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Visualizing SNVs to quantify allele-specific expression in single cells Marshall J Levesque, Paul Ginart, Yichen Wei, Arjun Raj Systems Biology Lab, UPenn Bioengineering Nature Methods (2013) doi:10.1038/nmeth.2589

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Page 1: Visualizing SNVs to quantify allele-specific expression in single cells

Visualizing SNVs to quantify allele-specific

expression in single cells

Marshall J Levesque, Paul Ginart, Yichen Wei, Arjun RajSystems Biology Lab, UPenn Bioengineering

Nature Methods (2013) doi:10.1038/nmeth.2589

Page 2: Visualizing SNVs to quantify allele-specific expression in single cells

Paternal RNA

Maternal RNA

Transcription Sites

Nucleus

Cytoplasm

Heterozygotic cellMaternal Paternal

Human cells produce RNA using two copies of a gene

Page 3: Visualizing SNVs to quantify allele-specific expression in single cells

Human cells produce RNA using two copies of a gene

Mutant Wild-type

Wild-type RNA

Mutant RNA

Transcription Sites

Nucleus

Cytoplasm

Heterozygotic cell

Page 4: Visualizing SNVs to quantify allele-specific expression in single cells

RNA FISH probes directly detect RNA in single cells

RNA

FISH: Fluorescence in situ Hybridization

Page 5: Visualizing SNVs to quantify allele-specific expression in single cells

How do we distinguish chromosomes and their RNA?

Chr. 19EEF2 mRNAEEF2 intronLevesque and Raj, Nature Methods doi:10.1038/nmeth.2372

Page 6: Visualizing SNVs to quantify allele-specific expression in single cells

Our probes are not sensitive to single nucleotide differences

RNA

Page 7: Visualizing SNVs to quantify allele-specific expression in single cells

Our probes are not sensitive to single nucleotide differences

RNA

Page 8: Visualizing SNVs to quantify allele-specific expression in single cells

Our probes are not sensitive to single nucleotide differences

RNA

Page 9: Visualizing SNVs to quantify allele-specific expression in single cells

A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C

Masked probes provide specificity thru a short “toehold” sequence

C T T T A G C T A G G T C T G T T GG A T C C A G A C A A C

probe

mask

toehold dye

RNA target

Page 10: Visualizing SNVs to quantify allele-specific expression in single cells

A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C

CTAGGTCTGTTG

GATCCAGACAAC

C T T T A G

Masked probes bind through DNA strand displacement

probe

mask

dye

RNA target

toehold

Page 11: Visualizing SNVs to quantify allele-specific expression in single cells

C T T T A G C T A G G T C T G T T G

G A T C C A G A C A A C

A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C

Masked probes bind through DNA strand displacement

probe

mask

dye

RNA target

toehold

Page 12: Visualizing SNVs to quantify allele-specific expression in single cells

A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C

CTAGGTCTGTTG

GATCCAGACAAC

C T T T A G

Competing masked probes distinguish single nucleotide variants

probe

mask

toehold

RNA target

dye

Page 13: Visualizing SNVs to quantify allele-specific expression in single cells

A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C

CTAGGTCTGTTG

GATCCAGACAAC

C T T T G G

RNA target

probe

mask

toehold

C T T T A G C T A G G T C T G T T GG A T C C A G A C A A C

dye

Competing masked probes distinguish single nucleotide variants

Page 14: Visualizing SNVs to quantify allele-specific expression in single cells

C T T T G G C T A G G T C T G T T GA G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C

toehold

RNA target

G A T C C A G A C A A C

mask

probe dye

C T T T A G C T A G G T C T G T T GG A T C C A G A C A A C

Competing masked probes distinguish single nucleotide variants

Page 15: Visualizing SNVs to quantify allele-specific expression in single cells

RNA

Single oligos produce false positives when bound to off-targets

Page 16: Visualizing SNVs to quantify allele-specific expression in single cells

RNA

Single oligos produce false positives when bound to off-targets

Page 17: Visualizing SNVs to quantify allele-specific expression in single cells

Single oligos produce false positives when bound to off-targets

Page 18: Visualizing SNVs to quantify allele-specific expression in single cells

Single oligo detection assay takes advantage of co-localization

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 19: Visualizing SNVs to quantify allele-specific expression in single cells

Single oligo detection assay takes advantage of co-localization

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 20: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH clearly shows genotype of melanoma cell lines

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 21: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH shows mRNA allelic imbalance in the cell population

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 22: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH shows mRNA allelic imbalance in single-cells

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 23: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH shows mRNA allelic imbalance in single-cells

p = 0.00017

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 24: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH shows mRNA allelic imbalance in single-cells

p = 0.083

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 25: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH shows mRNA allelic imbalance in single-cells

p = 0.83

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 26: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH + iceFISH distinguishes maternal from paternal chromosome

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589Levesque and Raj, Nature Methods doi:10.1038/nmeth.2372

Page 27: Visualizing SNVs to quantify allele-specific expression in single cells

SNP FISH + iceFISH distinguishes maternal from paternal chromosome

Levesque et al, Nature Methods, doi:10.1038/nmeth.2589

Page 28: Visualizing SNVs to quantify allele-specific expression in single cells

Acknowledgements

Members of the Raj LabUPenn Bioengineering

Herlyn Lab

Wistar Institute

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