J O S E P H P O W E L L

S I N G L E C E L L A N D C O M P U T A T I O N A L G E N O M I C S G R O U P

INSTITUTE FOR MOLECULAR BIOSCIENCE

Using ultra high-throughput single cell sequencing to understanding

cellular heterogeneity

Complex/Common disease

Environmental factors

Genetics

Background

Genetic control of gene expression and it’s role in human complex disease �  Most common diseases are highly polygenic (GWAS and quantitative genetics) �  Susceptibility variants are located throughout the genome, but not randomly

Using the transcriptome

How do genetic variants influence disease susceptibility? �  Most genetic variation underlying disease susceptibility is

located in regulatory regions �  By what means do most DNA variants alter cellular behavior and

ultimately contribute to differences in disease susceptibility?

Powell et al. PLoS Genetics, 2013 Westra et al. Nature Genetics, 2014 Lloyd-Jones et al. AJHG, 2016

Most genetic effects on transcription are tissue specific

�  GWAS -> eQTL -> Transcriptions -> Cell type specificity

Powell et al. Genome Research 2012 Powell et al. Genome Research 2014 McKenzie et al. BMC Genomics 2014 Hemani et al. Nature 2015 Zhu et al. Nature Genetics, 2016 Powell et al. Human Molecular Genetics 2016

Context Specific Effects

The genetic effect of a loci can vary under different biological or environmental conditions

Powell et al. Human Molecular Genetics 2016 Fung et al. Human Reproduction, 2017

Context-specific genetic association with differential gene expression in IFN-β signaling. (A) A local association (cis-eQTL) with IFNB1 expression for SNP (rs2275888) revealed after 2 hours of LPS stimulation of monocytes. (B) This genetic marker shows association with expression of 17 genes on different chromosomes (trans-eQTLs) after 24 hours of LPS stimulation, forming a gene network Fairfax et al. Science 2014

Technology in the lab

Throughput

Cost

Per

Cel

l

C1 – SMART-Seq

Chromium QX200

***FLOWSeq***

Dropseq

ASCEND - R Package

Analysis of Single Cell Expression, Normalisation and Differential expression

Biological questions

1.  Identification of specific subpopulations in iPSC cultures

2. Mapping transcriptional trajectory across a cell lineage 3. Detecting limited changes between cells

Single-cell transcriptome sequencing of 18,787 human induced pluripotent stem cells identifies differentially primed subpopulations

Under review

Single-cell transcriptome sequencing of 18,787 human induced pluripotent stem cells identifies differentially primed subpopulations

Selection of significant gene predictors for classifying each cluster using LASSO regression

Single-cell transcriptome sequencing of 18,787 human induced pluripotent stem cells identifies differentially primed subpopulations

Mapping changes in the transcriptome across a cell lineage

Day 0 Day 2 Day 5 Day 15 Day 30

Palpalnt et al. Nature Protocols, 2016

Single cell lineage data

Time Cells Day 0 10,952 Day 2 9,431 Day 5 9,282 Day 15 10,828 Day 30 9,896 Average mapped reads per cell 44,000 Median Genes detected per cell 3,143

Clustering

Day0 Day2 Day5

Day15Day30

Merged5days

Statistical models to identify significant changes across the lineage

Module-2: 62 genes Module-1: 74 genes Module-3: 4 genes

Module-4: 302 genes Module-5: 41 genes

•  483 genes are significantly associated with changes in differentiation time

•  The 483 genes are divided into five co-regulated gene expression modules

What subpopulations of cells are transitioning?

6701 5614 133034

2245 1994 1666

3850 2577 2474 926

3520 2783

4038 3001

Day2

Day0

Day5

Day15

Day30

What subpopulations of cells are transitioning?

6701 5614 133034

2245 1994 1666

3850 2577 2474 926

3520 2783

4038 3001

Day2

Day0

Day5

Day15

Day30

Can we detect viral infection in single cells?

�  Live CD45- from epidermis of: ¡  2 x control mice ¡  2x transgenic mice with inserted variably expressed viral

transcript in E7 gene

The total number of TG cells = 3539 The total number of TG cells expressing E7 = 726 = 20.51% of cells express E7

Expressing E7 viral transcript

Thank You

Group Members Sam Lukowski Quan Nguyen Emily Wong Anne Senabouth Rebecca Lim Jose Alquicira Anita Goldinger Keyi Jiang

Collaborators Nathan Palpant (UQ) Alice Pebay (Uni Melb) Alex Hewitt (Uni Melb / UTAS) Ian Frazer (UQ) Kelvin Tuong (UQ) Hanlee Ji (Stanford) --- Geoff Hill & Siok Tey (QIMR) Dan Chambers (Prince Charles) Ernst Wolvetang (UQ) Tonu Esko (Broad/Harvard)