Nature - USA (2019-07-18)

reSeArcH Article

(i) 50 bases at 5’ end mapped to SF3B1

(ii) 50 bases at 5’ end mapped to other transcripts

(ii) 2.2%

5’ 3’ (i) 97.8%

5’ 3’

SF3B1 Non SF3B1 GoT targeted loci

0

0.5

1

01000 2000

Pairwise difference of read lengths

in duplicate reads

Density

SF3B1 CALR NFE2

---

+--

++-

+++

MF05

Single cell colony genotyping (n = 94)

a

CALR (85%)

XBP1 (80%)

SF3B1 (24%)

NFE2 (60%)

JAK2 (7%)

0

1

2

3

4

1000 1500 2000

Distance from gene end

Mean per 10,000 UMI per cell

Linear GoT

Circularization GoT

(%)Avg genotyping efficiency

b

1

2

3

4

Log

(Number of cases) 10

04 1,000 2,000 3,000 ,000 5,000

NFE2.E261fs

CALR.K385fs*47

CALR.K367fs*46

XBP1 splice site

SF3B1.H662D

JAK2.V617F (5’)

JAK2.V617F (3’)

Distance from closest mRNA end (5’ or 3’)

Density

5’ end3’ end

Oncogene

Tumor Suppressor gene

Passenger

Oncogene

Tumor Suppressor gene

c

BRAF V600E

KRAS G12D

NRAS Q61K

TP53 R273H

Loci targeted

by GoT

0

25

50

75

ATAAGTGTC/GTCTCGC GC

-10-8-6- 4 -2 0 246810

Genomic position around the target (SF3B1 c.1894C>G)

100 CC TT

Determine read direction (by scanning both ends)

Trancript of interest

10X

CB

10X

UMI

GoT

targeted loci

5’ 3’

Trim P5, Read#1 primer and PCR#3 handle

Extract sequences for cell barcodes, UMI and region of interest

(1) Identify reads with expected priming

(2) Identify cell barcodes within whitelist

(3) Replace cell barcodes that are not whithin whitelist

(4) Deduplicate reads

(5) Analyze reads with cell barcodes also present in the 10X scRNA-seq data

Native ONT

adapter

ONT

BarcodeP5

Read#1

primer

10X

CB

10X

UMI

Native ONT

adapter

ONT

Barcode

PCR#3

handle

GoT

targeted loci

5’ 3’

3’ 5’

OR Trancript / region of interest

h

e

WT

MUT

WT MUT

Linear GoT

Circularization GoT

2.8%

44.1% 2.5%

50.6%

CALR

0

0.5

1

R^2 = 0.92

P < 10-10

00 .5 1

Mutant fraction per cell

Circularization GoT

Linear GoT

i

d

e

f

g

71%

5%

14%

10%

Extended Data Fig. 9 | Deciphering subclonal progenitor identities
using multiplex GoT, and targeting loci that are distant from transcript
ends using circularization GoT. a, Single-cell cloning assay of peripheral
blood cells from patient MF05 (Methods). b, Rate of targeted locus
capture (per cent) as a function of gene expression and the distance of the
targeted locus from the transcript ends. c, Distance of the mutation locus
from transcript ends for pan-cancer drivers, and their frequencies (based
on the number of times they are reported in the COSMIC database).
Mutations are annotated as oncogenes, tumour-suppressor genes or
passengers (as previously defined^60 ,^61 ). Relative density of each subclass
of mutations from the closer end (that is, 3′ or 5′) is shown in the top
panel. d, Schematic of analysis of ONT sequencing reads. e, Frequency
of SF3B1-mutant and wild-type reads of linear GoT amplicon library

sequenced with ONT. f, Analysis of SF3B1 amplicon reads sequenced by

ONT for inter-transcript PCR recombination by mapping 50  bp at the

opposite end of the targeted locus, showing only the 2.2% of fragments

that reflect inter-transcript recombination. g, Pairwise difference of

read lengths for duplicate reads (that is, reads with the same cell barcode

and UMI) of the SF3B1 amplicon library sequenced with ONT, showing

consistent read lengths of duplicate reads that support a low rate of intra-

transcript PCR recombination. h, Comparison of genotype assignment

for CALR in sample MF01 between linear GoT and circularization GoT

after downsampling reads to 300,000 with 10 iterations (n = 320 cells).

i, Comparison of CALR-mutant UMI fraction per cell in sample MF01

between linear GoT and circularization GoT after downsampling reads to

300,000 with 10 iterations (n = 320 cells, Pearson’s correlation, F-test).