Andrew Gentles CCSB NGS workshop September 2012 Youve slogged through QC trimming alignment realignment variant calling What next Mutational processes molding the genomes of 21 breast cancersThe life history of 21 breast cancers ID: 240502
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Slide1
Recent applications of NGS sequencing in cancer studies
Andrew Gentles
CCSB NGS workshop
September 2012Slide2
You’ve slogged through QC, trimming, alignment, realignment, variant calling
What next ?Slide3
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
unannotated
lincRNA
implicated in disease progression
Prensner
et al. (2011) Nat Biotech 29: 742-9Slide4
Companion papers from Cell May 2012Slide5
Whole genome sequencing of 21 Breast cancers
Sample
Age at first diagnosis
Previous histopathological diagnosis
Histo
patho
logical Grade
ER Status
PR StatusHER2 StatusBRCA mutationsPD385161DuctalIII++- PD389041DuctalIII---BRCA1PD390439DuctalIII++-BRCA2PD390534DuctalIII---BRCA1PD394559DuctalIII+--BRCA2PD400539DuctalIII---BRCA1PD400639DuctalIII---BRCA1PD408564DuctalIII++-PD408658DuctalIII---PD408832DuctalIII+--PD410346DuctalIII++-PD410733DuctalIII---BRCA1PD410967DuctalIII---PD411554DuctalIII++-BRCA2PD411632DuctalIII++-BRCA2PD4120*60DuctalII++-PD419270DuctalIII--+PD419443LobularIII+++PD419859DuctalIII+-+PD419959DuctalII--+PD424848DuctalII---
>30x coverage tumor and normal (188x for *)Slide6
Analysis outline
WGS sequencing to >30x coverage tumor/normal
~100
bp
paired-end reads
BWA alignment
Compare tumor/normal for variant calling
CaVEMan
,
PindelDetection of structural rearrangementsIn-house methodInference of copy number changesASCATSlide7
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, NCOR1Slide8
Higher rate in BRCA1/2
C>A most common
Mutational spectrum in breast cancerSlide9
Kataegis
: regions of enhanced mutation rateSlide10
Kataegis
is highly focal upon zooming inSlide11
Kataegis
associated with structural rearrangementsSlide12
A very deep look into mutation frequencies to reconstruct tumor evolutionSlide13
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 validationSlide14
Patterns of copy number alteration in PD4120a
Relatively few CNVs
Some sub-
clonalSlide15
D
C
B
A
1
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)2322Mutation frequencies show clusters representing major and minor clonesSlide16
5% 11% 19% 35%
15600
26762
Founder clone
“most-recent common ancestor”Slide17
D
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 del134Slide18
If SNVs close enough to SNPs, can be phased with them
2171 on chr13
756 can be phasedSlide19
Phasing of somatic mutations (Supp Fig 4)Slide20
Phasing of somatic mutations (Supp Fig 4)
Found 17 mutually exclusive, 76 examples of sub-
clonal
evolutionSlide21
Figure 3:
Reconstructed evolution of tumor
(see paper for details)Slide22
Sci
Trans Med 2012Slide23
Prospective separation of residual HSC from leukemic patientsSlide24
Residual HSC lack AML FLT3-ITD mutationsSlide25
Strategy for identifying pre-leukemic mutations in HSC
67-239x
exome
coverageSlide26
Occurrence of AML mutations in residual HSC
~25000x targeted coverageSlide27
Mutations in HSC or both HSC/LSCSlide28
HSC with the pre-leukemic mutations are capable of differentiating to produce functional immune cellsSlide29Slide30Slide31Slide32
Filtering to identify
ncRNAsSlide33Slide34
Enrichment of
histone
modification marks around transcripts
H3K4me2
H3K4me3
Figure 2Slide35
Novel transcripts are highly expressed in prostate cancerSlide36
PCAT-1 is highly expressed in metastatic/high-grade prostate cancer
PCAT-1 expression is mutually exclusive with EZH2
Figure 3f
Figure 4bSlide37
Relationship of PCAT-1 to EZH2/PRC complexSlide38
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)Slide39
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