massachusetts institute of technology - van...

1

Supplementary Information

A versatile genome-scale PCR-based pipeline for high-definition DNA FISH

Magda Bienko1–3,7, Nicola Crosetto1–3,7, Leonid Teytelman1–3, Sandy Klemm4, Shalev Itzkovitz1–3 & Alexander van Oudenaarden1–3,5,6

1Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 2Department

of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 3Koch Institute for

Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 4Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology,

Cambridge, Massachusetts, USA. 5Hubrecht Institute–KNAW (Royal Netherlands Academy of Arts and

Sciences). 6University Medical Center Utrecht, Utrecht, The Netherlands. 7These authors contributed equally to

this work. Correspondence should be addressed to A.v.O. ([email protected]).

Nature Methods: doi: 10.1038/nmeth.2306

2

Supplementary Item Title or Caption

Supplementary Figure 1 Distribution of HD-FISH amplicons along the human and

mouse genomes

Supplementary Figure 2 HD-FISH cost analysis

Supplementary Figure 3 Distribution of spot counts for HER2 probes of decreasing

size

Supplementary Figure 4 Spotting of chromosome 1 and 17 using HD-FISH probes

Supplementary Figure 5 Frequency distribution of HER2 mRNA counts

Supplementary Note

Supplementary Video 1 3D rendering of Chr17 in HME cells, visualized with ten HD-

FISH probes evenly spaced every 8 Mb and labeled with two

alternating fluorophores (green: AlexaFluor594; magenta:

AlexaFluor647). The nucleus displayed is the same as in the

Z-projection shown in Figure 3a (mid panel).

Supplementary Video 2 3D animation of Chr17 in HME cells, visualized with sixteen

HD-FISH probes spaced evenly every 5 Mb and labeled with

two alternating fluorophores (green: AlexaFluor594; magenta:

AlexaFluor647) together with a Chr17 paint probe (blue).


3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

50

100

150

200

250

300

Genomic Position Along Chromosome 1 (10MB)N

umbe

r of P

robe

s pe

r 100

KB

p36.

33p3

6.32

p36.

31p3

6.23

p36.

22p3

6.21

p36.

13p3

6.12

p36.

11p3

5.3

p35.

2p3

5.1

p34.

3p3

4.2

p34.

1p3

3p3

2.3

p32.

2p3

2.1

p31.

3p3

1.2

p31.

1

p22.

3p2

2.2

p22.

1p2

1.3

p21.

2p2

1.1

p13.

3p1

3.2

p13.

1p1

2p1

1.2

p11.

1q1

1

q12

q21.

1q2

1.2

q21.

3q2

2q2

3.1

q23.

2q2

3.3

q24.

1q2

4.2

q24.

3q2

5.1

q25.

2q2

5.3

q31.

1q3

1.2

q31.

3

q32.

1q3

2.2

q32.

3q4

1q4

2.11

q42.

12q4

2.13

q42.

2q4

2.3

q43

q44

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

50

100

150

200

250

300

Genomic Position Along Chromosome 2 (10MB)

Num

ber o

f Pro

bes

per 1

00KB

p25.

3p2

5.2

p25.

1p2

4.3

p24.

2p2

4.1

p23.

3p2

3.2

p23.

1p2

2.3

p22.

2p2

2.1

p21

p16.

3p1

6.2

p16.

1p1

5p1

4p1

3.3

p13.

2p1

3.1

p12

p11.

2p1

1.1

q11.

1q1

1.2

q12.

1q1

2.2

q12.

3q1

3q1

4.1

q14.

2q1

4.3

q21.

1q2

1.2

q21.

3q2

2.1

q22.

2q2

2.3

q23.

1q2

3.2

q23.

3q2

4.1

q24.

2q2

4.3

q31.

1q3

1.2

q31.

3q3

2.1

q32.

2q3

2.3

q33.

1q3

3.2

q33.

3q3

4q3

5q3

6.1

q36.

2q3

6.3

q37.

1q3

7.2

q37.

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p26.

3p2

6.2

p26.

1p2

5.3

p25.

2p2

5.1

p24.

3p2

4.2

p24.

1p2

3p2

2.3

p22.

2p2

2.1

p21.

33p2

1.32

p21.

31p2

1.2

p21.

1p1

4.3

p14.

2

p14.

1p1

3p1

2.3

p12.

2p1

2.1

p11.

2p1

1.1

q11.

1q1

1.2

q12.

1q1

2.2

q12.

3q1

3.11

q13.

12q1

3.13

q13.

2q1

3.31

q13.

32q1

3.33

q21.

1q2

1.2

q21.

3q2

2.1

q22.

2q2

2.3

q23

q24

q25.

1q2

5.2

q25.

31q2

5.32

q25.

33q2

6.1

q26.

2q2

6.31

q26.

32q2

6.33

q27.

1q2

7.2

q27.

3q2

8q2

9

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p16.

3p1

6.2

p16.

1p1

5.33

p15.

32p1

5.31

p15.

2

p15.

1

p14

p13

p12

p11

q11

q12

q13.

1

q13.

2q1

3.3

q21.

1q2

1.21

q21.

22q2

1.23

q21.

3q2

2.1

q22.

2q2

2.3

q23

q24

q25

q26

q27

q28.

1q2

8.2

q28.

3

q31.

1q3

1.21

q31.

22q3

1.23

q31.

3

q32.

1q3

2.2

q32.

3q3

3q3

4.1

q34.

2q3

4.3

q35.

1q3

5.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p15.

33p1

5.32

p15.

31p1

5.2

p15.

1p1

4.3

p14.

2p1

4.1

p13.

3p1

3.2

p13.

1p1

2p1

1q1

1.1

q11.

2

q12.

1q1

2.2

q12.

3q1

3.1

q13.

2q1

3.3

q14.

1q1

4.2

q14.

3

q15

q21.

1q2

1.2

q21.

3q2

2.1

q22.

2q2

2.3

q23.

1

q23.

2q2

3.3

q31.

1q3

1.2

q31.

3

q32

q33.

1q3

3.2

q33.

3

q34

q35.

1q3

5.2

q35.

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p25.

3p2

5.2

p25.

1p2

4.3

p24.

2p2

4.1

p23

p22.

3

p22.

2p2

2.1

p21.

33p2

1.32

p21.

31p2

1.2

p21.

1

p12.

3p1

2.2

p12.

1p1

1.2

p11.

1q1

1.1

q11.

2q1

2

q13

q14.

1q1

4.2

q14.

3q1

5

q16.

1q1

6.2

q16.

3

q21

q22.

1q2

2.2

q22.

31q2

2.32

q22.

33q2

3.1

q23.

2q2

3.3

q24.

1q2

4.2

q24.

3q2

5.1

q25.

2q2

5.3

q26

q27

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p22.

3p2

2.2

p22.

1

p21.

3

p21.

2p2

1.1

p15.

3p1

5.2

p15.

1p1

4.3

p14.

2p1

4.1

p13

p12.

3p1

2.2

p12.

1p1

1.2

p11.

1q1

1.1

q11.

21

q11.

22

q11.

23

q21.

11

q21.

12q2

1.13

q21.

2q2

1.3

q22.

1q2

2.2

q22.

3

q31.

1

q31.

2q3

1.31

q31.

32q3

1.33

q32.

1q3

2.2

q32.

3q3

3

q34

q35

q36.

1q3

6.2

q36.

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p23.

3p2

3.2

p23.

1

p22

p21.

3

p21.

2p2

1.1

p12

p11.

23p1

1.22

p11.

21p1

1.1

q11.

1q1

1.21

q11.

22q1

1.23

q12.

1q1

2.2

q12.

3q1

3.1

q13.

2q1

3.3

q21.

11q2

1.12

q21.

13q2

1.2

q21.

3

q22.

1

q22.

2q2

2.3

q23.

1q2

3.2

q23.

3q2

4.11

q24.

12

q24.

13

q24.

21

q24.

22

q24.

23

q24.

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p24.

3p2

4.2

p24.

1

p23

p22.

3p2

2.2

p22.

1p2

1.3

p21.

2

p21.

1

p13.

3p1

3.2

p13.

1p1

2p1

1.2

p11.

1q1

1

q12

q13

q21.

11q2

1.12

q21.

13q2

1.2

q21.

31q2

1.32

q21.

33q2

2.1

q22.

2q2

2.31

q22.

32q2

2.33

q31.

1

q31.

2q3

1.3

q32

q33.

1

q33.

2

q33.

3

q34.

11q3

4.12

q34.

13q3

4.2

q34.

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p15.

3p1

5.2

p15.

1

p14

p13

p12.

33p1

2.32

p12.

31p1

2.2

p12.

1p1

1.23

p11.

22p1

1.21

p11.

1q1

1.1

q11.

21

q11.

22q1

1.23

q21.

1

q21.

2

q21.

3

q22.

1q2

2.2

q22.

3

q23.

1

q23.

2q2

3.31

q23.

32q2

3.33

q24.

1q2

4.2

q24.

31q2

4.32

q24.

33q2

5.1

q25.

2q2

5.3

q26.

11q2

6.12

q26.

13

q26.

2

q26.

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p15.

5

p15.

4

p15.

3p1

5.2

p15.

1

p14.

3p1

4.2

p14.

1

p13

p12

p11.

2

p11.

12p1

1.11 q11

q12.

1q1

2.2

q12.

3q1

3.1

q13.

2q1

3.3

q13.

4q1

3.5

q14.

1

q14.

2q1

4.3

q21

q22.

1q2

2.2

q22.

3

q23.

1q2

3.2

q23.

3

q24.

1q2

4.2

q24.

3q2

5

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13.

33p1

3.32

p13.

31

p13.

2p1

3.1

p12.

3p1

2.2

p12.

1p1

1.23

p11.

22p1

1.21

p11.

1q1

1

q12

q13.

11q1

3.12

q13.

13q1

3.2

q13.

3q1

4.1

q14.

2q1

4.3

q15

q21.

1

q21.

2

q21.

31

q21.

32q2

1.33 q22

q23.

1

q23.

2

q23.

3

q24.

11q2

4.12

q24.

13q2

4.21

q24.

22q2

4.23

q24.

31

q24.

32

q24.

33

0 1 2 3 4 5 6 7 8 9 10 11

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13

p12

p11.

2

p11.

1q1

1q1

2.11

q12.

12q1

2.13

q12.

2q1

2.3

q13.

1q1

3.2

q13.

3

q14.

11q1

4.12

q14.

13q1

4.2

q14.

3

q21.

1

q21.

2q2

1.31

q21.

32

q21.

33

q22.

1q2

2.2

q22.

3

q31.

1

q31.

2

q31.

3

q32.

1q3

2.2

q32.

3q3

3.1

q33.

2q3

3.3

q34

0 1 2 3 4 5 6 7 8 9 10

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13

p12

p11.

2

p11.

1q1

1.1

q11.

2

q12

q13.

1q1

3.2

q13.

3

q21.

1

q21.

2

q21.

3

q22.

1q2

2.2

q22.

3

q23.

1

q23.

2

q23.

3q2

4.1

q24.

2

q24.

3

q31.

1q3

1.2

q31.

3

q32.

11q3

2.12

q32.

13

q32.

2

q32.

31q3

2.32

q32.

33

0 1 2 3 4 5 6 7 8 9 10

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13

p12

p11.

2

p11.

1q1

1.1

q11.

2

q12

q13.

1q1

3.2

q13.

3

q14

q15.

1q1

5.2

q15.

3

q21.

1

q21.

2

q21.

3

q22.

1q2

2.2

q22.

31q2

2.32

q22.

33 q23

q24.

1q2

4.2

q24.

3q2

5.1

q25.

2

q25.

3

q26.

1

q26.

2

q26.

3

a

1


4

0 1 2 3 4 5 6 7 8 9

0

50

100

150

200

250

300


umbe

r of P

robe

s pe

r 100

KB

p13.

3

p13.

2p1

3.13

p13.

12p1

3.11

p12.

3

p12.

2

p12.

1

p11.

2

p11.

1q1

1.1

q11.

2

q12.

1

q12.

2q1

3

q21

q22.

1

q22.

2q2

2.3

q23.

1

q23.

2

q23.

3

q24.

1q2

4.2

q24.

3

0 1 2 3 4 5 6 7 8

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13.

3

p13.

2

p13.

1

p12

p11.

2

p11.

1q1

1.1

q11.

2

q12

q21.

1q2

1.2

q21.

31

q21.

32

q21.

33 q22

q23.

1q2

3.2

q23.

3q2

4.1

q24.

2

q24.

3

q25.

1

q25.

2

q25.

3

0 1 2 3 4 5 6 7

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p11.

32

p11.

31

p11.

23p1

1.22

p11.

21

p11.

1q1

1.1

q11.

2

q12.

1

q12.

2

q12.

3

q21.

1

q21.

2

q21.

31

q21.

32

q21.

33

q22.

1

q22.

2

q22.

3

q23

0 1 2 3 4 5

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13.

3

p13.

2

p13.

13p1

3.12

p13.

11 p12

p11

q11

q12

q13.

11

q13.

12

q13.

13

q13.

2

q13.

31

q13.

32

q13.

33

q13.

41

q13.

42

q13.

430 1 2 3 4 5 6

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13

p12.

3

p12.

2

p12.

1

p11.

23

p11.

22

p11.

21

p11.

1

q11.

1

q11.

21

q11.

22

q11.

23 q12

q13.

11

q13.

12

q13.

13

q13.

2

q13.

31

q13.

32

q13.

33

0 1 2 3 4

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13

p12

p11.

2

p11.

1

q11.

1

q11.

2

q21.

1

q21.

2

q21.

3

q22.

11

q22.

12

q22.

13

q22.

2

q22.

3

0 1 2 3 4 5

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

p13

p12

p11.

2

p11.

1

q11.

1

q11.

21

q11.

22

q11.

23

q12.

1

q12.

2

q12.

3

q13.

1

q13.

2

q13.

31

q13.

32

q13.

33

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

50

100

150

200

250

300

Genomic Position Along X Chromosome (10MB)

Num

ber o

f Pro

bes

per 1

00KB

p22.

33p2

2.32

p22.

31

p22.

2

p22.

13p2

2.12

p22.

11p2

1.3

p21.

2p2

1.1

p11.

4

p11.

3p1

1.23

p11.

22p1

1.21

p11.

1q1

1.1

q11.

2q1

2q1

3.1

q13.

2q1

3.3

q21.

1

q21.

2q2

1.31

q21.

32q2

1.33

q22.

1q2

2.2

q22.

3

q23

q24

q25

q26.

1q2

6.2

q26.

3q2

7.1

q27.

2q2

7.3

q28

0 1 2 3 4 5

0

50

100

150

200

250

300

Genomic Position Along Y Chromosome (10MB)

Num

ber o

f Pro

bes

per 1

00KB

p11.

32p1

1.31

p11.

2

p11.

1q1

1.1

q11.

21

q11.

221

q11.

222

q11.

223

q11.

23 q12

a


5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

50

100

150

200

250

300


um

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qA

4

qA

5

qB

qC

1.1

qC

1.2

qC

1.3

qC

2

qC

3

qC

4

qC

5

qD

qE

1.1

qE

1.2

qE

2.1

qE

2.2

qE

2.3

qE

3

qE

4

qF

qG

1q

G2

qG

3

qH

1

qH

2.1

qH

2.2

qH

2.3

qH

3

qH

4

qH

5

qH

6

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qB

qC

1.1

qC

1.2

qC

1.3

qC

2

qC

3

qD

qE

1

qE

2

qE

3

qE

4

qE

5

qF

1

qF

2

qF

3

qG

1

qG

2

qG

3

qH

1

qH

2

qH

3

qH

4

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qB

qC

qD

qE

1

qE

2

qE

3

qF

1

qF

2.1

qF

2.2

qF

2.3

qF

3

qG

1

qG

2

qG

3

qH

1

qH

2

qH

3

qH

4

0 1 2 3 4 5 6 7 8 9 10

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qB

1

qB

2

qB

3.1

qB

3.2

qB

3.3

qC

qD

1

qD

2

qD

3

qE

1

qE

2

qE

3

qF

1

qF

2q

F3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qA

4

qA

5

qB

1

qB

2

qB

3

qC

1

qC

2

qC

3

qC

4

qC

5

qC

6

qC

7

qD

1

qD

2.1

qD

2.2

qD

2.3

qD

3

qE

1

qE

2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qB

1

qB

2

qB

3

qC

1

qC

2

qC

3.1

qC

3.2

qC

3.3

qD

qE

1

qE

2

qE

3

qE

4

qE

5

qF

qG

1.1

qG

1.2

qG

1.3

qG

2

qG

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3.1

qA

3.2

qA

3.3

qB

1

qB

2.1

qB

2.2

qB

2.3

qB

3

qC

1

qC

2

qC

3

qD

1

qD

2

qD

3

qE

1

qE

2

qE

3

qF

1

qF

2

qF

3

qG

1

qG

2

qG

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qB

1

qB

2

qB

3

qB

4

qB

5

qC

qD

1

qD

2

qD

3

qE

1

qE

2

qE

3

qF

1

qF

2

qF

3

qF

4

qF

5

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1.1

qA

1.2

qA

1.3

qA

2

qA

3

qA

4

qB

1.1

qB

1.2

qB

1.3

qB

2

qB

3.1

qB

3.2

qB

3.3

qC

1

qC

2

qC

3

qC

4

qC

5

qD

1

qD

2

qD

3

qE

1

qE

2

0 1 2 3 4 5 6 7 8 9 10 11 12

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qA

4

qA

5.1

qA

5.2

qA

5.3

qB

qC

qD

qE

1

qE

2

qE

3.1

qE

3.2

qE

3.3

qE

4

qF

1

qF

2

qF

3

qF

4

0 1 2 3 4 5 6 7 8 9 10 11 12

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qA

4

qB

1

qB

2

qB

3

qB

4

qB

5.1

qB

5.2

qB

5.3

qC

1

qC

2

qC

3

qD

1

qD

2

qD

3

0 1 2 3 4 5 6 7 8 9 10 11 12

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3.1

qA

3.2

qA

3.3

qA

4

qA

5

qB

1.1

qB

1.2

qB

1.3

qB

2

qB

3

qB

4

qB

5

qC

qD

qE

1

qE

2

0 1 2 3 4 5 6 7 8 9 10 11 12

0

50

100

150

200

250

300


Nu

mb

er

of

Pro

be

s p

er

10

0K

B

qA

1.1

qA

1.2

qA

1.3

qA

2

qA

3

qB

1

qB

2

qB

3

qC

1

qC

2

qC

3

qD

1

qD

2

qD

3

qE

qF

1

qF

2

0 1 2 3 4 5 6 7 8 9 10 11 12

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3.1

qA

3.2

qA

3.3

qA

4

qA

5

qB

1

qB

2

qB

3

qC

1

qC

2

qC

3

qD

1

qD

2.1

qD

2.2

qD

2.3

0 1 2 3 4 5 6 7 8 9 10 11 12

0

50

100

150

200

250

300


Num

ber

of P

robes p

er

100K

B

qA

1

qA

2

qA

3

qB

qC

1

qC

2

qC

3

qD

1

qD

2

qD

3

qE

1

qE

2.1

qE

2.2

qE

2.3

qE

3

qE

4

qE

5

b


6

Supplementary Figure 1: Density of unique HD-FISH amplicons along the human (a) and

mouse (b) genome. G-bands ideograms are displayed.

0 1 2 3 4 5 6 7 8 9

0

50

100

150

200

250

300


umbe

r of P

robe

s pe

r 100

KB

qA1

qA2

qA3

qB1

qB2

qB3

qB4

qB5

qC1.

1qC

1.2

qC1.

3

qC2

qC3.

1

qC3.

2

qC3.

3

qC4

0 1 2 3 4 5 6 7 8 9

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

qA1

qA2

qB1

qB2

qB3

qC qD1

qD2

qD3

qE1

qE2

qE3

qE4

0 1

0

50

100

150

200

250

300

Genomic Position Along Y Chromosome (10MB)

Num

ber o

f Pro

bes

per 1

00KB

qA1

qA2 qB qC1

qC2

qC3

qD qE

0 1 2 3 4 5 6 7 8 9

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

qA1

qA2

qA3.

1

qA3.

2

qA3.

3

qB1

qB2

qB3

qC qD

qE1.

1

qE1.

2

qE1.

3

qE2

qE3

qE4

qE5

0 1 2 3 4 5 6

0

50

100

150

200

250

300


Num

ber o

f Pro

bes

per 1

00KB

qA qB qC1

qC2

qC3

qD1

qD2

qD3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

0

50

100

150

200

250

300

Genomic Position Along X Chromosome (10MB)

Num

ber o

f Pro

bes

per 1

00KB

qA1.

1

qA1.

2qA

1.3

qA2

qA3.

1qA

3.2

qA3.

3

qA4

qA5

qA6

qA7.

1qA

7.2

qA7.

3 qB qC1

qC2

qC3

qD qE1

qE2

qE3

qF1

qF2

qF3

qF4

qF5

b


7

Supplementary Figure 2: HD-FISH cost analysis. (a) Cumulative cost as a function of the

number of tests per probe in three different scenarios. Costs include synthesis of PCR primers

and reagents for PCR, PCR purification, labeling, and preparation of hybridization solutions.

Costs for other consumables (e.g. gloves, pipette tips, etc.) as well as for instrument

depreciation and personnel are excluded. (b) Linear dependence of primer synthesis cost on

the number of probes and amplicons per probe.

a

cum

ulat

ive

cost

x 1

,000

(USD

)

0500 1000

48 amplicons / probe

0

50


150


# probes

b

0 200

400

600

80010

0012

0014

0016

0018

0020

0022

0024

0002468

1012141618202224

tests / probe

cum

ulat

ive

cost

x 1

,000

(USD

) 20 probes (48 amplicons / probe)

10 probes (24 amplicons / probe)

1 probe (24 amplicons / probe)


8

Supplementary Figure 3: Distribution of spot counts for HER2 probes of decreasing size in

HME cells. The number of amplicons from which each probe was derived is shown. Numbers

in brackets represents ETS values. n: number of cells analyzed.

0

10

20

30

40

50

# spots / nucleus

15 amplicons (3 kb)

51 2 3 4

n = 134

0

10

20

30

40

50

# spots / nucleus

19 amplicons (3.8 kb)

51 2 3 4

n = 92

0

10

20

30

40

50

# spots / nucleus

24 amplicons (4.8 kb)

51 2 3 4

n = 99

0 1 2 3 40

10

20

30

40

50

# spots / nucleus

10 amplicons (2 kb)

n = 75

rela

tive

frequ

ency

(%)


9

Supplementary Figure 4: (a) Genomic location of individual probes forming the spotting

probes for Chr1 and Chr17 described. Magenta: AlexaFluor647-labeled probes. Green:

AlexaFluor594-labeled probes. G-bands ideograms and band names are displayed. (b) Chr1

spotting with twenty-two alternatively labeled HD-FISH probes (green and magenta), and

simultaneous Chr1 painting (blue) in HME cells. (c) Inter-channel concordance for spot

counts shown in Figure 3b. The linear regression fit line of slope m is shown. (d) Single-cell

distribution of the difference between error estimates of HD-FISH spot counts measured

a

-12 -8 -4difference between AF647 and AF594 spot counts

0 4 8 12

n = 744

rela

tive

frequ

ency

(%)

20

25

10

0

15

5

2

6

10

1430

20

10

0

Alex

aFlu

or 5

94

AlexaFluor 647

3020100

# observations

m = 0.96

b c

d

e

p36.33p36.32p36.31p36.23p36.22p36.21p36.13p36.12p36.11p35.3p35.2p35.1p34.3

p34.2p34.1

p33

p32.3p32.2p32.1

p31.3

p31.2

p31.1

p22.3p22.2p22.1p21.3p21.2p21.1

p13.3

p13.2p13.1

p12p11.2p11.1

q11

q12

q21.1q21.2q21.3

q22q23.1q23.2q23.3q24.1q24.2q24.3q25.1q25.2

q25.3

q31.1q31.2q31.3

q32.1

q32.2q32.3

q41

q42.11q42.12q42.13q42.2q42.3

q43

q44

x 10

p13.3

p13.2

p13.1

p12

p11.2

p11.1q11.1

q11.2

q12

q21.1q21.2

q21.31

q21.32

q21.33

q22

q23.1q23.2q23.3q24.1q24.2

q24.3

q25.1q25.2

q25.3

x 10

p13.3

p13.2

p13.1

p12

p11.2

p11.1q11.1

q11.2

q12

q21.1q21.2

q21.31

q21.32

q21.33

q22

q23.1q23.2q23.3q24.1q24.2

q24.3

q25.1q25.2

q25.3

Chr17

Chr1

AF647 AF594

AF647 + AF594 PaintAF647 + AF594

Spotting Paint

f


10

simultaneously in the AlexaFluor 594 and 647 channels. These data reflect gene copy

number-invariant noise and demonstrate that the respective channel estimates are unbiased

relative to each other (median value = 0) and symmetric (quartile skewness = 0). n: number of

cells analyzed. (e) Representative field of view with HME cells simultaneously hybridized

with a Chr17 spotting probe consisting of sixteen HD-FISH probes (left) and with a paint

probe (right). Small panels: zoom-in view of the area marked by the white dashed square.

AF647: AlexaFluor647; AF594: AlexaFluor594. (f) Comparison of Chr17 spotting (green and

magenta) and painting (blue) in several Z-projections.


11

Supplementary Figure 5: Frequency distribution of HER2 mRNA counts in HME cells with

2 (purple), 3 (orange), or 4 (brown) HER2 loci detected by HD-FISH. Gaussian fits are

superimposed onto histograms. n: number of cells analyzed.

0 10 20 30 40# mRNA

Rel

ativ

e fre

quen

cy (%

)

20

25

10

15

0

5

n = 445


12

SUPPLEMENTARY NOTE

In order for HD-FISH to attract a wide audience (including diagnostics laboratories and

industrial parties), economic measures of its performance must be obtained in addition to

scientific ones. For this purpose, we have performed cost effectiveness analysis in the

following three scenarios. The first case is relevant to both research laboratories that

occasionally need to perform DNA FISH on loci for which there are no ready-to-use probes

commercially available, as well as to cytogenetics laboratories that wish to rapidly generate

probes for rare rearrangements for which no commercial probes are available. The second

case is relevant to diagnostics laboratories that perform a high volume of routine

hybridizations against a limited number of loci, and that wish to reduce reagents costs. The

third case is relevant to policy makers and healthcare systems organizers that are willing to

financially support initiatives aiming at improving the efficiency of high quality diagnostics

by cutting reagents costs and optimizing processes.

CASE 1. Let us imagine the case in which a particular locus (or a small number of loci) for

which no commercial probes are available needs to be analyzed for research or diagnostic

purposes. An investment of approx. $1,880 allows purchase of primers for 24 amplicons

(which allows robust signal quantification as shown in the new Supplementary Fig. 2a) and

for all the reagents needed for PCR, labeling, purification, and hybridization. This investment

enables approx. 900 tests (i.e. hybridizations with 20 ng of probe in 20 µL buffer) at no extra

costs, after which the cumulative cost grows in a pseudo-linear, step-wise manner at very

slow pace, mostly driven by the need to periodically replenish PCR and labeling reagents

(Supplementary Fig. 2a). An extra starting investment of $900, for example, enables ten

HD-FISH probes to be simultaneously prepared in the same cost-effective manner as outlined

above, allowing 90 tests per probe to be performed at no extra cost (Supplementary Fig. 2a).

In comparison, custom design and BACs-based synthesis of ready-to-use probes for the same

number of loci enabling at least 90 tests per probe would at minimum require $8,000-10,000

(data based on quote requests to several DNA FISH probes manufacturers, including Empire

Genomics, Kreatech Diagnostics, and Abnova).

CASE 2. Let us imagine the case in which a limited set of probes (e.g. 20) is routinely used

for diagnostic purposes. An investment of less than $5,800 allows purchasing primers sets for

20 probes, each consisting of 48 amplicons (which based on our results allows robust signal

quantification in cells as well as tissues), as well as reagents needed for PCR, labeling,


13

purification, and hybridization. After 45 initial tests per probe at no extra cost, the cumulative

cost rises in a pseudo-linear, step-wise manner, mostly driven by the need to periodically

replenish PCR and labeling reagents (Supplementary Fig. 2a). The cost effectiveness of this

approach is obvious by examining the cumulative cost (approx. $8,000) after 200 tests per

probe (i.e. 4,000 tests in total, assuming that each probe is used with the same frequency), a

figure perfectly within the range of average annual volume of tests performed by medium-

sized cytogenetics laboratories. In contrast, performing the same volume of tests for 20

different ready-to-use probes periodically purchased from existing commercial providers

would require a budget at least 5-fold higher (as an example, $2,500 is the price charged by a

top manufacturer for a ready-to-use probe for 50 tests. Even assuming a 20% discount from

the price list for large order volumes, purchase of probes for 20 different loci for 200

tests/probe would require $160,000).

CASE 3. Though surely cost effective in the cases depicted above, the start-up cost of HD-

FISH linearly grows with the number of primers (i.e. probes), quickly exceeding the financial

capabilities of a single laboratory (Supplementary Fig. 2b). Moreover, even at the lowest

possible synthesis scale, a large fraction of primers would remain unused, as it is basically

impossible for a single laboratory to reach the maximum number of tests (> 8 millions

hybridizations using 20 ng of probe in 20 µL buffer per test) that can be performed from a 96-

well plate containing 12 nmol primers per well. Instead, a consortium funded by public

resources and administered as a non-profit repository could build up a primers stock covering

the human and mouse genomes for less than $20 million. This budget is well within the

average funding that public agencies have been granting to such type of initiatives in the U.S.

as well as in the E.U. This would enable laboratories worldwide to purchase primers or even

pre-made PCR reactions at low price, therefore greatly facilitating high-quality FISH-based

research and diagnostics services, especially in emerging economies.


14

SUPPLEMENTARY VIDEO LEGENDS

All movies are in .mov format and can be played for example using QuickTime Player.

Supplementary Video 1: 3D rendering of Chr17 in HME cells, visualized with ten HD-FISH

probes evenly spaced every 8 Mb and labeled with two alternating fluorophores (green:

AlexaFluor594; magenta: AlexaFluor647). The nucleus displayed is the same as in the Z-

projection shown in Figure 3a (mid panel).

Supplementary Video 2: 3D animation of Chr17 in HME cells, visualized with sixteen HD-

FISH probes spaced evenly every 5 Mb and labeled with two alternating fluorophores (green:

AlexaFluor594; magenta: AlexaFluor647) together with a Chr17 paint probe (blue).


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