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Application of single cell analysis

Learn about downstream applications after single cell isolation

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QIAGEN has been cited in multiple scientific publications

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Cancer research

Circulating tumor cell research

Neuroscience research

Embryonic genetic research

Metagenomics and microbial single cell genomics

Infectious disease research

Single cell genomic research strategies

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Single cell genomics by QIAGEN, 2016

Sample to Insight

Advancing cancer research: an example of a milestone publication

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Chromothripsis from DNA damage in micronuclei (1)

(1) Zhang, C.-Z. et al. (2015) Chromothripsis from DNA damage in micronuclei. Nature 522, 179–184

“….we achieved the best overall coverage uniformity with this latest version of REPLI-g from Qiagen [REPLI-g Single Cell Kit]...)”

“…MDA gives better overall genome coverage than PCR-based methods ….”

“…Phi-29 polymerase has the highest processivity and the lowest error rate among existing polymerases”

“…the high processivity of Phi-29 polymerase consistently generates large amplicons above 10 kb”

• Selected individual cells based on live-cell imaging• single cell whole-genome sequencing

The superior performance of QIAGEN’s REPLI-g Single Cell Kit has been critical for this milestone research:

Molecular mechanism of chromothripsis identified

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Single cell genomics by QIAGEN, 2016

Sample to Insight

Rare cell analysis towards new liquid biopsy strategies

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Targeted resequencing of rare cells (1)

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(1) Yee, S. S et al (2016) A novel approach for next-generation sequencing of circulating tumor cells. Molecular Genetics & Genomic Medicine. doi: 10.1002/mgg3.210

• Breast cancer cells spiked into whole blood & isolated with DEPArray• Whole genome amplification (WGA) with REPLI-g Single Cell Kit• Targeted resequencing

A proof of concept for molecular analysis of small pools of CTCs

Study assesses• Compatibility of REPLI-g Single Cell Kit with targeted

resequencing◦ Mutations and copy number variations detected

successfully

• Compatibility of REPLI-g Single Cell Kit with blood sample preservation methods ◦ Q/C (Quality and quantity of WGA-DNA and total

depth of coverage) passed

Single cell genomics by QIAGEN, 2016

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Gene expression analysis and cell characterization

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(1) Lee, D. et al. (2015) Interplay between glucose and leptin signaling determines the strength of GABAergic synapses at POMC neurons. Nat. Commun 2015 Mar 26;6:6618(2) Jeong, J.H. (2015) Cholinergic neurons in the dorsomedial hypothalamus regulate mouse brown adipose tissue metabolism, MOLECULAR METABOLISM 4(3) Lee, D. et al. (2015) Apelin-13 Enhances Arcuate POMC Neuron Activity via Inhibiting M-Curren. PLoS ONE 10(3): e0119457.doi:10.1371/journal.pone.0119457

REPLI-g WTA Single Cell Kit overcomes the limits of low RNA quantity, allowing single cells to be analyzed using conventional molecular biology methods

• Generates a high-fidelity, complete copy of the input• Suitable for most downstream NGS and qPCR methods• Allows analysis of splice variants, gene expression and

sequence variations

single cell qRT-PCR analysis helped build signaling pathway models in neurons

Analyzing functional mechanisms in neurons (1, 2, 3)

• Single cell qRT-PCR◦ Single cell whole-transcriptome amplification (WTA) with REPLI-g WTA Single Cell Kit

◦ qPCR analysis

Single cell genomics by QIAGEN, 2016

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Breeding the perfect pony

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(1) Choi, Y.H. et al. (2015) Accuracy of preimplantation genetic diagnosis in equine in vivo recovered and in vitro-produced blastocysts. Reprod. Fertil. Dev. epub ahead of print

REPLI-g MDA technology* outperforms Illustra GenomiPhi

• REPLI-g results were accurate in 98.2% of total loci analyzed• An accuracy of only 25.8% was achieved with illustra

GenomiPhi.

* The authors obtained these results using the REPLI-g Midi Kit. Our recommended solution for very sensitive applications, when starting with a few cells only or even a single cell, is our single cell dedicated solution: the REPLI-g Single Cell Kit.

Screen embryos for specific mutations causing known genetic diseases

Screening equine embryos for preimplantation genetic diagnoses(1)

• Embryo biopsies (10-30 cells)• Whole genome amplification (WGA)• Multiplex PCR designed to detect a variety of heritable equine diseases

Single cell genomics by QIAGEN, 2016

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High-fidelity analysis of genetic aberrations

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REPLI-g is the ideal solution for pre-implantation genetic diagnosis and screening(1) Thornhill, A.R. et al. (2015) Karyomapping—a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan

syndrome, Journal of Assisted Reproduction and Genetics, 32(2) Wells, D. et al. (2015) Clinical utilization of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J Med Genet. 51:8(3) Xu, J. et al. (2015) Embryo Genome Profiling by single cell Sequencing for Preimplantation Genetic Diagnosis in a β-Thalassemia Family Clinical Chemistry 61:4(4) Wang, L. et al. (2014) Detection of Chromosomal Aneuploidy in Human Pre-implantation Embryos by Next Generation Sequencing, Biology of Reproduction

CGH arraySNP array

REPLI-g Single Cell

Kit

Blastomere Amplification Genomic Assay

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• Genome-wide SNP genotyping (1)

• NGS-based strategies for improved ◦ Aneuploidy research (1) ◦ Mutation analysis (2)

◦ Chromosome copy number change (3)

Multiple publications have used REPLI-g technology for embryonic genetic research

NGS on Illumina

sequencer

QIAseq FX Single Cell

DNA Library Kit

Blastomere NGS library prep NGS run

Recommended for aCGH or PCR-based analyses: Recommended for NGS-based analyses:

Consider our newest single cell dedicated solutions for embryo genetic research:

Single cell genomics by QIAGEN, 2016

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Metagenomics & microbial single cell genomics: a winning team

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High-quality draft genomes with metagenomics data mining & single cell sequencing (1)

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• Microbial single cell whole genome sequencing:◦ WGA of FACS-sorted cells using the REPLI-g Single Cell Kit◦ PCR screening for genomes from single cells matching C. Kryptonia SSU rRNA sequences◦ NGS on Illumina MiSeq

(1) Eloe-Fadrosh, E.A. et al. (2016) Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs. Nat. Commun. 7, doi:10.1038/ncomms10476

Discovered a novel bacterial candidate phylum (Candidatus kryptonia)

REPLI-g Single Cell Kit for microbial single cell NGS• Uniform coverage across the genome, for better genome

assembly.• Single cell optimized chemistry to start directly from a

bacterial cell• Controlled decontamination of buffers and reagents

ensuring the elimination of detectable residual DNA contamination

Single cell genomics by QIAGEN, 2016

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Virome determination

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REPLI-g Cell WGA & WTA Kit for total virus amplification (1)

• Multiplexed whole-genome sequencing:◦ DNA and RNA extraction from human samples

◦ rRNA depletion with GeneRead rRNA Depletion Kit

◦ WGA and WTA with REPLI-g Cell WGA and WTA Kit

◦ WGA and WTA fractions sequenced separately on IonTorrent PGM system

(1) Zoll, J. et al. (2015) Direct multiplexed whole genome sequencing of respiratory tract samples reveals full viral genomic information, Journal of Clinical Virology 66, 6–11

Virome determination enabled by parallel pre-amplification of viral DNA and RNA with the REPLI-g Cell WGA & WTA Kit

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A proof-of-concept for virome determination by NGS • Increases chances to detect causative viral agents• Provides rapid availability of highly detailed typing

information• Makes it possible to assemble almost complete viral

genomes potentially determine viral species in all cases

“In our opinion, direct-sequencing will become a serious alternative for current molecular diagnostic tools ….”

Single cell genomics by QIAGEN, 2016

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single cell technologies for infectious disease research

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Norovirus characterization (1)

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• Genotyping viral RNA using NGS:◦ RNA sample prep from norovirus-positive fecal samples

◦ Whole transcriptome amplification of RNA with REPLI-g WTA Single Cell Kit

◦ NGS followed by data analysis on CLC Genomics Workbench

(1) Bavelaar, H.H. (2015) Whole genome sequencing of fecal samples as a tool for the diagnosis and genetic characterization of norovirus. J. Clin. Virol. 72, 122

Proof of concept: detection & extended characterization of Norovirus genotypes

REPLI-g WTA Single Cell Kit overcomes the limits of low RNA quantity, enabling NGS analysis of low viral load samples

• Generates a high-fidelity, complete copy of the input• Suitable for downstream NGS• Comprehensive detection of variant genotypes

Single cell genomics by QIAGEN, 2016

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Accurate variant detection in rare cells – feasible and affordable

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Low pass sequencing enabled by REPLI-g …

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(1) Zhang, C.-Z. et al. (2015) Calibrating genomic and allelic coverage bias in single cell sequencing, Nat. Comm. 6, 6822

Single cell genomics by QIAGEN, 2016

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Increase variant detection & reduce false positives

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(1) Zhang, C.-Z. et al. (2015) Calibrating genomic and allelic coverage bias in single cell sequencing, Nat. Comm. 6, 6822

“For clinical samples with a limited number of cells, such as fine-needle aspirates or circulating tumor cells, the most interesting genetic variants are shared among the cells.…it is most efficient to perform ‘census-based variant detection’ from multiplexed sequencing of independently amplified single cell DNA libraries, each sequenced to modest depths (~1x ).“ (1)

Low-pass sequencing and census-based variant detection

… followed by census-based variant detection

Single cell genomics by QIAGEN, 2016

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Single Cell Analysis – Cellular Heterogeneity Uncovered

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Data analysis &

Interpretation

Single cell isolation

Single Cell DNA NGS library prep

Sequencing on Illumina sequencer

Insight

QIAseq FX Single Cell DNA Library Kit

QIAscout

Single Cell RNA NGS library prep

QIAseq FX Single Cell RNA Library Kit

Primary sample

WGA or WTA

REPLI-g Single Cell Kit

REPLI-g WTA Single Cell Kit

REPLI-g Cell WGA & WTA Kit*

PCR, microarray,

aCGH

CLC Genomics Workbench

Ingenuity Variant Analysis / Pathway

Analysis (IPA)

miRNA qPCR

analysismiScript Single Cell qPCR Kit

Single cell genomics by QIAGEN, 2016