Sites 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 with Erez Levanon, in preparation

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

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

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

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

18. Acknowledgements 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 Sequencing Yuan Gao Bin Xie Bob Steen Agilent Emily Leproust Wilson Woo George Church

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

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

21. Heterozygous Genotypes Correctly Called before after Homozygous wild type Heterozygous variation Homozygous variation

22. Methods in Comparison

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 Padlock Probes (BSP): CpG Methylation • 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-treated genome

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

29. Methods in Comparison

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