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 cell (human)PDX

Patient Derived Xenograft

Mix of Human and

Mouse readsRNAseq

Mix of Human and

Mouse reads

HUMAN-tumor Transcriptome or genome

MOUSE-stromaTranscriptome or genome

RNAseq

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

Mix of Human and

Concatenated Genome

1. SIMULTANEOUS MAPPING ON BOTH GENOMES

2. EVALUATION OF EACH READ’S ALIGNMENTS

Mix of Human and

Concatenated Genome

Read assigned to HUMAN

* mismatch

Mix of Human and

Concatenated Genome

Read assigned to MOUSE

* mismatch

Mix of Human and

Concatenated Genome

Discarded read

* mismatch

Mix of Human and

Concatenated Genome

Discarded read

Mix of Human and

Concatenated Genome

Discarded read

Mapping and specie assignment performance

Read simulation settings*

*fastq simulation using wgsim tool(Escalona et al., 2016)

DirectMapping

Xenome

Comparisons

Mapping and specie assignment performance

DirectMapping

Xenome

Ideal Mutations Mutations

& Errorsideal Mutations Mutations

& Errors

te 50 bp 100 bp

rate 50 bp 100 bp

0.25‰

0.50‰

Ideal Mutations Mutations& Errors

Ideal Mutations Mutations

& Errors

Estimating the impact of inter-specie gene homology

DirectMapping

Xenome

50 bp 100 bp

te>95 95-90 90-85 85-80 <80 >95 95-90 90-85 85-80 <80

Identity(in %)

0.25‰

0.50‰

0.75‰

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

Tumor cell (human)PDX Patient Derived Xenograft

Mix of Human and

Mouse reads

RNAseq

Pancreatic

Adenocarcinoma

Stroma signalCount matrix

Tumor signalCount matrix

Pathways analysis

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

Pathways analysis

Cell cycle

Pathways analysis

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

Pathways analysis

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

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

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

200400

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 %

TotTPFP

STAR-Fusion

fdr 5% fdr 1% fdr 0.1%

STAR_Fusion + SMAP-fuz

filter

Pancreatic

Adenocarcinoma

*inter-specie fusions

SMAP on Github https://remynicolle.github.io/SMAP/

Sup. Info

SMAP: sequencing patient-derived xenografts

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