SupInfo - MIT

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 1

Department 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. 4

Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology,

Cambridge, Massachusetts, USA.

5

Hubrecht Institute–KNAW (Royal Netherlands Academy of Arts and

6

Sciences). University 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

1

Supplementary Item Supplementary Figure 1

Title or Caption 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 HDFISH 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).

Nature Methods: doi: 10.1038/nmeth.2306

2

Nature Methods: doi: 10.1038/nmeth.2306 4 5 q15

6 q22

7 q23.3

q23.2

11

8

q33.3

q33.2

12

q34.11 q34.12 q34.13 q34.2 q34.3

q21

13

12

9

q27

q26

q25.3

q22.32 q22.33 q23.1 q23.2 q23.3 q24.1 q24.2 q24.3 q25.1 q25.2

q22.1 q22.2 q22.31

16

q24.33

0

q15

50

q16.1 q16.2 q16.3

q14.2 q14.3

q14.1

q13

p12.3 p12.2 p12.1 p11.2 p11.1 q11.1 q11.2 q12

p21.1

15

q24.32

q33.1

q31.3 q32

q31.2

q31.1

q21.2 q21.31 q21.32 q21.33 q22.1 q22.2 q22.31 q22.32 q22.33

q21.13

q13 q21.11 q21.12

11

q24.31

10

q24.11 q24.12 q24.13 q24.21 q24.22 q24.23

9

q23.1

8

q21.32 q21.33

q21.31

q21.2

q21.1

7

10

14

q29

q26.2 q26.31 q26.32 q26.33 q27.1 q27.2 q27.3 q28

q24 q25.1 q25.2 q25.31 q25.32 q25.33 q26.1

p12.3 p12.2 p12.1 p11.2 p11.1 q11.1 q11.2 q12.1 q12.2 q12.3 q13.11 q13.12 q13.13 q13.2 q13.31 q13.32 q13.33 q21.1 q21.2 q21.3 q22.1 q22.2 q22.3 q23

p13

p14.1

p24.2 p24.1 p23 p22.3 p22.2 p22.1 p21.33 p21.32 p21.31 p21.2 p21.1 p14.3 p14.2

p24.3

p26.3 p26.2 p26.1 p25.3 p25.2 p25.1

Number of Probes per 100KB

18

q26.3

3

9

13

17

q26.2

2 q12

p22.3 p22.2 p22.1 p21.33 p21.32 p21.31 p21.2

100

12

16

q26.1

0 150

11

15

q25.3

50 1 6

8

14

q25.2

100

200

10

13

q25.1

150

250

9

q23

200 0 5

7

12

q24.1 q24.2 q24.3

300

6

11

q22.31 q22.32 q22.33

10

8

q22.1 q22.2

Genomic Position Along Chromosome 14 (10MB) 4

10

q21.3

0 3

5

9

q21.2

50 4

7

q14.2 q14.3

100 2

6

q21.1

150 1

5

8

q14.1

200 0

4

7

q15.1 q15.2 q15.3

300

6

q13.11 q13.12 q13.13 q13.2 q13.3

13

5

q14

Genomic Position Along Chromosome 11 (10MB) 3

p13.3 p13.2 p13.1 p12 p11.2 p11.1 q11

0

3

q12

50

4

q12 q13.1 q13.2 q13.3

100 2

q11.2

150 1

p21.1

200 0

3

p11.23 p11.22 p11.21 p11.1 q11

Genomic Position Along Chromosome 8 (10MB)

q11.1

300

p23

14

2

p21.2

0

1

p21.3

50

0

p22.3 p22.2 p22.1

Genomic Position Along Chromosome 5 (10MB)

p12.1

100

300

2

p12.2

18

1

p12.3

150

Number of Probes per 100KB

200

p25.3 p25.2 p25.1 p24.3 p24.2 p24.1 p23

0

p13.2 p13.1

250

Number of Probes per 100KB

250

p24.3 p24.2

50

p24.1

100

p12

250

Number of Probes per 100KB

q35 q36.1 q36.2 q36.3 q37.1 q37.2 q37.3

150

p13.33 p13.32

q34 q35.1 q35.2 q35.3

200

0

p13.31

12 q24.3

q24.23

q31.1 q31.2 q31.3 q32.1 q32.2 q32.3 q33.1 q33.2 q33.3 q34

p11.2 p11.1 q11.1 q11.2 q12.1 q12.2 q12.3 q13 q14.1 q14.2 q14.3 q21.1 q21.2 q21.3 q22.1 q22.2 q22.3 q23.1 q23.2 q23.3 q24.1 q24.2 q24.3

250

300

p11.1

9 q24.22

q24.21

17

q25

q24.13

13

q24.3

q32 q33.1 q33.2 q33.3

q31.2 q31.3

q31.1

q23.3

q23.2

16

q24.2

q24.11 q24.12

q23.3

q23.1 q23.2

q22.3

q22.2

q22.1

q21.1 q21.2 q21.3 q22.1 q22.2 q22.3 q23.1

q15

q14.3

q12.1 q12.2 q12.3 q13.1 q13.2 q13.3 q14.1 q14.2

q11.2

Genomic Position Along Chromosome 2 (10MB)

p11.2

250

Number of Probes per 100KB

8 q24.1

11

q23.3

10

q23.1 q23.2

q22.3

q22.1 q22.2

q21

q14.3

q21.3

q21.11 q21.12 q21.13 q21.2

q12.1 q12.2 q12.3 q13.1 q13.2 q13.3

p13.1 p12 p11 q11.1

p13.2

p13.3

p15.1 p14.3 p14.2 p14.1

Number of Probes per 100KB

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

p13

7 12

q32.2

6 q14.2

q14.1

q13.5

q13.4

9

15

q32.31 q32.32 q32.33

5 11

q32.11 q32.12 q32.13

0 4 8

14

q31.3

50 3 10

q31.2

100 2 9

13

q31.1

150 1 7

12

q24.3

200 0 8

q24.2

300 6

11

q24.1

11 5 7

q23.3

Genomic Position Along Chromosome 13 (10MB) 6

10

q23.2

0 4

9

q23.1

50 3 5

8

q22.1 q22.2 q22.3

100 2

7

q21.3

150 1 4

9

q21.2

200 0

6

p11.12 p11.11 q11 q12.1 q12.2 q12.3 q13.1 q13.2 q13.3

300

5

8

q21.1

13 3

7

p11.2

Genomic Position Along Chromosome 10 (10MB)

6

q13.1 q13.2 q13.3

0 4

p12

50 2

p12

100 1

p11.23 p11.22 p11.21 p11.1 q11.1 q11.21 q11.22 q11.23

150 0

3

p13

200 2

5

q12

300

4

q11.2

15 p15.2

0

p22

50 1

3

p21.2 p21.1

100 0

p21.3

150 300

p23.1

Genomic Position Along Chromosome 4 (10MB)

p14.3 p14.2 p14.1

19

2

p15.1

0

1

p15.3 p15.2

200 p25.3 p25.2 p25.1 p24.3 p24.2 p24.1 p23.3 p23.2 p23.1 p22.3 p22.2 p22.1 p21 p16.3 p16.2 p16.1 p15 p14 p13.3 p13.2 p13.1 p12

50

p15.4

250

Number of Probes per 100KB

100

p15.33 p15.32 p15.31

150 0

p12

250

Number of Probes per 100KB

200

p23.3 p23.2

q44

Genomic Position Along Chromosome 1 (10MB)

p11.1 q11.1

250

Number of Probes per 100KB

q32.2 q32.3

q32.1

q41 q42.11 q42.12 q42.13 q42.2 q42.3 q43

250

p15.5

q35.1 q35.2

q32.1 q32.2 q32.3 q33 q34.1 q34.2 q34.3

Number of Probes per 100KB

300

p11.2

250

Number of Probes per 100KB

10 q26.3

12 q36.1 q36.2 q36.3

14

q26.2

q35

q34

q33

q28.3 q31.1 q31.21 q31.22 q31.23 q31.3

q21.1 q21.2 q21.3 q22 q23.1 q23.2 q23.3 q24.1 q24.2 q24.3 q25.1 q25.2 q25.3 q31.1 q31.2 q31.3

q12

p22.3 p22.2 p22.1 p21.3 p21.2 p21.1 p13.3 p13.2 p13.1 p12 p11.2 p11.1 q11

p31.1

p36.33 p36.32 p36.31 p36.23 p36.22 p36.21 p36.13 p36.12 p36.11 p35.3 p35.2 p35.1 p34.3 p34.2 p34.1 p33 p32.3 p32.2 p32.1 p31.3 p31.2

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

p13

q25.3

11 18

q34

9 q25.2

10 17

q33.3

8 13

q26.11 q26.12 q26.13

q31.1

12 16

q33.1 q33.2

7 15

q32.1 q32.2 q32.3

9 q31.2 q31.31 q31.32 q31.33 q32.1 q32.2 q32.3

11

q25.1

10 14

q31.3

q26

q25

q27 q28.1 q28.2

13

q31.2

6 q22.1 q22.2 q22.3

q21.12 q21.13 q21.2 q21.3

q21.11

12

q31.1

8

q23.2 q23.31 q23.32 q23.33 q24.1 q24.2 q24.31 q24.32 q24.33

7

q23.1

9

q22.1 q22.2 q22.3

5 11

q21.33

q11.23

8

q22.3

q13.3 q21.1 q21.21 q21.22 q21.23 q21.3 q22.1 q22.2 q22.3 q23 q24

10

q22.2

6

q22.1

q21.3

5

q21.2

7

q21.1

4 9

q21.32

4 q11.22

6

q21.2

5 8

q21.31

3 q13.2

7

q21.1

4 q13.1

q12

p14 p13 p12 p11 q11

6

q11.23

p13 p12.3 p12.2 p12.1 p11.2 p11.1 q11.1 q11.21

p15.1

p15.2

5

q11.22

3

8

q14.3

2 p14.1

3

7

q13.3

2

6

q14.12 q14.13 q14.2

1 4

p11.21 p11.1 q11.1 q11.21

1 p14.2

2

p12.1

Number of Probes per 100KB

3

p11.23 p11.22

p15.33 p15.32 p15.31

1

p15.3 p15.2 p15.1 p14.3

p16.3 p16.2 p16.1

Genomic Position Along Chromosome 7 (10MB)

0

5

q14.11

0 p21.2 p21.1

p21.3

Number of Probes per 100KB

2

4

q12.12 q12.13 q12.2 q12.3 q13.1 q13.2

0

3

q12.11

300 1

p13

300

p14

p22.3 p22.2 p22.1

0

2

p12.33 p12.32 p12.31 p12.2

Number of Probes per 100KB 300 1

p11.1 q11

p15.3 p15.2 p15.1

300 0

p11.2

Number of Probes per 100KB 300

p12

p13

a Genomic Position Along Chromosome 3 (10MB)

250

19

200

150

100

50

0

Genomic Position Along Chromosome 6 (10MB) 17

Genomic Position Along Chromosome 9 (10MB) 14

250

200

150

100

50

0

Genomic Position Along Chromosome 12 (10MB) 13

250

200

150

100

50

0

Genomic Position Along Chromosome 15 (10MB)

10

250

200

150

100

50

0

1

3

Nature Methods: doi: 10.1038/nmeth.2306 Genomic Position Along X Chromosome (10MB) 15

200

150

100

50

0 300 0 1 2 3 4

q23

q22.3

q22.2

q22.1

q21.33

6

q22.3

3

q22.2

q21.32

Number of Probes per 100KB

5

q22.13

q22.12

2

q21.31

q21.2

q21.1

q12.3

q12.2

q12.1

q11.2

p11.1 q11.1

p11.21

p11.22

p11.23

p11.31

p11.32

4

q22.11

q21.3

0

1

q21.2

50

q21.1

100

q11.2

150

0

q11.1

300

p11.1

Genomic Position Along Chromosome 20 (10MB)

p11.2

6

p12

200

Number of Probes per 100KB

250

p13

q25.3

q25.2

q25.1

q24.3

0

3

q12

14

q13.33

q24.2

50

q11.23

13

q13.32

100

2

q11.223

12

q13.31

q22

q23.3 q24.1

q23.1 q23.2

150

1

q11.222

11

q13.2

q13.13

q21.33

200

0

q11.221

10

q13.12

q21.32

q21.31

q21.1 q21.2

q12

q11.2

p11.1 q11.1

250

300

p11.1 q11.1 q11.21

9

q13.11

q12

q11.23

q11.22

q11.21

q11.1

p11.1

p11.21

p11.22

p11.2

p12

p13.1

Genomic Position Along Chromosome 17 (10MB)

p11.2

8

8

p11.31

250

Number of Probes per 100KB

7

7

p11.32

6

5

q28

5

q25

4

6

q26.3 q27.1 q27.2 q27.3

0 3

4

q26.1 q26.2

50 2

5

q24

100 1 3

q23

150 0

4

q22.1 q22.2 q22.3

200 300

2

q21.2 q21.31 q21.32 q21.33

5

3

q21.1

Genomic Position Along Chromosome 22 (10MB) p11.23

0

p11.3

50 2

p11.21 p11.1 q11.1 q11.2 q12 q13.1 q13.2 q13.3

100

p11.22

150 1

p11.23

200 0

p12.1

300

1

p11.4

5

p12.2

Genomic Position Along Chromosome 19 (10MB)

p21.1

0 0

p21.3 p21.2

50

p13.2

100

p13

150 300

p12.3

200

Number of Probes per 100KB

250

p13.3

9

p22.2

250

Number of Probes per 100KB

q24.2 q24.3

q24.1

q23.3

q23.2

q23.1

Genomic Position Along Chromosome 16 (10MB)

p22.13 p22.12 p22.11

250

Number of Probes per 100KB

q13.43

q13.42

q13.41

q21

q22.2 q22.3

q22.1

8

p22.33 p22.32 p22.31

4

q13.33

q13.32

q13.33

4

q13.2

q13.32

7

q13.31

3 q13.31

q13

q12.2

q12.1

6

q13.1

3

q13.2

q13.13

q13.12

q11.2

p11.1 q11.1

5

q12.3

2

q12.2

q13.11

2

q12

q11

p11.2

4

q12.1

q11.23

1 p11

p12.1

p12.2

p12.3

3

q11.22

1

p12

p13.11

p13.13 p13.12

p13.2

p13.3

2

q11.21

0

q11.1

0 p13.12 p13.11

p13.13

Number of Probes per 100KB

1

p11.1

300 p13.2

300 0

p11.2

p13.3

Number of Probes per 100KB 300

p12

p13

Number of Probes per 100KB

a Genomic Position Along Chromosome 18 (10MB) 7

250

200

150

100

50

0

Genomic Position Along Chromosome 21 (10MB) 4

250

200

150

100

50

0

Genomic Position Along Y Chromosome (10MB)

250

5

200

150

100

50

0

4

Nature Methods: doi: 10.1038/nmeth.2306 12

200

150

100

50

0 200

150

100

50

0

300 0 1 2 3 4 5 6 7

10

8 9

11

qF4

qF3

10

qF2

qF1

qB3

qG3

qG2

qG1

qF3

qF2

qF1

qE3

qE1 qE2

qD3

qD2

qD1

qC3

qC2

qC1

13

qF2

9

qE4

9

qE

qE2

qE3.3

qE3.2

qE3.1

qB1 qB2.1 qB2.2 qB2.3

12

qF1

8

qD3

qE1

qD

qC

qB

qA5.3

8

qD2

qD1

qC3

7

qC2

250 6

11

Number of Probes per 100KB 100

50

0

qH4

qH3

qH2

qH1

qG3

qG2

qG1

qF3

qF2.3

qF2.2

qF2.1

qF1

qE3

qE2

qE1

qD

qC

qB

qA3

qA2

150

qA1

qH4

qH3

qH2

qH1

14

11

qF2 qF3

11 5

7

10

13

qF1

Genomic Position Along Chromosome 14 (10MB) 4

6

9

12

qE3

0 3

8

11

qE2

50 2

5

10

qE1

100 1

4

7

9

qD3

150 0

6

8

qD2

200 300

5

7

qC

12

6

qD1

Genomic Position Along Chromosome 11 (10MB) qA5.2

0 3

qB3

50 2

4

qB2

100 1

5

qC1

150 0

3

qA5.1

200 300

4

qB1

13

qA4

0

2

qA3

50

1

qA2

100

qA2

150

0

qA3

12

qA1

200

300

qA3.1 qA3.2 qA3.3

15

qA2

250

Number of Probes per 100KB

Genomic Position Along Chromosome 5 (10MB)

qA1.3

11 qA1

Genomic Position Along Chromosome 9 (10MB)

qG3

qF3

qF2

0

qA1.2

250

Number of Probes per 100KB

Genomic Position Along Chromosome 8 (10MB) qG2

11

qE2

qE1

qG1.1 qG1.2 qG1.3

qG1 qG2 qG3

50

3

qB3.3

10

14

qA1.1

250

Number of Probes per 100KB

10

qE2

9

qE1

10

qD3

qF1

qE5

qE4

qE3

100

200

2

qB3.2

9 qD2

qD1

qF

qE5

qE4

qE3

qE2

qE2

qE1

qD

qC3

qC2

150

250

1

qB3.1

8 qD

qC

qC4 qC5

qC3

qC2

qC1

qE1

qD

qC3.2 qC3.3

qC1.2 qC1.3

200

0

qB2

7 qB5

qB4

qC2

qC1

qC3.1

qB

qC1.1

250

300

qB1

6 qB3.3

qB3.2

qB3.1

qB2

qB1.3

qB1.2

qB1.1

qB3

qA3

qA2

Genomic Position Along Chromosome 2 (10MB)

qA2

5 8

13

18

qA1

250

Number of Probes per 100KB

4 7

12

17

qE5

0

3 6 9

16

qE4

50

2 8

11

15

qE3

100

1 7

10

14

qE2.3

150

0 9

13

qE2.2

200

300

8

12

qE2.1

12

5

11

qE1

Genomic Position Along Chromosome 13 (10MB) 4 6

10

qD3

0 5

9

qD2

50 3

7

qB3

100 2

8

qD1

150 1

6

qB2

200 0 4

qB1.3

300

5

qC3

12

7

qC2

Genomic Position Along Chromosome 10 (10MB)

qA5

0 3

6

qB1.2

50 2

4

qB1.1

100 1

5

qC1

150 0

3

qA4

200 300

qA4

Genomic Position Along Chromosome 7 (10MB)

4

qB

15

2

qB2

0

3

qA3

50

2

qA3.3

100 1

qB1

150 0

qA3

300

qA2

Genomic Position Along Chromosome 4 (10MB)

qA2

15

qA1.2 qA1.3

0

1

qA3.2

200

qA1.1

Number of Probes per 100KB

50

qA1

100

qA2

250

Number of Probes per 100KB

150

qA1

qH6

qH5

qH4

qH3

200 0

qA3

11 qE2

qE1

qD3

qG1 qG2 qG3 qH1 qH2.1 qH2.2 qH2.3

qF

qE4

qE3

qE2.3

250

qA1

250

Number of Probes per 100KB

14

qF5

14

300

qA3.1

250

Number of Probes per 100KB

11 qF4

qD2.3

qD2.2

qD

qE2.2

qE2.1

Genomic Position Along Chromosome 1 (10MB)

qA2

10 qF3

13

19

qA1

250

Number of Probes per 100KB

9

13

qD3

10

qD2

12

18

qD2.3

8 qD2.1

12

17

qD2.2

9 qF2

11

qF1

qD1

qC7

qC6

qC5

11

16

qD2.1

7

15

qD1

8

14

qD1

10

qE3

9

qE2

10

qC3

7 qE1

9

13

qC3

6 qD3

8

12

qC2

8

qC1

qC4

qC5

qC4

qE1.1 qE1.2

11

qC2

6 qD2

qC3

qC3

qC2

qC1.2 qC1.3

qC1.1

qB

qA5

qA4

qA3

Number of Probes per 100KB

10

qC1

5 qD1

7

qB5.3

5 qC2

qC1

7

qB3

4 qC

6

qB5.1 qB5.2

4 qB3

6

qB4

5

9

qB2

3 qB2

qB1

5

qB5

4

qB4

4

8

qB1

2

7

qB3

3

qB2

3

6

qA5

3

qB3

qA5

qA4

qA2

qA1

5

qA4

1

2

qB1

1 qB2

2

qB1

1

4

qA3.3

0 qA3

2

qA4

0 qA2

qA1

Number of Probes per 100KB

1

qA3

0

3

qA3.2

300

2

qA3.1

300 0

qA2

qA1

300 1

qA3

Number of Probes per 100KB 300 0

qA2

qA1

Number of Probes per 100KB 300

qA2

qA1

Number of Probes per 100KB

b Genomic Position Along Chromosome 3 (10MB) 15

Genomic Position Along Chromosome 6 (10MB) 14

250

200

150

100

50

0

12

250

200

150

100

50

0

Genomic Position Along Chromosome 12 (10MB) 12

Genomic Position Along Chromosome 15 (10MB)

10

250

200

150

100

50

0

5

b Genomic Position Along Chromosome 16 (10MB)

Genomic Position Along Chromosome 17 (10MB)

Genomic Position Along Chromosome 18 (10MB)

8

9

250

200

150

100

50

300

250

200

150

100

50

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

5

6

7

8

9

150

100

50

qE4

qE3

16

Genomic Position Along Y Chromosome (10MB) 300

250

200

150

100

50

0

1

250

200

150

100

50

qF5

qF4

qF3

qF2

qF1

qE3

qE2

qE1

qD

qC3

qC2

qC1

qB

qA6

qA7.1 qA7.2 qA7.3

qA5

qA4

qA2

qA3.1 qA3.2 qA3.3

qA1.2 qA1.3

0

qA1.1

qD3

qD2

qD1

qC3

qC2

qC1

qB

qA

4

qE2

15

0

0

3

200

qA1

qE3

qE2

qE1.3

qE1.2

qD

qE1.1

qC

qB3

qB2

qB1

qA3.3

qA3.2

qA2 qA3.1

qA1

6

2

250

Genomic Position Along X Chromosome (10MB)

5

1

qE

4

Number of Probes per 100KB

3

qE5

2

qE4

1

Number of Probes per 100KB

0

0

0

qC4

qC3.3

qC2

qC3.2

qC3.1

qC1.3

qB5

qC1.2

qC1.1

qB4

qB3

qB2

qB1

qA3

qA2

0

qA1

Number of Probes per 100KB

7

300

Genomic Position Along Chromosome 19 (10MB) 300

6

qD

0

5

qE1

50

4

qD3

100

3

qC3

150

2

qC2

200

1

qC

250

0

qD2

300

qD1

9

qB3

8

qC1

7

qB2

6

qB1

5

qB

4

qA2

3

qA2

2

qA1

1

Number of Probes per 100KB

0

Number of Probes per 100KB

Number of Probes per 100KB

300

Supplementary Figure 1: Density of unique HD-FISH amplicons along the human (a) and mouse (b) genome. G-bands ideograms are displayed.

Nature Methods: doi: 10.1038/nmeth.2306

6

b 24 22 20 18 16 14 12 10 8 6 4 2 0

10 probes (24 amplicons / probe)

1 probe (24 amplicons / probe)

cumulative cost x 1,000 (USD)

20 probes (48 amplicons / probe)

0 20 0 40 0 60 0 80 10 0 0 12 0 0 14 0 0 16 0 0 18 0 0 20 0 0 22 0 0 24 0 00

cumulative cost x 1,000 (USD)

a

tests / probe

48 amplicons / probe 150 24 amplicons / probe 100 12 amplicons / probe 50

0 0

500

1000

# probes

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.

Nature Methods: doi: 10.1038/nmeth.2306

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relative frequency (%)

24 amplicons (4.8 kb) 50 40

19 amplicons (3.8 kb)

40

n = 92

40

10 amplicons (2 kb) 50

50

50 n = 99

15 amplicons (3 kb) n = 134

40

30

30

30

30

20

20

20

20

10

10

10

10

0

0

0

1 2 3 4 5 # spots / nucleus

1 2 3 4 5 # spots / nucleus

1 2 3 4 5 # spots / nucleus

0

n = 75

0 1 2 3 4 # spots / nucleus

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.

Nature Methods: doi: 10.1038/nmeth.2306

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a

p36.33 p36.32 p36.31 p36.23 p36.22 p36.21

p13.3

p13.3

p13.2

p13.2

p13.1

p13.1

c

b

p36.13

p32.3 p32.2 p32.1

p12

p11.2

p11.1 q11.1

p11.1 q11.1

q11.2

q11.2

q12

q12 q21.1 q21.2

q21.1 q21.2

q21.31

q21.31

q21.32

q21.32

q21.33

q21.33

q22

q22

p21.1

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

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

p13.3

q24.2

q24.2

p13.2 p13.1 p12 p11.2 p11.1 q11

q24.3

q24.3

q25.1

q25.1

q25.2

q25.2

p31.3 p31.2 p31.1

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

q25.3

m = 0.96

14

20

10 6

10

2

0 0

10

20

30

AlexaFluor 647 x 10

x 10

q25.3

30

# observations

p34.2 p34.1 p33

p12

p11.2

AlexaFluor 594

p36.12 p36.11 p35.3 p35.2 p35.1 p34.3

q12

d

q21.1 q21.2 q21.3 q22 q23.1 q23.2 q23.3 q24.1 q24.2 q24.3 q25.1 q25.2

relative frequency (%)

Chr17

q25.3 q31.1 q31.2 q31.3 q32.1 q32.2 q32.3 q41 q42.11 q42.12 q42.13

25 20

n = 744

15 10 5

q42.2 q42.3 q43

0

q44

-12 -8 -4 Chr1

0

4

8 12

difference between AF647 and AF594 spot counts

e Spotting

AF647 + AF594

Paint

AF647

AF594

AF647 + AF594

Paint

f

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 Nature Methods: doi: 10.1038/nmeth.2306

9

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.

Nature Methods: doi: 10.1038/nmeth.2306

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Relative frequency (%)

25 20

n = 445

15 10 5 0

0

10

20

30

40

# mRNA

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.

Nature Methods: doi: 10.1038/nmeth.2306

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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,

Nature Methods: doi: 10.1038/nmeth.2306

12

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 mediumsized 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 HDFISH 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 96well 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.

Nature Methods: doi: 10.1038/nmeth.2306

13

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 Zprojection shown in Figure 3a (mid panel). Supplementary Video 2: 3D animation of Chr17 in HME cells, visualized with sixteen HDFISH probes spaced evenly every 5 Mb and labeled with two alternating fluorophores (green: AlexaFluor594; magenta: AlexaFluor647) together with a Chr17 paint probe (blue).

Nature Methods: doi: 10.1038/nmeth.2306

14