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Recent applications of NGS sequencing in cancer studies

Recent applications of NGS sequencing in cancer studies. Andrew Gentles CCSB NGS workshop September 2012. You’ve slogged through QC, trimming, alignment, realignment, variant calling. What next ?.

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Recent applications of NGS sequencing in cancer studies

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  1. Recent applications of NGS sequencing in cancer studies Andrew Gentles CCSB NGS workshop September 2012

  2. You’ve slogged through QC, trimming, alignment, realignment, variant calling What next ?

  3. Mutational processes molding the genomes of 21 breast cancers/The life history of 21 breast cancers • Nik-Zainal et al. (2012) Cell 149(5):994-1007 • Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia • Jan et al. (2012) Sci Trans Med 4, 149ra118 • Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotatedlincRNA implicated in disease progression • Prensner et al. (2011) Nat Biotech 29: 742-9

  4. Companion papers from Cell May 2012

  5. Whole genome sequencing of 21 Breast cancers >30x coverage tumor and normal (188x for *)

  6. Analysis outline • WGS sequencing to >30x coverage tumor/normal • ~100 bp paired-end reads • BWA alignment • Compare tumor/normal for variant calling • CaVEMan, Pindel • Detection of structural rearrangements • In-house method • Inference of copy number changes • ASCAT

  7. Summary of somatic mutations • 183916 somatic mutations (SNVs) identified in total • 1372 missense, 117 nonsense, 2 stop-lost, 37 splice, 521 silent • Most frequent mutations in known cancer genes such as TP53, GATA3, PIK3CA, MAP2K4, SMAD4, MLL2, MLL3, NCOR1

  8. Higher rate in BRCA1/2 C>A most common Mutational spectrum in breast cancer

  9. Kataegis: regions of enhanced mutation rate

  10. Kataegis is highly focal upon zooming in

  11. Kataegis associated with structural rearrangements

  12. A very deep look into mutation frequencies to reconstruct tumor evolution

  13. PD4120a • 188x coverage – enables deep look at mutation frequencies • 70690 somatic substitutions • Some in <5% of reads • Mainly C>* in TpC context • High rate of validation

  14. Patterns of copy number alteration in PD4120a Relatively few CNVs Some sub-clonal

  15. Mutation frequencies show clusters representing major and minor clones 2 1 D 2 3 C 2 B A 35% of reads -> all tumor cells since tumor is 70% tumor (cluster D) Trisomy 1q early since few mutations with high read fraction – most are subclonal 3 major clusters of sub-clonal mutations (A,B,C)

  16. 15600 26762 5% 11% 19% 35% Founder clone “most-recent common ancestor”

  17. D 4 C B A Cluster C ~19% - more than half of tumor cells (since >1/2*35%) “Pigeonhole principle”: for any 2 mutations, at least one tumor cell must have both – must be on same part of phylogenetic tree If one such mutation in greater fraction than another, must have occurred earlier Cluster C must be on same phylogenetic branch as del13

  18. If SNVs close enough to SNPs, can be phased with them • 2171 on chr13 • 756 can be phased

  19. Phasing of somatic mutations (Supp Fig 4)

  20. Phasing of somatic mutations (Supp Fig 4) Found 17 mutually exclusive, 76 examples of sub-clonal evolution

  21. Figure 3: Reconstructed evolution of tumor (see paper for details)

  22. Sci Trans Med 2012

  23. Prospective separation of residual HSC from leukemic patients

  24. Residual HSC lack AML FLT3-ITD mutations

  25. Strategy for identifying pre-leukemic mutations in HSC 67-239x exome coverage

  26. Occurrence of AML mutations in residual HSC ~25000x targeted coverage

  27. Mutations in HSC or both HSC/LSC

  28. HSC with the pre-leukemic mutations are capable of differentiating to produce functional immune cells

  29. Filtering to identify ncRNAs

  30. Enrichment of histone modification marks around transcripts H3K4me2 H3K4me3 Figure 2

  31. Novel transcripts are highly expressed in prostate cancer

  32. PCAT-1 is highly expressed in metastatic/high-grade prostate cancer Figure 4b Figure 3f PCAT-1 expression is mutually exclusive with EZH2

  33. Relationship of PCAT-1 to EZH2/PRC complex

  34. RNA-seq discovers novel ncRNAs • PCAT-1 highly expressed in high grade/metastatic prostate cancer • PCAT-1 promotes proliferation • Hypothesized role with EZH2 (c.f. HOTAIR)

  35. Final items • Please fill out evaluation form! • Slides: • Available soon from http://ccsb.stanford.edu • Sequence answers forum: • http://seqanswers.com • Stanford discussion group • https://mailman.stanford.edu/mailman/listinfo/wgs_club_stanford

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