Innovations in Single-Cell genomics
Paul Lacaze, PhD
Single cells - What’s really going on?
How heterogeneous is my cell population?
Fibroblasts
90
80
70
60
50
40
30
20
10
0
Pexp S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30
Data – Dr. Jose Polo
Transcriptional heterogeneity – is it phenotypic?
Single-Cell Genomics
Critical populations:
Development
Neurology
Immunology
Stem Cells
Cancer
…
Single cells drive the biological system
Publications in Single-Cell Genomics
Number of cells
Single-Cell Genomics
average
Transcripts per cell
Variability in Housekeeping Gene Expression
ActB mRNA - mouse islet cells
Bengtsson, Ståhlberg, Rorsman, & Kubista (2005) Genome Research 15:1388-1392
How is single-cell analysis done?
Cell Isolation
Cell Preparation
Data Acquisition
Data Analysis
where the most technical variability exists
Select &
Enrich
Isolate
Image
Verify
Manipulate
- Consistently isolate single cells
- Start with very limited material
- Many cell replicates required
- Many genes must be measured
Extract &
Purify
Detect
Analyze
Challenges in Single Cell Genomics
• Isolating single cells consistently
• Starting with limited material
• Cell lysis
• Reverse transcription
• Amplification
• Transcriptional heterogeneity
• High number of cell replicates required per group
• High number of genes required to reflect biology
• New data analysis approaches required
A simplified workflow
Enrich
Load
&
Capture
Wash
&
Stain
Isolate
C1 Single-Cell Auto Prep
System
Image
Lyse, RT,
Pre-amp
&
Harvest
Transfer
Load
Amplify
& Detect
BioMark System
Fluidigm C1 AutoPrep System
-
Captures single cells in 96
individual capture sites
-
Automated lysis of single
cells
-
Automated reverse
transcription of single cells
-
Each cell ready for RT-PCR
or RNA-Seq analysis
Advantages of Fluidigm Technology
Network of microfluidic channels,
chambers, and valves
Automatically assemble thousands of
individual PCR reactions
Greatly decrease number of pipetting
steps
Decrease amount of sample and
reagent used
Automated cell capture using the Fluidigm C1 system
13
What is the C1™ System?
C1™ Single-Cell Auto Prep
Array
C1™ Single-Cell Auto Prep
Kit
C1™ Single-Cell Auto Prep
System
A proprietary array for capture
& highly paralleled preparation
of 96 individual cells
A pre-formulated reagent kit to
support cell suspension, lysis &
purification*
A breakthrough, bench-top
system that automates the
isolation, lysis and preamplification from single-cells
*Single Cells to Ct, controls,
stain & assays sold separately
Captured
and washed
cell
Live dead Staining
Ethidium homodimer-1
Cell Capture and Staining
Preamplification Implementation in Chip
4.5nl
9 nl
9 nl
9 nl
135 nl
135 nl
Target-Specific Preamplification with pooled primers
Advantages of the C1 System
- Input required only 500-1000 cells
- Automated cell lysis (no nucleic acid extraction required)
- Automated reverse transcription (nanolitre volume)
- Cells prepared for RT-PCR or RNA-seq analysis with minimal
hands on time
Pipetting Map for C1 System
Waste
Waste
Output
s
Output
s
#1
#2
#3
#4
#5
#6
#7
#8
C1 Harvest Reagent
C1 Cell Wash Buffer
Lysis Mix
C1 Harvest Reagent
LIVE/DEAD Staining Solution
RT Mix
Cell Input
C1 Preloading Reagent
PreAmp Mix
Cell Outlet
C1 Harvest Reagent
C1 Chip Products to Dynamic Array
Preamplified cDNA
in 96-well plate
sample
outlets
BioMark Gene Expression Workflow
96 cDNA Samples
96 Assays
(primer pairs)
9,216 Reactions on Fluidigm BioMark HD
BioMark HD™ Real-Time PCR System
High-throughput qPCR based microfluidic technology for DNA,
RNA, miRNA analysis and next-gen library prep
Heat Map of Single Cell Gene Expression
96 Genes
Ct
Live Cells
Live/Dead
Cells
No Cells
Bulk Tube Controls
RNA spikes
Heat Map of Single Cell Gene Expression
96 Genes
Ct
Live Cells
Bulk Tube Controls
No Cells
RNA spikes
Time to results
Fluidigm C1 – Day 1
Capture, Wash
Lysis, RT, Pre-amp &
Real-time
& Stain
Harvest
PCR
2 hrs.
+
6 hrs.
+
Cells to Data: 11-12 hrs.
3-4 hrs.
Fluidigm
BioMark HD
Day 2
Fluidigm BioMark vs 384 QPCR
Case Study – Dr. Jose Polo
6 different populations X 30 cells X 28 targets= 5040 X 3 = 15120
By 384 PCR = 40 plates
By Fluidigm BioMark = 2 plates (96x96)
Cell size is important
K562 8-11-11
K562
HL60
120
250
Leukemia cell
Ave. diam = 10 microns
200
150
100
80
60
100
Myelogenous Leukemia
cell
Ave. diam = 14
40
HL60
50
20
K562 8-11-11
0
0
BJ fibroblasts
BJ Fibroblasts
photoshoped direct selection
Keratinocytes 3-19-11
90
80
70
60
50
40
30
20
10
0
Fibroblast
Ave. diam = 16
microns
Keratinocytes 3-19-11 Av.
diam= 17.3 +/- 4.1 Max.
diam=
Min.
diam.=
Av.
diam42.8
= 17.3
+/4.1
Max
11 diam
Average
= 42.8
AR= 1.12 +/0.09
Max.= AR=
Min
diam
11 1.76 Min.
AR= 1 N=257
Average
AR= 1.12 +/-
0.09
Min AR =1
N=257
50
45
40
35
30
25
20
15
10
5
0
Fibroblast
BJ Fibroblasts 07-27-11
Ave. diam = 25 microns
Av. diam= 23.66 +/- 5.23
Max diam= 42.51
Min diam= 11.65
Average AR= 1.28+/- 0.22
Max AR= 2.02
Min AR=1
n= 251
Fluidigm C1 Capture Plates
Different C1 chips optimized to isolate cells by cell size:
5-10 micron new: for small cells such as stem cells
and WBC
10-17 micron: for medium cells such as iPS,
progenitor cells, neurons, and others
17-25 micron: for large cells such as fibroblasts,
keratinocytes, cardiomyocytes, smooth muscle cells,
and others
Commercial Application Roadmap
Targeted Gene Expression (STA):
Q3 2012
mRNA sequencing:
Q1 2013
Whole genome amplification (DNA Sequencing):
mid 2013
A simplified workflow for Single Cell mRNA-Seq Library Prep
Enrich
Load
&
Capture
Wash
&
Stain
Isolate
C1 Single-Cell Auto Prep System
Clontech
SMRTer
Nextera
Lyse, RT
&
Amplify
Prepare
Library
Sequence
Analyze
Any Illumina System
mRNA Amplification
SMARTer (Clontech)
Template-Switching Method
• Works directly from
cell lysate
• No RNA Fragmentation
• PolyA+ RNA
• Produces long ds cDNA
RNASeq Data
13 hours from cells to sequence-ready libraries with only 3
hours of hands on time
Capture & prepare
cells
C1 Reagent Kit & C1 IFC
2 hours
Reverse transcribe &
amplify
Clontech SMARTer Kit & C1 IFC
9 hours
Prepare sequence
library
Nextera XT Kit
2 hours
Library preparation
Nextera XT (Illumina)
Transposase-based Method
• Simple workflow
cDNA
• No mechanical
fragmentation
• Integrated barcoding
RNASeq Data
Number of Detected RefSeq Transcripts
Detected RefSeq Transcripts
20,000
0
48 Individual Cells (K562)
% Ribosomal Reads
1.00 %
% of reads
0%
Thank you
Paul Lacaze, PhD - [email protected]
Open Nanoflex Valve
Control Line
Fluid Line
Closed Nanoflex Valve
Control Line
Fluid Line
Fluid Line
C1 AutoPrep Array Architecture
Load 500-1000
cells in suspension
Automated cell
capture using
microfluidics
Each cell confined
to its own reaction
chamber
The workflow is fast and easy
Prime
Pipette
Load
15 min
30 min
90 min
PCR/Scan
90 min
Opportunities for Improvement
Cell Selection
FACS
or
Manual
Method
Large number
of cells required
or
labor intensive
RT-STA*
Single Cell
RNA
No verification.
0, 1, >1 cell?
cDNA
STA
Expensive Chemistry;
“only” 96 gene STA;
Labor intensive
* STA, or Specific Target Amplification,
pre-amplifies target cDNA
Specific Target Amplification (STA)
Multiplex
Primer Pool
(0.2X or 500 nM)
+
DNA sample
(low conc)
+
14 cycles
Preamplified
DNA
Dilution
Done in 96 or 384-well plates
“off-chip”
Multiplex PCR
Master Mix
How do we analyze single cell data?
Violin plots of expression levels
Distribution of
gene expression
for individual
genes
Hierarchical Cluster analysis
C1 Single-Cell AutoPrep System
Single-cell precision
Easy to use
Single Cells to Ct in <24 hrs
Verify # cells & live/dead
Forward Compatible
Thank you!
Paul Lacaze
[email protected]