www.sygnis.com

Abstract

Gene frequencies

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1

10

100

1

10

100

1

10

100

AB

L1A

LKA

PC

ATM

BR

AF

CD

H1

CD

KN

2AC

TN

NB

1E

GF

RE

GF

R,E

GF

R−A

S1

ER

BB

2E

RB

B4

FB

XW

7F

GF

R1

FG

FR

2F

GF

R3

FLT

3G

NA

11G

NA

QH

NF

1AH

RA

SJA

K2

JAK

3K

DR

KIT

KR

AS

ME

TN

OT

CH

1N

PM

1N

RA

SP

DG

FR

AP

IK3C

AP

TE

NP

TP

N11

RB

1R

ET

SM

AD

4S

MA

RC

B1

ST

K11

TP

53V

HL

Freq

uenc

y

100 pg amplified cfD

NA

NA

cfDN

Atum

or biopsy

Target-derived output even at 1 fg1 ng input DNA 1 pg input DNA

1 fg input DNA 1 pg input DNA, random primed

Conclusions• Advantages of the TruePrimeTM amplification technology - excellent sensitivity and reproducibility, no primer artifacts, excellent coverage with little bias • TruePrimeTM Liquid Biopsy preserves SNVs and SNV frequencies even across different sequencing methods (Illumina and IonTorrent amplicon approach and Illumina exome capturing) • TruePrimeTM Liquid Biopsy works reliably at 100pg DNA level

In conclusion, TruePrimeTM LiquidBiopsy is a highly promising approach to enhance sensitivity of cfDNA analyses.The method is sensitive, cfDNA can be amplified to a level that allows sequencing, and SNV recovery is good.

Recommended approach: Targeted exome capturing (e.g Agilent SureSelect Focused Exome) with high sequencing depth for SNV detection and WGS for CNV estimations.

Colorectal carcinoma case I - Amplicon

We amplified 100 pg of plasma DNA from a colorectal adenocarcinoma case (T4a M1a L1 G4) and compared this to non-amplified DNA from a tumor biopsy and healthy tissue. The GATC amplicon panel (230 amplicons) with digital PCR (Raindance) was used on Illumina sequencer. TOP LEFT: SNV frequency correlation between tumor biopsy and TruePrimeTM 100pg amplified sample. TOP CENTER: position and frequencies of KRAS SNVs in TruePrimeTM 100pg amplified sample and tumor biopsy. Note the high overlap between the two samples. TOP RIGHT: number of identical COSMIC key pathogenic mutations in specific genes in TruePrimeTM 100pg amplified sample and tumor biopsy. Note the high number of KRAS mutations between the two samples. LEFT: Venn diagramm showing overlap of identical SNVs from the TruePrimeTM 100pg amplified sample (blue) and tumor biopsy sample (red).

We amplified 100 pg of plasma DNA from a colorectal adenocarcinoma case (T3 N0 Mo G3) and compared this to non-amplified DNA from a tumor biopsy and healthy tissue. The Agilent SureSelect Human All Exome V6 capture kit was used on Illumina sequencer.TOP LEFT: Circos plot of all exome samples. Bin size was chosen optimal for

exome mapping. Note the evenness of coverage for the TruePrimeTM 100pg amplified sample.TOP CENTER: Venn diagramm showing overlap of identical SNVs from the TruePrimeTM 100pg amplified sample (blue) and tumor biopsy sample (red). TOP RIGHT: SNV frequencies of two key pathogenic KRAS mutations (A59T and Q61P). Note the high similarity between the three samples. RIGHT: SNV frequency correlation between tumor biopsy and TruePrimeTM 100pg amplified sample. The number of SNV frequencies at certain values is color coded. Note the high similarity of frequency overlap.

Tumor biopsy

100 pg amplified cfDNA

186

52

503

TruePrime™ is the name of a novel technology dedicated to the amplification of genomic DNA. While the current

gold standard MDA (multiple displacement amplification) relies on short oligonucleotides to start off the

amplification, TruePrime™ is based on a combination of Phi29 DNA polymerase with the recently discovered

primase/polymerase TthPrimPol. In this setup, TthPrimPol synthesizes the DNA primers needed for Phi29 DNA pol in

the course of the reaction, which allows for the exponential amplification of genomic DNA. Key advantages of the

TruePrime™ technology for amplification of single cell genomes include complete absence of primer artefacts,

superior sensitivity down to the femtogram range, high reproducibility, little bias in genome coverage, and superior

variant detection. Moreover, the TruePrime™ workflow is easy and reaction products work well with major NGS

platforms. We are currently tuning the TruePrime™ technology towards the amplification of cell-free DNA from

plasma and serum with the intention to improve sensitivity and reliability of the method. Results on the feasibility of

this approach are presented.

TruePrime™ Liquid Biopsy: Tuning whole genome amplification towards improving the sensitivity of circulating tumor DNA analysis

Human genomic DNA was 10-fold serially diluted, and amplified using the TruePrimeTM protocol, and sequenced using Ion Torrent technology. (~20 000 reads). Reads were BLAST-compared to the EMBL databases. Even at 1 fg input amount the vast majority of output sequences is target derived (>95% of sequences from human), demonstrating the high quality of components of the TruePrimeTM reaction, and the robustness of the protocol.

TruePrimeTM reaction overview1. TthPrimPol binds denatured DNA at different sites

2. TthPrimPol synthesizes short DNA primers

3. Phi29 DNA pol displaces TthPrimPol and begins polymerization

4. Phi29 DNA pol performs strand displacement

5. TthPrimPol binds to newly formed DNA and synthesizes new DNA primers

6. Phi29 DNA pol displaces TthPrimPol, binds DNA primers and begins polymerization

Angel J. Picher1, Bettina Budeus2, Luis Blanco3, Simon Joose4, Laura Muinelo5, Trinidad Caldés6, Klaus Pantel4 and Armin Schneider2

Preservation of allele frequencies

Human99,19%

Human97,41%

Human75,84%

Human95,40%

Other0,03%

Other0,10%

Other0,10%Other

0,13%

Unknown0,78%

Unknown2,49%

Unknown19,92%

Unknown1,91%E.coli

2,35%

Vectors0,21%

Invertebrates4,11%

Colorectal carcinoma case II - Exome

00-09

10-19

20-29

30-39

40-49

50-59

60-69

70-79

80-89

90-99

TOP: Circos plots depict genome coverage pattern in blue, SNV allele frequencies as a color-coded band (color legend at right), number of detected SNVs per genome segment as histogram (black), and finally a CNV plot showing chromosomal aberrations in the Hek293 line we studied (blue = haploid, green = diploid, red = triploid and above).LEFT: Preservation of SNV allele frequencies is shown in a scatter plot from chromosome1(diploid in our Hek293 cells), where all common heterozygous SNV allele frequencies (BAF, B allele frequencies) are shown with a density. The TruePrime™ amplified genome shows the greatest density in the central intersection of the 50% frequencies, while the commercial RP-MDA sample shows a broader dispersion of these

Non-amplified TruePrime™ Commercial RP MDA

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0.0

0.5

1.0

1.5

2.0

0 0.5 10 1.5 2.0log10 (SNV frequencies - Tumor biopsy)

log 1

0 (SN

V fr

eque

ncie

s - 1

00 p

g am

plifi

ed c

fDN

A)

440

395

983

Tumor biopsy

100 pg amplified cfDNA

0102030

A P C

C D H 1

E GF R

E G F R ,E G F R −A S 1

F B XW7

F G F R 3

H R A SJ A K 3

KIT

NR A S

P DGF RA

P IK3CA

S MAD4

TP53

PTEN

KRAS

CDKN2A

ATM

4 Institut für Tumorbiologie, UKE Hamburg, Germany3 CBMSO, Madrid, Spain1 SYGNIS Biotech SLU, Madrid, Spain 2 SYGNIS Bioscience GmbH & Co KG, Heidelberg, Germany5 Fundación Ramón Domínguez, Complejo Hospitalario Santiago de Compostela, Spain 6 Hospital Clínico San Carlos, Madrid, Spain

ProteinPosition

AminoAcids

TumorBiopsy

100 pga cfDNA

6159*3120151413

Q/PA/TE/QT

G/SV/AG/S

xxxx

xxx x

xx

x

x

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0.0

0.5

1.0

1.5

2.0

0.0 0.5 1.0 1.5 2.0log10(SNV frequiencies - Tumor biopsy)

log 1

0(SN

V fr

equi

enci

es -

100p

g am

plifi

ed c

fDN

A)

log10(frequency -100 pg amplified cfDNA)

log 1

0(fre

quen

cy -

Tum

or b

iops

y)

1

7

55

400

0 1 2 3 4

4

3

2

1

0

A59T Q61P

10

20

30

40

50

60

70

80

90

100

0

Freq

uenc

y100 pg amplified cfDNA

Tumor biopsyHealthy tissue

100 pg amplified cfDNA

Tumor biopsyHealthy biopsy

Direct plasma amplified

RIGHT: SNV frequencies of TruePrimeTM 100pg amplified sample, tumor biopsy and non-amplified cfDNA sorted by corresponding gene with entry in Cosmic. [Amplicon sequencing - colorectal carcinoma case III] Note the high overlap between the tumor biopsy and the amplified sample.

LEFT: SNV frequency correlation between tumor biopsy and TruePrimeTM 100pg amplified sample (shown are only those SNV frequencies which are identical in TruePrimeTM 100pg amplified sample, tumor biopsy, healthy tissue and non-amplified cfDNA. [Amplicon sequencing - colorectal carcinoma case I]

* KRAS codon 59 point mutations lead to constant activation of the protein, and render Anti-EGFR therapies ineffective