Update on an investigation of somatic mutations in eMERGE datasets

1. Update on an investigation of somatic mutations in eMERGE datasets Ken Kaufman CCHMC 6-20-19

2. Somatic Mutations RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues Keren Yizhak, François Aguet, Jaegil Kim, et al Science 07 Jun 2019: Vol. 364, Issue 6444, eaaw0726

3. Mutations • Germline Somatic +/- +/+ +/+ +/- +/- +/- +/+ +/+ +/- +/- +/- +/- +/- +/- 50% 25%

4. Germ Line

5. Somatic

6. Somatic Mutation Pipeline • Samtools pileup • Ref and alt alleles detected • Each base sequenced • Calculate ratio alt/depth allele • Filter • Ratio 1% to 30% • Amino-Acid altering • Depth of 10 • Not in a Duplicated regions

7. Samples processed • eMERGE 3 set A • 16,170 samples screened • 801 candidate somatic mutations in 773 samples • PGX ~10,000 samples processed • Initially 2798 samples 4403 variations • Filtered • 555 candidate somatic mutations in 541 samples • 66 samples with 2 or more candidates (5 highest) • 58 candidates found in 2 or more samples (4 highest). • 419 candidates found in 1 sample • 252 of the 555 candidate somatic mutations are found in EXAC database • MAF 0.009 to 8.2x10-6

8. Characteristics of Somatic Mutations • 178 genes • 488 type –A variants • 483 Non-syn • 2 Ins • 3 Del • 61 type –B (loss of function) • 24 Stopgain • 1 Stoploss • 21 Frameshift • 3 Init codon • 12 Splicing

9. Functional Predictions

10. Genes with Most Somatic Mutations

11. GATK 150 555 75

12. Alt allele ratio Number of Variants Average Ratio

13. Validation • Obtained DNA for 11 samples from Vanderbilt and Northwestern (Thank you Very Much!) • Sanger Sequence PCR amplified product (with controls) • Real-time PCR • Digital droplet PCR

14. Validation Results

15. Validation • PGX • 9142 samples screened (6.1%) • 555 candidate somatic mutations in 541 samples • eMERGE 3 set A • 16,170 samples screened (5.0%) • 801 candidate somatic mutations in 773 samples • eMERGE 3 set B • In process downloading bam files from DNAnexus

16. Validation Strategy(iGENOMX Riptide) SNP Adapter Sequence  biotinylated ddNTP

17. Strand displacing Extension SNP Adapter Barcode Random Sequence

18. Low cycle PCR SNP

19. Sequencing • Samples from 96 to 960 • Sequencers • MiSeq 25M reads • 96 samples • 96 targets • ~270 X coverage • HiSeq 900M reads (3 Lane) • 960 samples • 960 targets • ~100 X coverage

20. Sample Status

21. Current Status • Process remaining eMERGE 3 data set • Obtain samples for validation • Finalize validation strategy. • Contact Ken Kaufman (Kenneth.Kaufman@cchmc.org) or Paul Gecaine (Paul.Gecaine@cchmc.org) to participate.

22. Acknowledgements DNAnexus • Andrew Carroll • John Didion • eMERGE consortium • Vanderbilt • Northwestern • University of Washington • Baylor (Richard Gibbs group) • CCHMC • John Harley • Scott Richards • Paul Gecaine • Beth Cobb • Cindy Prows • Bahram Namjou-Khales Contact Ken Kaufman (Kenneth.Kaufman@cchmc.org) or Paul Gecaine (Paul.Gecaine@cchmc.org) to participate

23. Strategy • VCF files only have data where a variant was called. • Bam files have data at every position sequenced. • Ratio of Ref to Alternate allele skewed

24. Comparison other Programs • Mosaic Hunter • Tested against candidate samples • 56 somatic candidates • 3 overlapped • Somaticsniper • Normal vs tumor • Failed in our application • Most somatic mutation detection approaches require optimization for each data set.