[PPT] - Chromosome tracing with OligoFISSEQ Marc A. Marti-Renom CNAG-CRG PowerPoint Presentation

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Chromosome tracing with OligoFISSEQ

Nature Methods (2020) 17 p822

Marc A. Marti-Renom

CNAG-CRG · ICREA Huy Nguyen Shyamtanu Chattoraj David Castillo in collaboration with the Wu Lab (HMS)

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Targets per cell Number of cells

OligoSTORM OligoFISSEQ

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5’ Genome homology 3’ LIT primer site LIT barcode SIT primer site SIT barcode HIT bridge sites

OligoFISSEQ

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OligoFISSEQ

From tens of kb to Mb

Min. of few 100s oligos/target

At least a Mb between targets

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OligoFISSEQ

5’ Genome homology 3’ LIT primer site LIT barcode SIT primer site SIT barcode HIT bridge sites

Ligation based Identification of Targets Synthesis based Identification of Targets

C G A T

5’ OH 3’

ATCA

Pol T

5’ 3’ OH

ATCA

5’ 3’

ATCA

T

90.8 ± 5.6%

Hybridization based Identification of Targets

10 µm

91.6 ± 3.8%

barcode B barcode A barcode B barcode A barcode B barcode A

10 µm

92.1± 5.7%

1st Base 2nd Base A C T G A C T G

T G N N N Z Z Z

5’ PO4 3’

ACTGTGAATCGC

5’ 3’

TGNNNZZZ ACTGTGAATCGC

Ligase

5’ 3’

TGNNN ACTGTGAATCGC Z Z Z

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# of targets = FN

F = # of fluorophores N = # of seq. rounds

# of targets = F*N

F = # of fluorophores N = # of seq. rounds

OligoFISSEQ scales exponentially!

Barcode sequencing Sequential hybridization

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OligoFISSEQ scales exponentially!

Sequential hybridization vs. Barcode sequencing

4500 9000 13500 18000 1 2 3 4 5 6 7 Number of rounds Number of targets

16,384 24

Genome coverage @500kb 360 6,000 4 16 64 256 1,024 4,096

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600kb-1Mb/target (876 kb average) 5,000 oligos/target 7-70Mb between targets

Proof-of-principle

chrX chr19 chr16 chr5 chr3 chr2

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Detecting a given target

Round 1 Round 2 Round 3 Round 4

X 19 16 5 3 2

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OligoFISSEQ "Manhattan plot”

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In OligoFISSEQ every pixel matters & make “patches”

4 rounds / 4 channels

chr2

2qR2

75 pixels ~20𝞶m 0.265𝞶m/pixel

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In OligoFISSEQ every pixel matters & make “patches”

chr2

2pR1 2pR2 2qR3 2qR2 2qR1 2pR3

2pR1 2pR2 2pR3 2qR1 2qR2 2qR3

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OligoFISSEQ barcode efficiency

5𝞶m 5𝞶m

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10𝞶m

OligoFISSEQ is high throughput!

~2 days of image acquisition ~1,000 cells ~5,000 complete chromosomes ~150 cells with complete chromosomes

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Single cell homolog resolved tracing of chromosomes

Cell #1. 97% detection

2 p R 1

1
2

p R 2

1
2

p R 3

1
2

q R 1

1
2

q R 2

1
2

q R 3

1
2

p R 1

2
2

p R 2

2
2

p R 3

2
2

q R 1

2
2

q R 2

2
2

q R 3

2
3

p R 1

1
3

p R 2

1
3

p R 3

1
3

q R 1

1
3

q R 2

1
3

q R 3

1
3

p R 1

2
3

p R 2

2
3

p R 3

2
3

q R 1

2
3

q R 2

2
3

q R 3

2
5

p R 1

1
5

p R 2

1
5

p R 3

1
5

q R 1

1
5

q R 2

1
5

q R 3

1
5

p R 1

2
5

p R 2

2
5

p R 3

2
5

q R 1

2
5

q R 2

2
5

q R 3

2
1

6 p R 1

1
1

6 p R 2

1
1

6 p R 3

1
1

6 q R 1

1
1

6 q R 2

1
1

6 q R 3

1
1

6 p R 1

2
1

6 p R 2

2
1

6 p R 3

2
1

6 q R 1

2
1

6 q R 2

2
1

6 q R 3

2
1

9 p R 1

1
1

9 p R 2

1
1

9 p R 3

1
1

9 q R 1

1
1

9 q R 2

1
1

9 q R 3

1
1

9 p R 1

2
1

9 p R 2

2
1

9 p R 3

2
1

9 q R 1

2
1

9 q R 2

2
1

9 q R 3

2
X

p R 1

1
X

p R 2

1
X

p R 3

1
X

q R 1

1
X

q R 2

1
X

q R 3

1

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Do OligoFISSEQ tracing maps show known features?

Hi-C contact maps & Radial position of chromosomes Are the chromosomes randomly located inside the nucleus? Are there preferred configurations in the cell population?

OligoFISSEQ n=691 Hi-C n = 1x106+ cells

r2 = 0.71

2pR1 2pR2 2pR3 2qR1 2qR2 2qR3 3pR1 3pR2 3pR3 3qR1 3qR2 3qR3 5pR1 5pR2 5pR3 5qR1 5qR2 5qR3 16pR1 16pR2 16pR3 16qR1 16qR2 16qR3 19pR1 19pR2 19pR3 19qR1 19qR2 19qR3 XpR1 XpR2 XpR3 XqR1 XqR2 XqR3

X 19 16 5 3 2

n=1,108

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OligoFISSEQ tracing of (almost) entire chromosomes

46 Plex in chromosome X

1𝞶m

5 rounds 445 kb/probe 2,000 Oligopaints/probe 2 Mb between loci

5𝞶m

chrX

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OligoFISSEQ tracing of (almost) entire chromosomes

46 Plex in chromosome X

n=146

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OligoFISSEQ tracing of (almost) entire chromosomes

46 Plex in chromosome X

Cluster 1 (n=156) Cluster 2 (n=20)

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OligoFISSEQ beyond chromosome tracing

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chr2

OligoFISSEQ pipelined with OligoSTORM

OligoSTROM 1 round (2h/round)

1

OligoFISSEQ 2 round (3h/round)

2

Decoding OligoFISSEQ

3

Mapping OligoSTROM

4

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72% 100%

OligoFISSEQ for multiple loci detection

HESS: 97 oligos, 11kb ABL: 454 oligos, 45kb FLI1: 499 oligos, 39kb DXZ4: 617 oligos, 136kb BCR: 1,015 oligos, 100kb MMP2: 297 oligos, 27kb

X 22 16 11 9 1

Efficiency 0.0 1.0

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OligoFISSEQ + protein immunofluorescence

X 19 16 5 3 2

tubulin

GAPDH Tomm20 WGA

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Is a set of technologies for in-situ genome mapping
Is highly versatile: mainstreet and backstreet

OligoFISSEQ

5’ Genome homology 3’ LIT primer site LIT barcode SIT primer site SIT barcode HIT bridge sites

Used with wide-field microscopy permits the analysis of thousands of cells.
Identifies sub-clusters with specific conformational characteristics
Can be pipelined with other approaches
OligoSTORM
Protein immunofluorescence
RNA…

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http://marciuslab.org http://3DGenomes.org @marciuslab @mamartirenom

.: Our current sponsors :.

David Castillo Yasmina Cuartero Silvia Galan Rodrigo Jara Iana Kim Maria Marti-Marimon Francesca Mugianesi Julen Mendieta Aleksandra Sparavier

Marco Di Stefano Irene Farabella Mike Goodstadt Juan A. Rodriguez

In collaboration with the Wu Lab – Ting Wu, Huy Nguyen & Shyamtanu Chattoraj