Nature - USA (2019-07-18)

reSeArcH Article

Oncogene

TSG

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

VAF in WES

I

II

III

IV

V

VAF in RNAseq

WES’s VAF = 0

RNA-seq’s VAF = 0

WES’s VAF = RNA-seq’s VAF

WES’s VAF < RNA-seq’s VAF

WES’s VAF > RNA-seq’s VAF

I.

II.

III.

IV.

V.

II

IV

V

BRCA

0.0092%

13.61%

0.28%

55.89%

30.21%

I.

II.

III.

IV.

V.

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

VAF in WES

VAF in RNAseq

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0. 81

TSG Oncogene

II

IV

V

LUAD

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

VAF in WES

VAF in RNAseq

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

TSG Oncogene

0.0058%

21.98%

0.16%

59.39%

18.47%

I.

II.

III.

IV.

V.

II

IV

V

STAD

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

VAF in WES

VAF in RNAseq

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0. 81

TSG Oncogene

0.0015%

20.95%

0.22%

53.67%

25.14%

I.

II.

III.

IV.

V.

II

IV

V

KIRC

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

VAF in WES

VAF in RNAseq

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0. 81

TSG Oncogene

0.036%

12.60%

0.43%

55.40%

31.53%

I.

II.

III.

IV.

V.

II

IV

V

HNSC

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

VAF in WES

VAF in RNAseq

0

0.2

0.4

0.6

0.8

1

00 .2 0.40.6 0.81

TSG Oncogene

0.0068%

8.74%

0.099%

67.65%

23.50%

I.

II.

III.

IV.

V.

Poly(dT)UMICBRead1

TSO Poly(dT)UMICBRead1

SpikeInPCR#1

PCR#2fw

PCR#3 PCR#2rv

RPI-x handle

SpikeIn PCR#1

TSO

SI-PCR

SI-PCR

PartialRead#1

Gibson or U- overhang handle

GoT (3’ kit)

Circularization GoT (3’ kit)

Read1CBUMI

Poly(dT)

TSO

PCR#2rv PCR#1 SpikeIn

PCR#2fw

PCR#3

RPI-x handle

PCR#1 SpikeIn

SI-PCR

PartialRead#1

Gibson or U- overhang handle

GoT (5’ kit)

Circularization GoT (5’ kit)

Poly(dT)

Read1CBUMITSO

SI-PCR

Guide RT handle

Non-poly(dT)

Non-poly(dT)

JAK2 V617F (c.1849G>T) (Circularization GoT - 5’ kit)

5’-CTGCAGGAAGGAGAGAGGAAGAGGAGCAGAAGGGGGC [... >2kb ...] GATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAATATTCTGGTTCAGGAG

TTTGTAAAATTTGGATCACTAGATACATATCTGAAAAAGAATAAAAATTGTATAAATATATTATGGAAACTTGAAGTTGCTAAACAGTTGGCATGGGCCATGCATTTTCTAGAAGAAAACACCCTTATTCATGGG

AATGTATGTGCCAAAAATATTCTGCTTATCAGAGAAGAAGACAGGAAGACAGGAAATCCTCCTTTCATCAAACT.....-3’

PCR#2-fw

PCR#2-rv PCR#3

PCR#1

Spike In

5’-...ACAAAGGCAGCAGAGAAACAAATGAAGGACAAACAGGACGAGGAGCAGAGGCTTAAGGAGGAGGAAGAAGACAAGAAACGCAAAGAGGAGGAGGAGGCA [... ~670bp ...] -3’

PCR#1

CALR L367fs*46 (c.1099_1150del) (GoT - 3’ kit)

5’-...GAGGCTTAAGGAGGAGGAAGAAGACAAGAAACGCAAAGAGGAGGAGGAGGCAGAGGACA{TTGTC}AGGA [... ~670bp ...] -3’

PCR#1

CALR K385fs*47 (c.1154_1155insTTGTC) (GoT - 3’ kit)

5’-...GCCCTGGGCATTCCTTCTTTATTGCCCTTCTTAAAAGCTGTGTGCAAAAGCAAGAAGTCCTGGCAAGCGAGACACACTGGTATTAAGATTGTACAACA [...>1.5 kb ...]- 3’

Spike In PCR#1

SF3B1 H662D (c.1984C>G) (GoT - 3’ kit)

NFE2 E261fs*3 (c.782_785delAGAG) (GoT - 3’ kit)

5’-...GATTGTCAACTTGCCGGTAGATGACTTTAATGAGCTATTGGCAAGGTACCCGCTGACAGAGAGCCAGCTAGCGCTA [... ~580bp ...]- 3’

PCR#1

XBP1 - Splice site (GoT - 3’ kit)

5’-...AGCCAAGGGGAATGAAGTGAGGCCAGTGGCCGGGTCTGCTGAGTCCGCAGCACTCAGACTACGTGCACCTCTGCAGCAGGTGCAG [... ~1.3kb ...]- 3’

PCR#1

5’-...CCTCTGCCCTGGGCATTCCTTCTTTATTGCCCTTCTTAAAAGCTGTGTGCAAAAGCAAGAAGTCCTGGCAAGCGAGACACACTGGTATTAAGATTGTACAACAGATAGCTATTCTTATGGGCTGTGC

CATCT [... >1.5kb ...] CTGTGAGCACCAAGTACCACTAGATGAATAAAACGTATTATATCTAAA- 3’

Spike In

PCR#1

PCR#3

PCR#2-rv PCR#2-fw

SF3B1 H662D (c.1984C>G) (Circularization GoT - 3’ kit)

5’-...ATACGCTGAGGAGTTTGGCAACGAGACGTGGGGCGTAACAAAGGCAGCAGAGAAACAAATGAAGGACAAACAGGACGAGGAGCAGAGGCTTAAGGAGGAGGAAGAAGACAAGAAACGCAAAGAGGAGGAGGAGGCAGAGG

ACA{TTGTC}AGGAGGATGATGAGGACAAAGATGAGGATGAGGAGGATGAGGAGGACAAG [... ~600bp] GGCTCACACTGAGAATGTAAGAACTACAAACAAAATTTCTATTAAATTAAATTTTGTGTCTC- 3’

CALR K385fs*47 (c.1154_1155insTTGTC) (Circularization GoT - 3’ kit)

PCR#1

PCR#2-fw

PCR#3

PCR#2-rv

5’-...TTTTAAAAGTTCTGGATAAAGCACACAGAAACTATTCAGAGTCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAAT

ATTCTGGTTCAGGAGTTTGTAAAATTTGGATCACTAGATACATATC [... >2.8kb ...] GTGTTATTTATACAAAACTTAAAATACTTGCTGTTTTGATTAAAAAGAAAATAGTTTCTTACTTTA-3’

PCR#1

PCR#2-fw

PCR#3

PCR#2-rv

JAK2 V617F (c.1849G>T) (Circularization GoT - 3’ kit)

a

b

c

Extended Data Fig. 1 | Comparison of VAF between WES and RNA-
seq, and primer sequences and positions of linear and circularization
GoT. a, Pie charts show the fraction of variants, which are categorized as
described in the top panel. The distribution of the mutant allele fraction
is annotated as oncogene or tumour-suppressor gene (TSG) (according
to previously published definitions^60 ,^61 ). Diagonal dashed lines indicate
an equal allelic fraction between WES and RNA-seq. Yellow density

contours represent driver distributions. BRCA, breast invasive carcinoma;

HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal

clear cell carcinoma; LUAD, lung adenocarcinoma; STAD, stomach

adenocarcinoma. b, Schematic localization of primers for linear GoT

and circularization GoT for 3′ and 5′ libraries. c, Primer positions and

sequences of the regions targeted by GoT and circularization GoT.