TruePrime™, a novel technology for whole genome amplification€¦ · EMBO | EMBL Symposium:...
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EMBO | EMBL Symposium: Frontiers in Stem Cells & Cancer SYGNIS Bioscience GmbH, INF 515, 69120 Heidelberg www.sygnis.com TruePrime™, a novel technology for whole genome amplification 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 TruePrime™ is the name of a novel technology dedicated to the amplification of va- rious DNA or RNA species for a multitude of applications.TruePrime™ is based on a combination of Phi29 DNA polymerase with the recently discovered primase/po- lymerase 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 the recently discovered family of enzymes named PrimPol. TthPrimPol is a monomeric enzyme (34 kDa) that displays a potent primase activity, preferring dNTPs as substrates unlike conventional primases. This DNA primase activity can be activated by magnesium or manganese ions, having wide sequence specificity for template recognition. Key advantages of the TruePrime™ technology include absence of primer artefacts, insensitivity to DNA contaminations, reduced amplification bias compared to me- thods using random synthetic primers and an exquisite reproducibility. Moreover, TruePrime™ shows superior sensitivity, is easy to use and works perfectly well with all commonly used NGS platforms and already tested for Illumina and IonTorrent workflows. We believe that TruePrime™ will help to advance genomic studies in cancer and stem cell research when working with minute DNA amounts or single cells. 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. Repriming of DNA synthesis at stalled replication forks by human PrimPol. Nature Structural & Molecular Biology, 2013, 20(12), 1383-9. 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.The- reafter, 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 seri- ally diluted, and amplified using the True- Prime TM protocol. A, The absolute input sensitivity as shown in the 3 h amplification experiment is at least 1 fg, the DNA content of 20 λ pha- ges. B, Amplification for 6 h yields 5 µg of DNA also for 1 fg, implying an amplifica- tion factor of 10 10 -fold. At 100 ag there appears still to be amplification although with high variance as dilution can result in Poisson-like distribution effects. Human genomic DNA was 10-fold serially diluted, and amplified using the TruePrime TM pro- tocol, 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 TruePrime TM reaction, and the robustness of the protocol. 1 pg of human genomic DNA (~ 1/6 of the content of one human/mammalian cell) has been amplified using either TruePrime™ (TthPrimPol-based MDA) or random primed MDA reac- tions. Random primed reactions contain 20% of sequences that cannot be mapped to any organism in sequence databases, whereas TruePrime TM -amplified DNA contains only target-derived se- quences. TruePrime TM is a novel, primer-free multiple-displacement type amplification technology 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 • Reduced amplification bias in genome coverage • 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 TruePrime 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 negative (no template) controls. 3 h amplification time yields sufficient DNA for most downstream applications in- cluding sequencing. • Library prep illumina TruSeq • Sequencing by Illumina HiSeq • Sampling of 5 Mio read pairs • Excellent mapping results (~92% mapped read pairs) • Overall base coverage deviates by only 12% from an ideal Poisson distribution Shown is the a scheme of the TruePrime™ multiple displacement amplification (MDA) re- action. Red squares: TthPrimPol, blue circles: Phi29 polymerase, red lines: DNA primer syn- thesized by TthPrimPol. End product is a multibranched DNA structure that can be sub- jected to downstream processing. Abstract TruePrime TM workflow TruePrime TM sensitivity Target-derived output even at 1 fg Absence of primer artefacts Conclusions Single cell amplification TruePrime TM reaction overview 1. TthPrimPol binds denatured DNA at diffe- rent sites 2. TthPrimPol synthesizes short DNA pri- mers 3. Phi29 DNA pol displaces TthPrimPol and begins polymerization 4. Phi29 DNA pol performs strand displace- ment 5. TthPrimPol binds to newly formed DNA and synthesizes new DNA primers 6. Phi29 DNA pol displaces TthPrimPol, binds DNA primers and begins polymerization A B 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 Random-Primer mediated MDA Human 75,84% Unknown 19,92% Invertebrates 4,11% Synthetic 0,10% Human 99,34% Unknown 0,62% Invertebrates 0,04% TruePrime TM DNA from 4 single human HEK293 cells was amplified following the TruePrime TM protocol for 3 or 6 h, subjected to library prep and sequencing (Illumina HiSeq), and 5 million read pa- ris were mapped onto the human genome using CLCBio software. Shown are comparisons of chromosome coverage to published data (Lin et al., 2014, Nature Communications) on HEK293 sequence using Complete Genomics technology. Note the evenness of coverage, and the high similarity between single cells. Chromosome coverage Chromosome 3 #158 Chromosome 4 0,00E+00 5,00E+08 1,00E+09 1,50E+09 2,00E+09 2,50E+09 0 1 2 3 4 5 6 7 8 9 10 1d observed cov expected External contamination was simulated using a 1000-fold excess of yeast DNA (non-denatu- red) together with 1 pg of human denatured DNA. While random primers accept also non- denatured DNA to some degree, TthPrimPol is less sensitive to these contaminations that come in after the initial denaturation step (e,.g. handling, pipets, air etc.). Contamination with environmental DNA is a significant problem in amplification processes. Insensitivity to contaminations Yeast 53,43% Human 37,76% Unknown 6,40% Invertebrates 1,61% Synthetic 0,50% Others 0,14% Environmental 0,14% Human 89,57% Yeast 7,48% Unknown 2,90% Synthetic 0,04% + M + + + - - - - DNA input Random-Primer mediated MDA TruePrime TM
Transcript of TruePrime™, a novel technology for whole genome amplification€¦ · EMBO | EMBL Symposium:...
EMBO | EMBL Symposium: Frontiers in Stem Cells & Cancer
SYGNIS Bioscience GmbH, INF 515, 69120 Heidelberg
www.sygnis.com
TruePrime™, a novel technology for whole genome amplificationÁngel 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, Germanyc, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, Madrid 28049, Spain
TruePrime™ is the name of a novel technology dedicated to the amplification of va-rious DNA or RNA species for a multitude of applications. TruePrime™ is based on a combination of Phi29 DNA polymerase with the recently discovered primase/po-lymerase 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 the recently discovered family of enzymes named PrimPol. TthPrimPol is a monomeric enzyme (34 kDa) that displays a potent primase activity, preferring dNTPs as substrates unlike conventional primases. This DNA primase activity can be activated by magnesium or manganese ions, having wide sequence specificity for template recognition. Key advantages of the TruePrime™ technology include absence of primer artefacts, insensitivity to DNA contaminations, reduced amplification bias compared to me-thods using random synthetic primers and an exquisite reproducibility. Moreover, TruePrime™ shows superior sensitivity, is easy to use and works perfectly well with all commonly used NGS platforms and already tested for Illumina and IonTorrent workflows. We believe that TruePrime™ will help to advance genomic studies in cancer and stem cell research when working with minute DNA amounts or single cells.
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. Repriming of DNA synthesis at stalled replication forks by human PrimPol. Nature Structural & Molecular Biology, 2013, 20(12), 1383-9.
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. The-reafter, 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 seri-ally diluted, and amplified using the True-PrimeTM protocol.
A, The absolute input sensitivity as shown in the 3 h amplification experiment is at least 1 fg, the DNA content of 20 λ pha-ges.
B, Amplification for 6 h yields 5 µg of DNA also for 1 fg, implying an amplifica-tion factor of 1010-fold. At 100 ag there appears still to be amplification although with high variance as dilution can result in Poisson-like distribution effects.
Human genomic DNA was 10-fold serially diluted, and amplified using the TruePrimeTM pro-tocol, 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.
1 pg of human genomic DNA (~ 1/6 of the content of one human/mammalian cell) has been amplified using either TruePrime™ (TthPrimPol-based MDA) or random primed MDA reac-tions. Random primed reactions contain 20% of sequences that cannot be mapped to any organism in sequence databases, whereas TruePrimeTM-amplified DNA contains only target-derived se-quences.
TruePrimeTM is a novel, primer-free multiple-displacement type amplification technology 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
• Reduced amplification bias in genome coverage
• 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 TruePrimeTM 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 negative (no template) controls. 3 h amplification time yields sufficient DNA for most downstream applications in-cluding sequencing.
• Library prep illumina TruSeq• Sequencing by Illumina HiSeq• Sampling of 5 Mio read pairs• Excellent mapping results (~92% mapped read
pairs)• Overall base coverage deviates by only 12%
from an ideal Poisson distribution
Shown is the a scheme of the TruePrime™ multiple displacement amplification (MDA) re-action. Red squares: TthPrimPol, blue circles: Phi29 polymerase, red lines: DNA primer syn-thesized by TthPrimPol. End product is a multibranched DNA structure that can be sub-jected to downstream processing.
Abstract
TruePrimeTM workflow TruePrimeTM sensitivity
Target-derived output even at 1 fg Absence of primer artefacts
Conclusions
Single cell amplification
TruePrimeTM reaction overview1. TthPrimPol binds denatured DNA at diffe-rent sites
2. TthPrimPol synthesizes short DNA pri-mers
3. Phi29 DNA pol displaces TthPrimPol and begins polymerization
4. Phi29 DNA pol performs strand displace-ment
5. TthPrimPol binds to newly formed DNA and synthesizes new DNA primers
6. Phi29 DNA pol displaces TthPrimPol, binds DNA primers and begins polymerization
A
B
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
Random-Primer mediated MDA
Human75,84%
Unknown19,92%
Invertebrates4,11%
Synthetic0,10%
Human99,34%
Unknown0,62%
Invertebrates0,04%
TruePrimeTM
DNA from 4 single human HEK293 cells was amplified following the TruePrimeTM protocol for 3 or 6 h, subjected to library prep and sequencing (Illumina HiSeq), and 5 million read pa-ris were mapped onto the human genome using CLCBio software. Shown are comparisons of chromosome coverage to published data (Lin et al., 2014, Nature Communications) on HEK293 sequence using Complete Genomics technology. Note the evenness of coverage, and the high similarity between single cells.
Chromosome coverageChromosome 3
#158
Chromosome 4
0,00E+00
5,00E+08
1,00E+09
1,50E+09
2,00E+09
2,50E+09
0 1 2 3 4 5 6 7 8 9 10
1d
observed cov expected
External contamination was simulated using a 1000-fold excess of yeast DNA (non-denatu-red) together with 1 pg of human denatured DNA. While random primers accept also non-denatured DNA to some degree, TthPrimPol is less sensitive to these contaminations that come in after the initial denaturation step (e,.g. handling, pipets, air etc.). Contamination with environmental DNA is a significant problem in amplification processes.
Insensitivity to contaminations
Yeast53,43%
Human37,76%
Unknown6,40%
Invertebrates1,61%
Synthetic0,50%
Others0,14%
Environmental0,14%
Human89,57%
Yeast7,48%
Unknown2,90%
Synthetic0,04%
+M + + +- - - - DNA input
Random-Primer mediated MDA TruePrimeTM
