smap: sequencing patient-derived xenografts

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


Tumor cell (human)PDX

Patient Derived Xenograft

Mix of Human and

Mouse readsRNAseq

Goal




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


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


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




Mix of Human and

Mouse readsSequencing







Mix of Human and


Mouse Genome Human Genome

Concatenated Genome

+

SMAP





Mix of Human and



Concatenated Genome

+

1. SIMULTANEOUS MAPPING ON BOTH GENOMES

2. EVALUATION OF EACH READ’S ALIGNMENTS

SMAP





Mix of Human and



Concatenated Genome

+



Read assigned to HUMAN

*

*

**

* mismatch

SMAP





Mix of Human and



Concatenated Genome

+




*

*

**

Read assigned to MOUSE

* *

*

*

* mismatch

SMAP





Mix of Human and



Concatenated Genome

+




*

*

**


* *

*

*

*

*

Discarded read

* mismatch

SMAP





Mix of Human and



Concatenated Genome

+

SMAP




*

*

**


* *

*

*

*

*

Discarded read





Mix of Human and



Concatenated Genome

+



SMAP




*

*

**


* *

*

*

*

*

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


Tumor cell (human)PDX Patient Derived Xenograft

Mix of Human and

Mouse reads

RNAseq



Pancreatic

Adenocarcinoma

n=30

SMAP



Tumor cell (human)PDX Patient Derived Xenograft

Mix of Human and

Mouse reads

RNAseq



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




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



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


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


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


2 1 0 -1 -2


SMAPStromal cell (mouse)



Mix of Human and



Concatenated Genome

+




*

*

**


* *

*

*

*

*

Discarded read

* mismatch




SMAPStromal cell (mouse)



Mix of Human and



Concatenated Genome

+




*

*

**


* *

*

*

*

*

Discarded read

* mismatch



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


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

smap: sequencing patient-derived xenografts

Documents