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SMAP: a tool for patient-derived xenografts sequencing data analysis
CIT SÉMINAIRE – 05/10/17
PDX: Patient Derived Xenograft
Stromal cell (mouse)
Tumor cell (human)
PDX Patient Derived Xenograft
Human primary
tumor
Goal
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsRNAseq
Goal
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse reads
HUMAN-tumor Transcriptome or genome
MOUSE-stromaTranscriptome or genome
RNAseq
Existing methods
Existing methods
• Naïve approachHuman Genome
Discarded reads
Kept reads
mapping
Existing methods
• Naïve approachHuman Genome
Discarded reads
Kept reads
Assigned to human
discarded read
mapping
Human Genome Kmer
Mouse Genome Kmer
Read Kmers
• Xenome (Conway et al. 2012)or
Assigned to mouse
Existing methods
• Naïve approachHuman Genome
Discarded reads
Kept reads
mapping
Mouse Genome
Human Genomemapping
mapping
• S3 (Chivukula et al. 2015)
Assigned to mouse
Assigned to human
Ambiguous mapping score comparison
Assigned to human
discarded readHuman
Genome Kmer
Mouse Genome Kmer
Read Kmersor
Assigned to mouse• Xenome (Conway et al. 2012)
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
HUMAN-tumor Transcriptome or genome
MOUSE-stromaTranscriptome or genome
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
SMAP
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
SMAP
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
Read assigned to HUMAN
*
*
**
* mismatch
SMAP
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
Read assigned to HUMAN
*
*
**
Read assigned to MOUSE
* *
*
*
* mismatch
SMAP
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
Read assigned to HUMAN
*
*
**
Read assigned to MOUSE
* *
*
*
*
*
Discarded read
* mismatch
SMAP
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
SMAP
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
Read assigned to HUMAN
*
*
**
Read assigned to MOUSE
* *
*
*
*
*
Discarded read
SMAP: Overall approach
Stromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
HUMAN-tumor Transcriptome or genome
MOUSE-stromaTranscriptome or genome
SMAP
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
Read assigned to HUMAN
*
*
**
Read assigned to MOUSE
* *
*
*
*
*
Discarded read
Mapping and specie assignment performance
Read simulation settings*
*fastq simulation using wgsim tool(Escalona et al., 2016)
DirectMapping
Xenome
S3
SMAP
Comparisons
Mapping and specie assignment performance
DirectMapping
Xenome
S3
SMAP
Fals
e P
osit
ives
Tru
e P
osit
ives
Ideal Mutations Mutations
& Errorsideal Mutations Mutations
& Errors
50%
60%
70%
80%
90%
Un
iqu
ely
ma
pp
ed
re
ad
ra
te 50 bp 100 bp
96%
98%
100%
Un
iqu
ely
map
ped
read
rate 50 bp 100 bp
5%
10%
15%
0‰
0.25‰
0.50‰
Ideal Mutations Mutations& Errors
Ideal Mutations Mutations
& Errors
Estimating the impact of inter-specie gene homology
DirectMapping
Xenome
S3
SMAP
Tru
e P
os
itiv
es
50 bp 100 bp
50%
60%
70%
80%
90%
100%
Un
iqu
ely
ma
pp
ed
re
ad
ra
te>95 95-90 90-85 85-80 <80 >95 95-90 90-85 85-80 <80
Identity(in %)
Un
iqu
ely
ma
pp
ed
re
ad
ra
te
Fa
lse
Po
sit
ive
s
10%
20%
30%
40%
0‰
0.25‰
0.50‰
0.75‰
1‰
50 bp 100 bp
>95 95-90 90-85 85-80 <80 >95 95-90 90-85 85-80 <80
Homology
Application to pancreatic cancer xenograft gene expression analysis
Stromal cell (mouse)
Tumor cell (human)PDX Patient Derived Xenograft
Mix of Human and
Mouse reads
RNAseq
HUMAN-tumor Transcriptome or genome
MOUSE-stromaTranscriptome or genome
Pancreatic
Adenocarcinoma
n=30
SMAP
Application to pancreatic cancer xenograft gene expression analysis
Stromal cell (mouse)
Tumor cell (human)PDX Patient Derived Xenograft
Mix of Human and
Mouse reads
RNAseq
HUMAN-tumor Transcriptome or genome
MOUSE-stromaTranscriptome or genome
Pancreatic
Adenocarcinoma
n=30
SMAP
Stroma signalCount matrix
Tumor signalCount matrix
Pathways analysis
PD
AC
001.T
PD
AC
003.T
PD
AC
004.T
PD
AC
005.T
PD
AC
006.T
PD
AC
007.T
PD
AC
008.T
PD
AC
009.T
PD
AC
032.T
PD
AC
011.T
PD
AC
012.T
PD
AC
013.T
PD
AC
014.T
PD
AC
015.T
PD
AC
016.T
PD
AC
017.T
PD
AC
018.T
PD
AC
019.T
PD
AC
020.T
PD
AC
021.T
PD
AC
022.T
PD
AC
023.T
PD
AC
024.T
PD
AC
025.T
PD
AC
026.T
PD
AC
027.T
PD
AC
028.T
PD
AC
029.T
PD
AC
030.T
PD
AC
031.T
PD
AC
001.S
PD
AC
003.S
PD
AC
004.S
PD
AC
005.S
PD
AC
006.S
PD
AC
007.S
PD
AC
008.S
PD
AC
009.S
PD
AC
032.S
PD
AC
011.S
PD
AC
012.S
PD
AC
013.S
PD
AC
014.S
PD
AC
015.S
PD
AC
016.S
PD
AC
017.S
PD
AC
018.S
PD
AC
019.S
PD
AC
020.S
PD
AC
021.S
PD
AC
022.S
PD
AC
023.S
PD
AC
024.S
PD
AC
025.S
PD
AC
026.S
PD
AC
027.S
PD
AC
028.S
PD
AC
029.S
PD
AC
030.S
PD
AC
031.S
CytokeratinsNucleotide excision repairDNA replicationRNA transportCell cycleHomologous recombinationMismatch repairSpliceosomePyrimidine metabolismBase excision repairRibosome biogenesis in eukaryotesRNA degradationUbiquitin mediated proteolysisHistidine metabolismSteroid biosynthesisPeroxisomeGlycerophospholipid metabolismBiosynthesis of unsaturated fatty acidsVitamin digestion and absorption
LysosomeB cell receptor signaling pathwayHematopoietic cell lineageCell adhesion molecules (CAMs)Natural killer cell mediated cytotoxicityChemokine signaling pathwayCytokine-cytokine receptor interactionRegulation of actin cytoskeletonECM-receptor interactionCalcium signaling pathwayLeukocyte transendothelial migrationToll-like receptor signaling pathwayFocal adhesionVascular smooth muscle contraction
Tumor/human Stroma/murine
0.4
0.2
0
-0.2
-0.4
+
-
Pathways analysis
PD
AC
001.T
PD
AC
003.T
PD
AC
004.T
PD
AC
005.T
PD
AC
006.T
PD
AC
007.T
PD
AC
008.T
PD
AC
009.T
PD
AC
032.T
PD
AC
011.T
PD
AC
012.T
PD
AC
013.T
PD
AC
014.T
PD
AC
015.T
PD
AC
016.T
PD
AC
017.T
PD
AC
018.T
PD
AC
019.T
PD
AC
020.T
PD
AC
021.T
PD
AC
022.T
PD
AC
023.T
PD
AC
024.T
PD
AC
025.T
PD
AC
026.T
PD
AC
027.T
PD
AC
028.T
PD
AC
029.T
PD
AC
030.T
PD
AC
031.T
PD
AC
001.S
PD
AC
003.S
PD
AC
004.S
PD
AC
005.S
PD
AC
006.S
PD
AC
007.S
PD
AC
008.S
PD
AC
009.S
PD
AC
032.S
PD
AC
011.S
PD
AC
012.S
PD
AC
013.S
PD
AC
014.S
PD
AC
015.S
PD
AC
016.S
PD
AC
017.S
PD
AC
018.S
PD
AC
019.S
PD
AC
020.S
PD
AC
021.S
PD
AC
022.S
PD
AC
023.S
PD
AC
024.S
PD
AC
025.S
PD
AC
026.S
PD
AC
027.S
PD
AC
028.S
PD
AC
029.S
PD
AC
030.S
PD
AC
031.S
CytokeratinsNucleotide excision repairDNA replicationRNA transportCell cycleHomologous recombinationMismatch repairSpliceosomePyrimidine metabolismBase excision repairRibosome biogenesis in eukaryotesRNA degradationUbiquitin mediated proteolysisHistidine metabolismSteroid biosynthesisPeroxisomeGlycerophospholipid metabolismBiosynthesis of unsaturated fatty acidsVitamin digestion and absorption
LysosomeB cell receptor signaling pathwayHematopoietic cell lineageCell adhesion molecules (CAMs)Natural killer cell mediated cytotoxicityChemokine signaling pathwayCytokine-cytokine receptor interactionRegulation of actin cytoskeletonECM-receptor interactionCalcium signaling pathwayLeukocyte transendothelial migrationToll-like receptor signaling pathwayFocal adhesionVascular smooth muscle contraction
Tumor/human Stroma/murine
0.4
0.2
0
-0.2
-0.4
+
-
Pathways analysis
PD
AC
001.T
PD
AC
003.T
PD
AC
004.T
PD
AC
005.T
PD
AC
006.T
PD
AC
007.T
PD
AC
008.T
PD
AC
009.T
PD
AC
032.T
PD
AC
011.T
PD
AC
012.T
PD
AC
013.T
PD
AC
014.T
PD
AC
015.T
PD
AC
016.T
PD
AC
017.T
PD
AC
018.T
PD
AC
019.T
PD
AC
020.T
PD
AC
021.T
PD
AC
022.T
PD
AC
023.T
PD
AC
024.T
PD
AC
025.T
PD
AC
026.T
PD
AC
027.T
PD
AC
028.T
PD
AC
029.T
PD
AC
030.T
PD
AC
031.T
PD
AC
001.S
PD
AC
003.S
PD
AC
004.S
PD
AC
005.S
PD
AC
006.S
PD
AC
007.S
PD
AC
008.S
PD
AC
009.S
PD
AC
032.S
PD
AC
011.S
PD
AC
012.S
PD
AC
013.S
PD
AC
014.S
PD
AC
015.S
PD
AC
016.S
PD
AC
017.S
PD
AC
018.S
PD
AC
019.S
PD
AC
020.S
PD
AC
021.S
PD
AC
022.S
PD
AC
023.S
PD
AC
024.S
PD
AC
025.S
PD
AC
026.S
PD
AC
027.S
PD
AC
028.S
PD
AC
029.S
PD
AC
030.S
PD
AC
031.S
CytokeratinsNucleotide excision repairDNA replicationRNA transportCell cycleHomologous recombinationMismatch repairSpliceosomePyrimidine metabolismBase excision repairRibosome biogenesis in eukaryotesRNA degradationUbiquitin mediated proteolysisHistidine metabolismSteroid biosynthesisPeroxisomeGlycerophospholipid metabolismBiosynthesis of unsaturated fatty acidsVitamin digestion and absorption
LysosomeB cell receptor signaling pathwayHematopoietic cell lineageCell adhesion molecules (CAMs)Natural killer cell mediated cytotoxicityChemokine signaling pathwayCytokine-cytokine receptor interactionRegulation of actin cytoskeletonECM-receptor interactionCalcium signaling pathwayLeukocyte transendothelial migrationToll-like receptor signaling pathwayFocal adhesionVascular smooth muscle contraction
Tumor/human Stroma/murine
Cell cycle
0.4
0.2
0
-0.2
-0.4
+
-
Pathways analysis
PD
AC
001.T
PD
AC
003.T
PD
AC
004.T
PD
AC
005.T
PD
AC
006.T
PD
AC
007.T
PD
AC
008.T
PD
AC
009.T
PD
AC
032.T
PD
AC
011.T
PD
AC
012.T
PD
AC
013.T
PD
AC
014.T
PD
AC
015.T
PD
AC
016.T
PD
AC
017.T
PD
AC
018.T
PD
AC
019.T
PD
AC
020.T
PD
AC
021.T
PD
AC
022.T
PD
AC
023.T
PD
AC
024.T
PD
AC
025.T
PD
AC
026.T
PD
AC
027.T
PD
AC
028.T
PD
AC
029.T
PD
AC
030.T
PD
AC
031.T
PD
AC
001.S
PD
AC
003.S
PD
AC
004.S
PD
AC
005.S
PD
AC
006.S
PD
AC
007.S
PD
AC
008.S
PD
AC
009.S
PD
AC
032.S
PD
AC
011.S
PD
AC
012.S
PD
AC
013.S
PD
AC
014.S
PD
AC
015.S
PD
AC
016.S
PD
AC
017.S
PD
AC
018.S
PD
AC
019.S
PD
AC
020.S
PD
AC
021.S
PD
AC
022.S
PD
AC
023.S
PD
AC
024.S
PD
AC
025.S
PD
AC
026.S
PD
AC
027.S
PD
AC
028.S
PD
AC
029.S
PD
AC
030.S
PD
AC
031.S
CytokeratinsNucleotide excision repairDNA replicationRNA transportCell cycleHomologous recombinationMismatch repairSpliceosomePyrimidine metabolismBase excision repairRibosome biogenesis in eukaryotesRNA degradationUbiquitin mediated proteolysisHistidine metabolismSteroid biosynthesisPeroxisomeGlycerophospholipid metabolismBiosynthesis of unsaturated fatty acids
LysosomeB cell receptor signaling pathwayHematopoietic cell lineageCell adhesion molecules (CAMs)Natural killer cell mediated cytotoxicityChemokine signaling pathwayCytokine-cytokine receptor interactionRegulation of actin cytoskeletonECM-receptor interactionCalcium signaling pathwayLeukocyte transendothelial migrationToll-like receptor signaling pathwayFocal adhesionVascular smooth muscle contraction
Tumor/human Stroma/murine
Vitamin digestion and absorption
0.4
0.2
0
-0.2
-0.4
+
-
Pathways analysis
PD
AC
001.T
PD
AC
003.T
PD
AC
004.T
PD
AC
005.T
PD
AC
006.T
PD
AC
007.T
PD
AC
008.T
PD
AC
009.T
PD
AC
032.T
PD
AC
011.T
PD
AC
012.T
PD
AC
013.T
PD
AC
014.T
PD
AC
015.T
PD
AC
016.T
PD
AC
017.T
PD
AC
018.T
PD
AC
019.T
PD
AC
020.T
PD
AC
021.T
PD
AC
022.T
PD
AC
023.T
PD
AC
024.T
PD
AC
025.T
PD
AC
026.T
PD
AC
027.T
PD
AC
028.T
PD
AC
029.T
PD
AC
030.T
PD
AC
031.T
PD
AC
001.S
PD
AC
003.S
PD
AC
004.S
PD
AC
005.S
PD
AC
006.S
PD
AC
007.S
PD
AC
008.S
PD
AC
009.S
PD
AC
032.S
PD
AC
011.S
PD
AC
012.S
PD
AC
013.S
PD
AC
014.S
PD
AC
015.S
PD
AC
016.S
PD
AC
017.S
PD
AC
018.S
PD
AC
019.S
PD
AC
020.S
PD
AC
021.S
PD
AC
022.S
PD
AC
023.S
PD
AC
024.S
PD
AC
025.S
PD
AC
026.S
PD
AC
027.S
PD
AC
028.S
PD
AC
029.S
PD
AC
030.S
PD
AC
031.S
CytokeratinsNucleotide excision repairDNA replicationRNA transportCell cycleHomologous recombinationMismatch repairSpliceosomePyrimidine metabolismBase excision repairRibosome biogenesis in eukaryotesRNA degradationUbiquitin mediated proteolysisHistidine metabolismSteroid biosynthesisPeroxisomeGlycerophospholipid metabolismBiosynthesis of unsaturated fatty acidsVitamin digestion and absorption
B cell receptor signaling pathwayHematopoietic cell lineage
Natural killer cell mediated cytotoxicityCytokine-cytokine receptor interactionRegulation of actin cytoskeletonECM-receptor interaction
Leukocyte transendothelial migrationToll-like receptor signaling pathwayFocal adhesionVascular smooth muscle contraction
Tumor/human Stroma/murine
0.4
0.2
0
-0.2
-0.4
+
-
Immune populations (MCPcounter tool, Becht et al. 2016)
CD8 T cells
Cytotoxic lymphocytes
T cells
NK cells
B lineage
Neutrophils
Monocytic lineage
Endothelial cells
Fibroblasts
PD
AC
00
1.T
PD
AC
00
3.T
PD
AC
00
4.T
PD
AC
00
5.T
PD
AC
00
6.T
PD
AC
00
7.T
PD
AC
00
8.T
PD
AC
00
9.T
PD
AC
03
2.T
PD
AC
01
1.T
PD
AC
01
2.T
PD
AC
01
3.T
PD
AC
01
4.T
PD
AC
01
5.T
PD
AC
01
6.T
PD
AC
01
7.T
PD
AC
01
8.T
PD
AC
01
9.T
PD
AC
02
0.T
PD
AC
02
1.T
PD
AC
02
2.T
PD
AC
02
3.T
PD
AC
02
4.T
PD
AC
02
5.T
PD
AC
02
6.T
PD
AC
02
7.T
PD
AC
02
8.T
PD
AC
02
9.T
PD
AC
03
0.T
PD
AC
03
1.T
PD
AC
00
1.S
PD
AC
00
3.S
PD
AC
00
4.S
PD
AC
00
5.S
PD
AC
00
6.S
PD
AC
00
7.S
PD
AC
00
8.S
PD
AC
00
9.S
PD
AC
03
2.S
PD
AC
01
1.S
PD
AC
01
2.S
PD
AC
01
3.S
PD
AC
01
4.S
PD
AC
01
5.S
PD
AC
01
6.S
PD
AC
01
7.S
PD
AC
01
8.S
PD
AC
01
9.S
PD
AC
02
0.S
PD
AC
02
1.S
PD
AC
02
2.S
PD
AC
02
3.S
PD
AC
02
4.S
PD
AC
02
5.S
PD
AC
02
6.S
PD
AC
02
7.S
PD
AC
02
8.S
PD
AC
02
9.S
PD
AC
03
0.S
PD
AC
03
1.S
Tumor/human Stroma/murine
2 1 0 -1 -2
SMAP: Overall approach
SMAPStromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
Read assigned to HUMAN
*
*
**
Read assigned to MOUSE
* *
*
*
*
*
Discarded read
* mismatch
HUMAN-tumor Transcriptome or genome
MOUSE-stromaTranscriptome or genome
SMAP: Overall approach
SMAPStromal cell (mouse)
Tumor cell (human)PDX
Patient Derived Xenograft
Mix of Human and
Mouse readsSequencing
Mouse Genome Human Genome
Concatenated Genome
+
1. SIMULTANEOUS MAPPING ON BOTH GENOMES
2. EVALUATION OF EACH READ’S ALIGNMENTS
Read assigned to HUMAN
*
*
**
Read assigned to MOUSE
* *
*
*
*
*
Discarded read
* mismatch
HUMAN-tumor Transcriptome or genome
MOUSE-stromaTranscriptome or genome
SMAP-fuzSMAP-fuz
SMAP-fuz: Fusion detection strategy
Spurious fusions (human-
murine fusions)
Spanning: null distribution(Negative binomial)
Junction: null distribution (Negative Binomial)
human-human fusions
RAW FUSIONS
P-value for each human-
human fusion spanning count
P-value for each human-
human fusion junction count
• Combining P-value
• FDR correction
50bp 100bp
0
200400
Nu
mb
er
of
fus
ion
s
Fusion detection performance
Read and fusions simulation settings
• 50M reads human
transcriptome
• 20M reads mouse
transcriptome
• 500 000 from 100 simulated
human fusions transcripts.
Reads were simulated using the Mutations rate 0.01 and errors 0.1 %
100
TotTPFP
FN
Application to pancreatic cancer xenograft gene expression analysis
0%
5%
10%
15%
all
STAR-Fusion
fdr 5% fdr 1% fdr 0.1%
Fa
lse
Fu
sio
ns
(in
%)*
STAR_Fusion + SMAP-fuz
filter
Pancreatic
Adenocarcinoma
n=30
*inter-specie fusions
SMAP on Github https://remynicolle.github.io/SMAP/
Sup. Info