TruePrime™ Liquid Biopsy: Tuning whole genome amplification ... · and compared this to...
Transcript of TruePrime™ Liquid Biopsy: Tuning whole genome amplification ... · and compared this to...
www.sygnis.com
Abstract
Gene frequencies
●
●
●
●
●
●
●●
●
●
●●●●●●● ●
●
●●
●●●
●
●
●
●●
●
●
●●
●
●
●●
●
●
●
●●●
●●●
●●●● ●
●
●●
●●●●
●●●
●
●●
●
●●●
●●
●
●●●●
●
●
●●
●
●●●
●
●
●
●●●
●
●●●
●●● ●
●●●●●●●●
●
●●
●●
●
●
●●●●●
●●●● ●●
●
●
●
●●
●●●
●
●
●
●●●●●
●
●
●●
●●
●●
●
●
●
●
●
●●●
●●
●
●
●●●●
●●
●
●●
●
● ●
●●●
●
●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●●
●●●
●
●●
●
●
●
●●●●● ●
●●
●●
●
●●●
●
●
●●●●●●
●
●
●●●
●
●
●
●●
●●●
●
●●●
●
●
●●
●
●
●
●●●●●
●
●
●
●● ●
●
●
●
●●●
●●●●●●●●●
●
●● ●●●
●●●●
●
●
●
●●
●
●
●●●
●
●●
●
●●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●●
●
● ●
●
●
●●
●
●●
●
●
●●
●
●
●●
●
●
●
●●
●●
●●●
●
●
●
●
●
●
●●
●
●●
●
● ●●●●
●
●●
●
●●
●
●
●
●
●●
●●●
●
●●●●●
●●
●
●
●
●●●
●
●●●
●
●
●●
●
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
●
●●
●
●
●
●●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●●●
●
●
●
●
●
●
●●●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●●●
●
●
●
●
●
●●
●●●
●●●
●●●●
●
●
●●
●
●
●
●
●
●
●●
●●
●
●
● ●●
●
●
●
●
●●
●
●●
●
● ● ●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●●
●●
●
●
●
●●
●
●
●
●●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●●
●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●● ●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●●●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●●
●●
●●●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●●●
●
●
●
●●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●●
●
●●
●●●
●
●
●
●
●
●●
●●
●
●
●●●●●
●
●●
●●
●
●
●
●
●
●
●
●
●
●●
●●●●●
●
●
●●
●
●
●
●
●
●●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●●
●
●
●
●
●
●
●
●
●●
● ●
●
●●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●●●
●●
●
●●●
●
●
●●●
●
●
●●
●
●
●
●
●●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●●●
●
●●
●
●
●
●
●
●
●●●
●
●
●●
●●
●
●●
●
●●
●
●
●
●
●
●
●●●●●
●
●
●
●●
●
●
● ●
●●
●
●●
●
●●
●
●
●●●●
●●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●●●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●●
●●
●
●●
●●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●●●
●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●
●●●
●●●
●
●
●
●
●
●
●
●
●●
●
●
●●
●●
●●
●
● ●●
●●
●●●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●●
●●
●
●
●●●
●
●
●
●●
●
●
●
●
●
●●
●
●
●●●
●
●●
●
●
●
●●
●
●
●
●
●
●
●●●
●
●
●
●
●
●
●●●
●
●
●
●●
●
●
●
●
●
●
●
●●
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
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
● ●
●
●
●
●●●
●
●
●
●
●●
●●●
●
●
●
●●
●●
●
●
●
●
●
●●
●
●●
●
●
●
●
●●●
●
●
●
●●
●●
●
● ●
●●
●
●
●●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●
●
●
●
●
●●
●●●●●
●
●
●
●
●
●●
●●● ●●
●●
●
●●
●
●
●●
●●
●
●
●●
●●
●
●
●
●
●●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●●
●●
●
●
●●●
●●
●
●●●
●
●●●●
●●●
●●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
● ●
●●
●
●
●●
●
●
●
●●
● ●
●
●
●
●
●
●
●●●
●
●
●
●
●
●
●
●
●
●●●●●●
●●
●●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●●
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