TruePrime™, a novel technology for whole genome amplifica€¦ · SYGNIS Bioscience GmbH, INF...
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SYGNIS Bioscience GmbH, INF 515, 69120 Heidelberg www.sygnis.com ESHG Glasgow TruePrime™, a novel technology for whole genome amplifica- tion from single cells and limited material Ángel J. Picher a Patricia Garrido a Armin Schneider b Oliver Wafzig b Luis Blanco c a, Sygnis Biotech S.L.U., Parque Científico de Madrid, Cantoblanco, Madrid 28049, Spain b, Sygnis Bioscience GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany c, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, Madrid 28049, Spain Shown is the simple workflow for setting up the TruePrime™ DNA amplification. One or a few cells are added to the lysis and denaturation buffer and incubated for 10 min on ice. Thereafter, this is neutralized, the reaction components added and incubated for 3-6 h at 30°C. The agarose gel image on the right shows the high molecular weight fragments produced by TruePrime TM amplification of 6 pg of DNA (equivalent of one human cell DNA content), and the absence of any DNA amplification in negative controls. Human genomic DNA was 10-fold serially diluted, and amplified using the TruePrime TM protocol, and se- quenced 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 TruePrime TM reaction, and the robustness of the protocol. TruePrime TM is a novel, primer-free multiple-displacement type amplification tech- nology based on TthPrimPol and Phi29 DNA polymerase. This novel amplification technology demonstrates the following features: • No primer artefacts • Insensitive to external, non-denatured DNA contaminations • Excellent sensitivity • Exquisite reproducibility when amplifying from single mammalian cells • Excellent genome coverage with little bias • Ideally suited for next generation sequencing, tested for Illumina and Ion Torrent workflows • Easy and reliable Single human HEK293 cells were isolated by dilution, manual picking and visual inspection, and subjected to the True- Prime TM amplification protocol for 3 and 6 h. DNA yield obtained was quantified by PicoGreen incorporation. Note the low variation between individual cells amplified, and the absence of any significant DNA amplification in the nega- tive (no template) controls. 3 h amplification time yields sufficient DNA for most downstream applications including sequencing. Red squares: TthPrimPol, blue circles: Phi29 polymerase, red lines: DNA primer synthesized by TthPrimPol. TruePrime™ is a novel technology dedicated to the amplification of whole genomes and DNA from various sources. TruePrime™ is based on the combination of the highly processive Phi29 DNA polymerase with the recently disco- vered primase/polymerase TthPrimPol. In this setup, TthPrimPol synthesizes the DNA primers needed for Phi29 DNA pol, which allows for the exponential amplification of target DNA. TthPrimPol is a thermostable member of a recently discovered family of enzymes named PrimPol. TthPrimPol is a monomeric enzyme (34 kDa) that displays a potent pri- mase activity, preferring dNTPs as substrates unlike conventional primases. This DNA primase activity can be activated by magnesium or manganese ions, having a wide sequence specificity for template recognition. Key advantages of the TruePrime™ technology include complete absence of primer artefacts, insensitivity to external DNA contaminations, reduced amplification bias compared to methods using random synthetic primers, and an exqui- site reproducibility when amplifying from single cells or minute DNA amounts. Moreover, TruePrime™ shows superior sensitivity, is easy to use and works perfectly well with commonly used NGS platforms such as Illumina or IonTorrent. We believe that TruePrime™ will advance human genetic analyses from single cells or otherwise limited input materi- al. Selected references 1. S. Garcia-Gomez, A. Reyes, M.I. Martinez-Jimenez, E.S. Chocrón, S. Mouron, G. Terrados, C. Powell, E. Salido, J. Mendez, I.J. Holt and L. Blanco. PrimPol, an Archaic Primase/Polymerase Operating in Human Cells. Molecular Cell, 2013, 52(4), 541-53. 2. S. Mouron, S. Rodriguez-Acebes, M.I. Martinez-Jimenez, S. Garcia-Gomez, E.S. Chocrón, L. Blanco and J. Mendez. Re- priming of DNA synthesis at stalled replication forks by human PrimPol. Nature Structural & Molecular Biology, 2013, 20(12), 1383-9. Abstract TruePrime TM workflow Target-derived output even at 1 fg Conclusions Single cell amplification TruePrime TM reaction overview 1. TthPrimPol binds denatured DNA at different sites 2. TthPrimPol synthesizes short DNA primers 3. Phi29 DNA pol displaces TthPrimPol and begins po- lymerization 4. Phi29 DNA pol performs strand displacement 5. TthPrimPol binds to newly formed DNA and synthe- sizes new DNA primers 6. Phi29 DNA pol displaces TthPrimPol, binds DNA primers and begins polymerization 99,19% Unknown 0,78% Other 0,03% Human 97,41% Unknown 2,49% Other 0,10% Human 95,40% Enterobacteriacea Unknown 1,91% 0,21% Other 0,13% E. coli 2,35% Human Vectors 1 ng input DNA 1 fg input DNA 1 pg input DNA DNA from 4 single hu- man HEK293 cells was amplified following the TruePrime TM protocol for 3 or 6 h, subjected to library prep and se- quencing (Illumina HiS- eq), and 5 million read paris were mapped onto the human genome using CLCBio software. Shown are comparisons of chro- mosome coverage to non-amplified HEK293 cell DNA from the same cells. Note the evenness of coverage, and the high similarity between single cells. Reproducibility + M + + + - - - - DNA input PS14.105 Excellent overall mapping data for TruePrime™-amplified DNA, closest to non-amplified DNA coverage reached. Exactly 12 mio read pairs were mapped to the human genome. Shown on the top left is a comparison of chromo- somal coverage between TruePrime™ and non-amplified DNA. The closeness of distribution is also highlighted by a comparison of the read depth per base. The graph on the right shows the coverage pattern after mapping on chromo- some 3. Note the extreme similarity between non-amplified and TruePrime TM . Coverage Sample TruePrime™ (1c) Random primers Competitor R Competitor G Non- amplified DNA Mapped reads 96.86% 97.42% 99.64% 98.62% 99.53% Reads in pairs 86.77% 59.07% 91.50% 37.94% 92.03% Fraction human genome covered 49% 28% 38% 1% 53% Calculated ideal (Poisson) coverage 57% 44% 59% 31% 59% Deviation observed from expected 13,8% 35,76% 35.4% 96,7% 10.2% 1 10 100 1000 10000 100000 1000000 10000000 00000000 1E+09 1E+10 0 50 100 150 200 250 depth non-amplified depth TruePrime Non-amplified DNA TruePrime™ Competitor R Competitor G Random primers Chromosome 3 coverage (12 Mio read pairs) Chromosome 3 Chromosome 4 Non-amplified DNA TruePrime™: 1c TruePrime™: 1d TruePrime™: 1g TruePrime™: 1i Orange: Non-amplified DNA Blue: TruePrime™ (1c) 12 Mio read pairs 0 10 20 30 40 50 60 70 80 90 100 chr1 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr2 chr20 chr21 chr22 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chrM chrX chrY Coverage (%)
Transcript of TruePrime™, a novel technology for whole genome amplifica€¦ · SYGNIS Bioscience GmbH, INF...
SYGNIS Bioscience GmbH, INF 515, 69120 Heidelberg
www.sygnis.com
ESHG Glasgow
TruePrime™, a novel technology for whole genome amplifica-tion from single cells and limited material
Ángel J. Pichera Patricia Garridoa Armin Schneiderb Oliver Wafzigb Luis Blancoc
a, Sygnis Biotech S.L.U., Parque Científico de Madrid, Cantoblanco, Madrid 28049, Spainb, Sygnis Bioscience GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany
c, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, Madrid 28049, Spain
Shown is the simple workflow for setting up the TruePrime™ DNA amplification. One or a few cells are added to the lysis and denaturation buffer and incubated for 10 min on ice. Thereafter, this is neutralized, the reaction components added and incubated for 3-6 h at 30°C.The agarose gel image on the right shows the high molecular weight fragments produced by TruePrimeTM amplification of 6 pg of DNA (equivalent of one human cell DNA content), and the absence of any DNA amplification in negative controls.
Human genomic DNA was 10-fold serially diluted, and amplified using the TruePrimeTM protocol, and se-quenced 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 is a novel, primer-free multiple-displacement type amplification tech-nology based on TthPrimPol and Phi29 DNA polymerase.
This novel amplification technology demonstrates the following features:
• No primer artefacts
• Insensitive to external, non-denatured DNA contaminations
• Excellent sensitivity
• Exquisite reproducibility when amplifying from single mammalian cells
• Excellent genome coverage with little bias
• Ideally suited for next generation sequencing, tested for Illumina and Ion Torrent workflows
• Easy and reliable
Single human HEK293 cells were isolated by dilution, manual picking and visual inspection, and subjected to the True-PrimeTM amplification protocol for 3 and 6 h. DNA yield obtained was quantified by PicoGreen incorporation. Note the low variation between individual cells amplified, and the absence of any significant DNA amplification in the nega-tive (no template) controls. 3 h amplification time yields sufficient DNA for most downstream applications including sequencing.
Red squares: TthPrimPol, blue circles: Phi29 polymerase, red lines: DNA primer synthesized by TthPrimPol.
TruePrime™ is a novel technology dedicated to the amplification of whole genomes and DNA from various sources. TruePrime™ is based on the combination of the highly processive Phi29 DNA polymerase with the recently disco-vered primase/polymerase TthPrimPol. In this setup, TthPrimPol synthesizes the DNA primers needed for Phi29 DNA pol, which allows for the exponential amplification of target DNA. TthPrimPol is a thermostable member of a recently discovered family of enzymes named PrimPol. TthPrimPol is a monomeric enzyme (34 kDa) that displays a potent pri-mase activity, preferring dNTPs as substrates unlike conventional primases. This DNA primase activity can be activated by magnesium or manganese ions, having a wide sequence specificity for template recognition. Key advantages of the TruePrime™ technology include complete absence of primer artefacts, insensitivity to external DNA contaminations, reduced amplification bias compared to methods using random synthetic primers, and an exqui-site reproducibility when amplifying from single cells or minute DNA amounts. Moreover, TruePrime™ shows superior sensitivity, is easy to use and works perfectly well with commonly used NGS platforms such as Illumina or IonTorrent. We believe that TruePrime™ will advance human genetic analyses from single cells or otherwise limited input materi-
al.
Selected references
1. S. Garcia-Gomez, A. Reyes, M.I. Martinez-Jimenez, E.S. Chocrón, S. Mouron, G. Terrados, C. Powell, E. Salido, J. Mendez, I.J. Holt and L. Blanco. PrimPol, an Archaic Primase/Polymerase Operating in Human Cells. Molecular Cell, 2013, 52(4), 541-53.
2. S. Mouron, S. Rodriguez-Acebes, M.I. Martinez-Jimenez, S. Garcia-Gomez, E.S. Chocrón, L. Blanco and J. Mendez. Re-priming of DNA synthesis at stalled replication forks by human PrimPol. Nature Structural & Molecular Biology, 2013, 20(12), 1383-9.
Abstract
TruePrimeTM workflow Target-derived output even at 1 fg
Conclusions
Single cell amplification
TruePrimeTM reaction overview1. TthPrimPol binds denatured DNA at different sites
2. TthPrimPol synthesizes short DNA primers
3. Phi29 DNA pol displaces TthPrimPol and begins po-lymerization
4. Phi29 DNA pol performs strand displacement
5. TthPrimPol binds to newly formed DNA and synthe-sizes new DNA primers
6. Phi29 DNA pol displaces TthPrimPol, binds DNA primers and begins polymerization
Primates99,19%
Unknown0,78%
Other0,03%
HumanPrimates97,41%
Unknown2,49%
Other0,10%
Human
Primates95,40%
Enterobacteriaceae
2,35%
Unknown1,91%
Synthetic0,21%
Other0,13%
E. coli2,35%
Human
Vectors
1 ng input DNA
1 fg input DNA
1 pg input DNA
DNA from 4 single hu-man HEK293 cells was amplified following the TruePrimeTM protocol for 3 or 6 h, subjected to library prep and se-quencing (Illumina HiS-eq), and 5 million read paris were mapped onto the human genome using CLCBio software. Shown are comparisons of chro-mosome coverage to non-amplified HEK293 cell DNA from the same cells. Note the evenness of coverage, and the high similarity between single cells.
Reproducibility
+M + + +- - - - DNA input
PS14.105
Excellent overall mapping data for TruePrime™-amplified DNA, closest to non-amplified DNA coverage reached.Exactly 12 mio read pairs were mapped to the human genome. Shown on the top left is a comparison of chromo-somal coverage between TruePrime™ and non-amplified DNA. The closeness of distribution is also highlighted by a comparison of the read depth per base. The graph on the right shows the coverage pattern after mapping on chromo-some 3. Note the extreme similarity between non-amplified and TruePrimeTM.
Coverage
Sample TruePrime™ (1c)
Random primers
Competitor R
CompetitorG
Non-amplified
DNA
Mapped reads 96.86% 97.42% 99.64% 98.62% 99.53%
Reads in pairs 86.77% 59.07% 91.50% 37.94% 92.03%
Fraction human genome covered 49% 28% 38% 1% 53%
Calculated ideal (Poisson)coverage
57% 44% 59% 31% 59%
Deviation observed from
expected13,8% 35,76% 35.4% 96,7% 10.2%
1
10
100
1000
10000
100000
1000000
10000000
100000000
1E+09
1E+10
0 50 100 150 200 250
depth non-amplified depth TruePrime
Non-amplified DNA
TruePrime™
Competitor R
Competitor G
Random primers
Chromosome 3 coverage (12 Mio read pairs)
Chromosome 3 Chromosome 4
Non-amplified DNA
TruePrime™: 1c
TruePrime™: 1d
TruePrime™: 1g
TruePrime™: 1i
Orange: Non-amplified DNA
Blue: TruePrime™ (1c)
12 Mio read pairs
0
10
20
30
40
50
60
70
80
90
100
chr1 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr2 chr20 chr21 chr22 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chrM chrX chrY
Cove
rage
(%)
