Single-Cell Analysis - Powered by REPLI-g: Single Cell Analysis Series Part 1

Sample to Insight

Single-Cell Analysis – From Sample to Insight

Single Cell Analysis - From Sample to Insight, Hilden, Oct. 2015 1

Courtney NadeauSr. Global Product Manager, NGS Life Sciences, QIAGEN Hilden, Germany

Sample to Insight

2Single Cell Analysis - From Sample to Insight, Hilden, Oct. 2015

Legal disclaimers

The QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

Sample to Insight

Overview


Overview• Why study single cells

o Scarce sampleo Genome heterogeneityo Transcriptome heterogeneityo Statistical power

• Basic parts of a single-cell workflowo Cell isolationo WGA or WTAo Analytical techniqueso Data analysis

• QIAGEN products for single cell analysis

Sample to Insight

Studying limited amounts of material


Scarce Sample Material• Allows the analysis of cells

from small and precious starting materials:

o Circulating tumor cells (CTCs)

o Cells from small biopsies o Cells from in vitro

fertilized embryos o Microorganisms from

environmental samples

Standard NGS Library Prep

Input:100-1000ng

Bacterium Mammalian cell

200 µl Blood

1 µg

1 ng

1 pg

1 fg

Average DNA content

Sample to Insight

Genome heterogeneity


Genome Heterogeneity• Allows investigation into genomic

heterogeneity between individual cells:

o Copy number alterationso Structural alterations and

rearrangementso Single nucleotide mutationso Variability in transposable

element or viral genome integration

Zhengwen Jiang et al. Nucl. Acids Res. 2005;33:e91

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Transcriptome heterogeneity

Single Cell Analysis - From Sample to Insight, Hilden, Oct. 2015

Transcriptome Heterogeneity• Investigate differences in

transcript expression and gene regulation in individual cells:

o Differences in transcript abundance

o Usage of alternate transcription initiation and polyadenylation sites

o Alternative splicing and differential expression of transcript isoforms

Zeisel A. et al. (2014) Brain Structure. Cell types of the cortex and hippocampus revealed by single-cell RNA-seq, Science 347(6226):1138-42

Sample to Insight

Averaging averages


The basic unit of research we are often interested in is the cell. But we usually analyze populations of cells and this can:• Lead to false positives from underestimating biological variability• Miss important biological divisions

0 0

0 3

0 0

0 0

0 0

0 6

0 6

0 0

0,938

Biological Sample 1

Biological Sample 2Population

Mean 2

1

Single Cell Analysis

Population Mean 1

Mean=0,969Stdev=1,470

Sample Size=32SEM=0,260

1 1

1

1 1

1 1

1 1

1 1

1 1

1 1

1

Mean=0,969Stdev=0,048

Sample Size=2SEM=0,031

Bulk Approach

Sample to Insight

Title, Location, Date 8

Wide array of applications for single-cell analysis

WGAor

WTA

Whole Genome Sequencing• Detect variability in genome sequence (SNV, microsatellites, etc.)• Variability in genome structure (CNV, structural rearrangements,

aneuploidy)• De novo sequencing of new, unidentified and unculturable organisms

Targeted Resequencing• Detect variability in a target set of genes or region of the genome

Microarrays• Use SNP-chips to genotype thousands of loci

mRNA-seq• Detect variability in transcript abundance for all expressed genes• Detect variability in isoform structure and abundance

qRT-PCR profiling• Profile gene expression for a targeted set of transcripts• Accurately quantify specific splice-junctions, isoforms or other structural

features

Sample to Insight

Single cell workflow overview


In general, single cell molecular biology experiments follow a general workflow:• Obtain primary sample• Detect and isolate cell of interest• Lyse cell (often integrated with WGA or

WTA)• Whole genome or whole transcriptome

amplification (for DNA/RNA studies)• Analytical technique of choice (NGS

library prep and sequencing, gene panels, real-time PCR, microarrays, sanger sequencing)

• Data analysis and interpretation

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In general, single cell molecular biology experiments follow a general workflow• Obtain primary sample• Detect and isolate cell of interest• Lyse cell (often integrated with WGA or



library prep and sequencing, gene panels, real-time PCR, microarrays, sanger sequencing)


Sample to Insight

Single cell isolation: cell suspensions


Isolating single cells:• Cells in suspension?

Dilution (cell suspension) • Cheap, but labor intensive • Negative wells• Mild conditions, easy on cells

Micromanipulation (cell culture) • Transfer pipet required • Mild conditions, easy on cells• Labor intensive• Obtain specific cell of interest

FACS (Fluorescence-activated cell sorting)• Efficient for isolating large numbers of cells,

needs high number of input cells• Expensive instrumentation required• Sorting alters RNA expression profiles

Microfluidics• Efficient for isolating large numbers of

cells, needs high number of input cells• Expensive instrumentation required

Image adapted from: Genome Biology. 2014 : 15(452): . doi:10.1186/s13059-014-0452-9

Sample to Insight

Single cell isolation: tissue


Isolating single cells:• Cells in suspension?• Solid Sample

Laser-capture microdissection (tissue)• Direct isolation of single (or multiple) cells• Loss of genomic information – slicing of nuclei• Mounting and LCM both disturb gene expression

Tissue dissociation• Mechanical, detergent and enzymatic disruption• Produce a suspension from a solid tissue• Potential for drastic alterations in gene expression

Image adapted from: Genome Biology. 2014 : 15(452): . doi:10.1186/s13059-014-0452-9

Sample to Insight

Single cell isolation: required throughput


Type of Primary sample?• Solid or Suspension?• Viability and Cell Stress• Scope and throughput:

• Depends on application• Depends on cells• Depends on budget

Cell-to-cellvariation

Biologicalvariation

Technicalvariation

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Single cell isolation: considerations


Type of Primary sample?• Solid or Suspension?• Viability and Cell Stress• Scope and throughput• Cell Storage and Transport

o Uninterrupted cold chaino Minimize storage time

when possibleo Keep in mind small

volumes

Sample to Insight

Single cell isolation: contamination


Type of Primary sample?• Solid or Suspension?• Viability and Cell Stress• Scope and throughput• Cell Storage and Transport• Contamination

o With intact cellso With DNA

Figure is taken from: FEMS Microbiol Rev. 2013 May ; 37(3): . doi:10.1111/1574-6976.12015.

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library prep and sequencing, gene panels, real-time PCR, microarrays, Sanger sequencing)


Sample to Insight

Whole genome or transcriptome amplification


Whole Genome (WGA) and Whole Transcriptome (WTA) Amplification • Uses one of several techniques to amplify

the DNA or RNA from a single cell to a level amenable to downstream protocols

PCR-Based

-Degenerative oligo-primer PCR (DOP-PCR)

-Multiple annealing and looping based amplification

cycles (MALBAC)

PCR-Free

-Multiple Displacement Amplification (MDA)

-Single Primer Isothermal Amplification (SPIA)

Whole Genome/Transcriptome Amplification Technologies

Sample to Insight

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Analytical techniques


Depends on the research topic:• PCR and Sanger sequencing for:

o Genes or regions of interest• qPCR (real-time or digital) single or

multiplex assays for:o Specific point mutations o Aneuploidy or structural rearrangementso Quantification of specific transcripts or

transcript variants• Microarrays for:

o RNA quantificationo SNP-chips

• NGS for:o Point mutations, aneuploidy and

structural rearrangementso Copy number variationo RNA quantification, splicing analysis

Sample to Insight

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Data analysis and interpretation


Depends on the research topic:• CLC:

o Sequence Viewero Genomics Workbencho Biomedical Workbench

• Ingenuity:o Variant Analysiso Pathway Analysis

Sample to Insight

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QIAGEN products for single-cell analysis


Complete Sample to Insight Solutions for Single Cell Applications

WGA, WTA or both• REPLI-g portfolio

Cell isolation• Coming soon

Analytical techniques• REPLI-g NGS Library Prep kits• GeneRead Panels• RT2 Profiler PCR arrays• Wide variety of available tools

Data Analysis and Interpretation• CLC bioinformatics software• Ingenuity variant and pathway analysis

Sample to Insight

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The PCR-free REPLI-g protocol


REPLI-g is QIAGEN’s MDA technology, and is incorporated into both WGA and WTA products:• Highly specific phi29 Polymerase• PCR-free isothermal amplification (30°C)• 1000-fold higher fidelity than Taq• Generates long fragments (2–70 kb)• Minimal sequence bias

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REPLI-g for WGA, WTA or both


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Single Cell Multiple Cells

TissueBloodgDNA RNA

Single-cell DNA Sequencing

Single-cell RNA sequencing

REPLI-g Single Cell DNA

Library Kit

REPLI-g Single Cell RNA

Library Kit

NGSLibrary

NGS

Single-cell DNA analysis

Single-cell RNA analysis

Comparative analysis of DNA and RNA

(25+ cells)

REPLI-g Single Cell

Kit

REPLI-g WTA Single

Cell Kit

REPLI-g Cell WGA & WTA

Kit

Amplified WTA- DNA or

WGA-DNA

NGS

Microarray

qPCR

Choosing a REPLI-g Single Cell Kit for your application

Starting material Application Q solution Kit output Analysis


Sample to Insight

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Lower background with REPLI-g


Bacterial DNA (2000 copies) was spiked into REPLI-g sc Reaction Buffer, which was then decontaminated using the standard procedure for all buffers and reagents provided with the REPLI-g Single Cell Kit. In subsequent real-time PCR, no bacterial DNA was detectable.

The PCR-free REPLI-g kits offer:• Minimal background:

o Kits are produced to exceptionally high standards and reagents undergo a unique manufacturing process which virtually eliminates any chance of contamination

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High yield: wide range of applications including archival


The PCR-free REPLI-g kits offer:• Minimal background• High Yield:

o Kits produce 10 µg or more of amplified cDNA or gDNA from a single cell

o Library prep kits produce 2-4 nM of PCR-free sequencer-ready whole genome or RNAseq library

Starting Material Typical YieldREPLI-g Single Cell RNA Library Prep Single cell or purified total RNA (50 pg-100 ng) 2-4 nM PCR-free NGS Library

REPLI-g Single Cell DNA Library Prep Single cell or purified gDNA (10 pg-10 ng) 2-4 nM PCR-free NGS Library

REPLI-g Single Cell Single cell or purified gDNA (1-10 ng) 40 µg amplified gDNA

REPLI-g WTA Single Cell Single cell or purified total RNA (10 pg-100 ng) 40 µg amplified poly(A+) cDNA

REPLI-g Cell WGA & WTA 25+ cells

WTA: 10-20 µg, depending on protocolWGA: 20 µg

Sample to Insight

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Completely PCR-free NGS workflows


The PCR-free REPLI-g kits offer:• Minimal background• High Yield• Integration with PCR-free NGS library prep:

o REPLI-g single cell DNA and RNA library kits produce NGS-ready libraries from a single cell in as little as 5.5 hours

REPLI-g Single Cell DNA Library Kit

Cell lysis15 min

WGA3 h

Shearing and purification30-60 min

End-repair50 min

A-addition40 min

Adapter ligation10 min

Cleanup and size selection

15 min

REPLI-g Single Cell RNA Library Kit

Cell lysis15 min Sequencing

Data AnalysisInterpretation

gDNA Removal10 min

Reverse Transciption

1 hLigation35 min

WTA2 h

One-tube

One-tube

One-tube

Sample to Insight

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Even coverage in whole genome sequencing


The PCR-free REPLI-g kits offer:• Minimal background• High Yield• Integration with PCR-free NGS library prep• Even Coverage:

o Superior genome coverage due to even amplification: fewer drop-outs, missed loci and more accurate quantification

o Important for NGS as well as traditional applications

1 pg DH10B DNA, amplified with either REPLI-g Single Cell Kit or by MALBAC, sequenced on MiSeq Illumina (V2, 2x150nt.)

Sample to Insight

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Fewer dropped loci


The PCR-free REPLI-g kits offer:• Minimal background• High Yield• Integration with PCR-free NGS library prep• Even Coverage

Repli-g 　 coverage Max 3,000

MALBAC 　 coverage Max 3,000

REPLI-g 　Max 153

MALBACMax 4284

Sample to Insight

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Higher transcript discovery rates


The PCR-free REPLI-g kits offer:• Minimal background• High Yield• Integration with PCR-free NGS library prep• Even Coverage

Sample to Insight

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Higher fidelity: fewer sequencing errors


The PCR-free REPLI-g kits offer:• Minimal background• High Yield• Integration with PCR-free NGS

library prep• Even Coverage• Fewer sequence errors:

o Polymerase has ~1000x better proofreading activity than Taq

o Lack of PCR means errors introduced aren’t propagated

Sample to Insight

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Key for evaluating SNV


The PCR-free REPLI-g kits offer:• Minimal background• High Yield• Integration with PCR-free NGS

library prep• Even Coverage• Fewer sequence errors

o Polymerase has ~1000x better proofreading activity than Taq

o Lack of PCR means errors introduced aren’t propagated

o ~10x better error rate than MALBAC(1); essential for SNV analysis

REPLI-g SC MALBAC

Total Reads 3 187 060 3 327 084

Mapped reads 3 176 341 (99,66%)

3 276 090 (98,47%)

Not mapped 10 719 (0,34%) 50 994 (1,53%)

Broken read pairs 284 017 (8,91% of total reads)

314 550 (9,45% of total reads)

Covered bases in Reference

98,69% 95,82%

Insertions 6 3

Deletions 0 6

Single-nucleotide variation

0 222

(1) Bourcy et al. (2014) PLoS ONE 9(8): e105585. doi:10.1371/journal.pone.0105585

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Summary

Advantages of single-cell analysis over bulk data:• Analyze scarce materials• Account for genomic and transcriptomic heterogeneity

Parts of a single-cell workflow:• Obtaining primary sample, detecting and isolating cells of interest• Lysis, WGA or WTA, and variety of molecular biology methods• Data analysis and interpretation

QIAGEN products for single-cell analysis

REPLI-g enables single-cell applications via:• Minimal background• High yield• Integration with PCR-free NGS library prep• Even coverage (manifests as better assembly, fewer drop-outs, better

transcript detection)• Fewer sequence errors


Sample to Insight

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Questions

Q&A

Single-Cell Analysis – From Sample to Insight


Single-Cell Analysis - Powered by REPLI-g: Single Cell Analysis Series Part 1

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