Nature - USA (2019-07-18)

reSeArcH Article

Number of UMI for reads with CALR locus of interest 01234 >5

ET01

10X 3’ v2 GoT

MF01

10X 3’ v2 GoT

ET05

10X 3’ v3 GoT

MF05

10X 3’ v2 GoT

ET02

10X 3’ v2 GoT

MF02

10X 3’ v3 GoT

ET03

10X 3’ v2 GoT

MF03

10X 3’ v3 GoT

ET04

10X 3’ v2 GoT

MF04

10X 3’ v3 GoT

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

1

4

16

64

10x 10x GoT

Ta rgeted CALR

locus

CALR gene

0

UMI counts per cell

UMI counts per cell

ET01

3’ v2

ET04

3’ v2

ET03

3’ v2

ET02

3’ v2

ET05

3’ v3

MF01

3’ v2

MF04

3’ v3

MF03

3’ v3

MF02

3’ v3

MF05

3’ v2

a

b

ET01

ET02

ET03

ET04

ET05

MF01

MF02

MF03

MF04

MF05

Essential thrombocythemia

Essential thrombocythemia

Essential thrombocythemia

Essential thrombocythemia

Essential thrombocythemia

Myelofibrosis

Myelofibrosis

Myelofibrosis

Myelofibrosis

Myelofibrosis

BM

BM

BM

BM

BM

PB

PB

PB

PB

PB

L367Tfs*46 (Type 1)

K385Nfs*47 (Type 2)

L367Tfs*46 (Type 1)

L367Tfs*46 (Type 1)

A382Rfs*58

K385Nfs*47 (Type 2)

L367Tfs*46 (Type 1)

L367Tfs*46 (Type 1)

K385Nfs*47 (Type 2)

K385Nfs*47 (Type 2)

6811

1135

3587

5131

2217

965

2631

3840

3657

8475

90%

82%

90%

92%

82%

51%

94%

90%

92%

74%

XBP1 splice site

XBP1 splice site

NFE2 (E261Afs*3), SF3B1 (H662D)

Patient ID Diagnosis Source CALR variant Cell #% genotyped Other targets

c

d

MF05

MF01

ET04

ET03

ET02

ET01

Healthy control

Number of genes per cell

n = 7,927

0 4000300020001000 5000 0 20,00040,00060,000

Number of UMI per cell

n = 6,811

n = 1,135

n = 3,587

n = 5,131

n = 965

n = 8,475

Cell #

Extended Data Fig. 3 | GoT captures genotyping information of single
cells through cDNA. a, Percentage of cells by number of UMIs with the
CALR-mutation locus captured in standard 10x data (left panels) and GoT
data (right panels) (see c for cell numbers in each sample). b, Number of
UMIs per cell of CALR transcript from standard 10x data (blue shading)
or targeted CALR locus from standard 10x or GoT (pink shading) (see
c for cell numbers in each sample). c, Summary of clinical, pathological
and GoT data from patients with CALR-mutated myeloproliferative

neoplasms. BM, bone marrow; PB, peripheral blood. d, Number of genes

per cell (left) and number of UMIs per cell (right) from published standard

10x data of healthy control CD34+ cells and 10x data from 3′ v.2 chemistry

of CD34+ cells from patient samples that underwent concurrent GoT, after

random downsampling of the reads from each sample to 50  million reads

× 3 iterations, showing that the extra cycle of PCR and portioning a small

aliquot from the 10x cDNA library for GoT using 3′ v.2 chemistry does not

compromise scRNA-seq data.