in fluorogenic pyrosequencing , each nucleotide species is labeled at the terminal phosphate position with an identical dye moiety ( fig . upon incorporation of a tplfn into an immobilized dna template by dna polymerase , native dna is generated and a labeled polyphosphate molecule is released . 1a , b ) that is trapped in resealable pdms microreactors and detected by a ccd camera . this process is repeated for all four tplfns , each of which has the same label . upon incorporation of a tplfn , the detectable species released by dna polymerase diffuses away , destroying the spatial correspondence between base incorporation and the presence of fluorophores .. to solve this problem , dna templates are spatially arranged in individual , sealable microreactors fabricated using soft lithography in pdms ( fig . these reactors can be sealed reversibly against a coverglass , isolating individual sequencing reactions and trapping the generated fluorophores . the microreactors , each of which contains 5,000 copies of a primed dna template , are cooled to a temperature where dna polymerase is relatively inactive and loaded with dna polymerase , phosphatase , and one of the four species of tplfns . tplfns are excellent substrates for dna polymerase , and because tplfns are non - fluorescent ( fig . 1b ) , they can be present in the sample at relatively high concentrations without concern for fluorescence background . the microreactors are then sealed , and the temperature of the sample is raised to allow nucleotide incorporation . if the introduced tplfn is complementary to the dna template base adjacent to the primer , fluorophores will be generated and trapped in the microreactor ( fig 1c ) . in homopolymeric regions with multiple , identical bases in the dna template adjacent to the primer , primer extension will continue until the identity of the template base changes . hence , the number of fluorophores generated ought to be proportional to the length of the homopolymer . after the nucleotide incorporation reaction has gone to completion , the microreactor array is imaged with a one - color fluorescence microscope , unsealed , and washed . this cycle can be automated and repeated within a large array to sequence multiple dna templates in parallel . each sequencing cycle results in the generation of physically trapped fluorophores in the microreactors which can be detected with high sensitivity at any time , obviating the need for real - time monitoring . tpln synthesis is simple compared to that of many other nucleotide analogs , and the same conjugation strategy can be applied to all four nucleotides . although tplfns , in which the label is attached to the -phosphate , are rather poor substrates for dna polymerase , extension of the phosphate chain to increase the distance between the labeling moiety and the polymerase active site , and recover the native charge of a nucleotide triphosphate has led to significant improvements . to demonstrate fluorogenic pyrosequencing , we synthesized a set of four -labeled nucleotide tetraphosphates ( fig . we conjugated all four nucleotides to 3-o - methyl-5(6)-carboxyfluorescein , which is a fluorogenic dye excitable at 476 nm with extremely high fluorogenic contrast ( figure 1b ) that does not diffuse appreciably into pdms under the conditions of our experiment . it is easily synthesized from the commercially available fluorophore 5(6)-carboxyfluorescein , and can be phosphorylated with standard phosphoramidite chemistry allowing conjugation to all four dntps ( supplementary note 1 , supplementary figs . 1 and 2 ) to generate tplfns ( fig . we used bst dna polymerase ( large fragment ) because it incorporates tplfns efficiently , has good strand displacement activity , a very temperature - sensitive incorporation rate ( high above 50 c but low below 5 c ) , and no exonuclease activity . after the microreactors are sealed , we trigger primer extension by raising the temperature to 55c , driving tplfn incorporation and release of 3-o - methyl-5(6)-carboxyfluorescein triphosphate to completion within two minutes . we introduce calf intestinal alkaline phosphatase ( ciap ) along with the polymerase to rapidly digest the released 3-o - methyl-5(6)-carboxyfluorescein triphosphate , generating fluorescent product . fluorogenic pyrosequencing occurs within a flow cell that facilitates rapid fluid exchange while minimizing sample volume . the instrumentation required for fluorogenic pyrosequencing consists of three modules : imaging , fluidics , and temperature control ( fig . we generated arrays of micron - sized pillars on a silicon wafer using conventional photolithography ( see online methods ) , and used this silicon master to generate pdms microreactor arrays with soft lithography . the regular array format has the potential for greatly simplified data acquisition and analysis while providing a straightforward means of increasing sample density and throughput . pdms is largely transparent to visible light and has low autofluorescence relative to other polymers used in microfluidics , making it compatible with fluorescence microscopy . although pdms is largely inert , it can be treated with oxygen plasma to form reactive groups for functionalization . this feature allows biotinylation of pdms microreactors , facilitating capture of streptavidin - coated beads to which dna templates are bound ( see online methods ) . similar platforms involving silica microreactors sealed to a silicone gasket have also been applied to single molecule enzymology . we constructed pdms microreactor flow cells consisting of a microreactor array - containing slab , a spacer , and a glass coverslip coated with a thin layer of pdms ( fig . sealing is accomplished by applying vacuum to the outlet of the flow cell , causing the elastomeric pdms slab that forms the top of the flow cell to deform and seal to the pdms coating on the glass surface ( see online methods , supplementary fig . this process is repeated in every sequencing cycle to facilitate microreactor loading , fluorescent product trapping , and washing . a subset of the fluorescence images obtained during a 60-cycle fluorogenic pyrosequencing run are shown in fig . 3a , with each image corresponding to the measured fluorescence generated after one tplfn reaction mixture was introduced to the sample . a fraction of the microreactors were loaded with 1 m polystyrene beads ( not visible in the fluorescence images ) each with 5,000 copies of immobilized dna template . each of the beads was decorated with one of four different dna oligo test sequences ( supplementary table 1 ) . 3b ) , and the other three are randomers with very different sequences ( r1 , r2 , and r3 ) ( fig . 4 , supplementary table 1 ) . data presented in figs the hl oligo sequence consists of a series of poly - t homopolymers ( up to five bases long ) interrupted by single g or c bases.unlike reversible terminator chemistry , in which primer extension can not proceed beyond the first base in a homopolymer within a single cycle , primer extension continues through the whole length of a homopolymer in pyrosequencing . the fidelity of fluorogenic pyrosequencing depends critically on the ability to quantitatively differentiate the levels of signal generated at homopolymers of different lengths . ideally , the signal is directly proportional to homopolymer length . homopolymer sequencing accuracy is one of the major challenges of methods with a pyrosequencing - type workflow such as fluorogenic pyrosequencing . the system achieved a single read raw accuracy of > 99% , and 80% of these hl sequencing traces are error - free . the origins of this accuracy can be visualized from the average and standard deviations ( fig . 3b , grey points ) of the signal generated in each cycle for all hl oligo - containing microreactors analyzed from this run . in addition , a histogram of the signal distribution for all analyzed hl oligo - containing microreactors across all sequencing cycles shows that the signal levels corresponding to zero , one , two , three , four , and five bases are well - separated ( fig . , we designed three quasi - random dna templates with very different sequence characteristics , and generated individual sequencing traces from the three oligos ( r1 in fig . 4a , r2 in fig . 4b , and r3 in fig 4c ) . a global analysis of all analyzed traces for all three random oligos sequenced in this experiment ( 36 traces , 12 for each sequence , fig . 4d ) demonstrates that our current system maintains a mean single read raw accuracy of at least 99% ( that is an error rate of 1% or less ) to a read length of 30 bases . 4d ) is due to signal decay and dephasing which occur in many clonal sequencing systems . data presented in figs . 3 and 4 were background subtracted and corrected for signal decay ( see online methods , supplementary fig . however , we can reasonably expect that substantial improvements in read length and accuracy could be achieved by more sophisticated means of data analysis . in fluorogenic pyrosequencing , each nucleotide species is labeled at the terminal phosphate position with an identical dye moiety ( fig . upon incorporation of a tplfn into an immobilized dna template by dna polymerase , native dna is generated and a labeled polyphosphate molecule is released . 1a , b ) that is trapped in resealable pdms microreactors and detected by a ccd camera . this process is repeated for all four tplfns , each of which has the same label . upon incorporation of a tplfn , the detectable species released by dna polymerase diffuses away , destroying the spatial correspondence between base incorporation and the presence of fluorophores .. to solve this problem , dna templates are spatially arranged in individual , sealable microreactors fabricated using soft lithography in pdms ( fig . these reactors can be sealed reversibly against a coverglass , isolating individual sequencing reactions and trapping the generated fluorophores . the microreactors , each of which contains 5,000 copies of a primed dna template , are cooled to a temperature where dna polymerase is relatively inactive and loaded with dna polymerase , phosphatase , and one of the four species of tplfns . tplfns are excellent substrates for dna polymerase , and because tplfns are non - fluorescent ( fig . 1b ) , they can be present in the sample at relatively high concentrations without concern for fluorescence background . the microreactors are then sealed , and the temperature of the sample is raised to allow nucleotide incorporation . if the introduced tplfn is complementary to the dna template base adjacent to the primer , fluorophores will be generated and trapped in the microreactor ( fig 1c ) . in homopolymeric regions with multiple , identical bases in the dna template adjacent to the primer , primer extension will continue until the identity of the template base changes . hence , the number of fluorophores generated ought to be proportional to the length of the homopolymer . after the nucleotide incorporation reaction has gone to completion , the microreactor array is imaged with a one - color fluorescence microscope , unsealed , and washed . this cycle can be automated and repeated within a large array to sequence multiple dna templates in parallel . each sequencing cycle results in the generation of physically trapped fluorophores in the microreactors which can be detected with high sensitivity at any time , obviating the need for real - time monitoring . tpln synthesis is simple compared to that of many other nucleotide analogs , and the same conjugation strategy can be applied to all four nucleotides . although tplfns , in which the label is attached to the -phosphate , are rather poor substrates for dna polymerase , extension of the phosphate chain to increase the distance between the labeling moiety and the polymerase active site , and recover the native charge of a nucleotide triphosphate has led to significant improvements . to demonstrate fluorogenic pyrosequencing we conjugated all four nucleotides to 3-o - methyl-5(6)-carboxyfluorescein , which is a fluorogenic dye excitable at 476 nm with extremely high fluorogenic contrast ( figure 1b ) that does not diffuse appreciably into pdms under the conditions of our experiment . it is easily synthesized from the commercially available fluorophore 5(6)-carboxyfluorescein , and can be phosphorylated with standard phosphoramidite chemistry allowing conjugation to all four dntps ( supplementary note 1 , supplementary figs . 1 and 2 ) to generate tplfns ( fig . we used bst dna polymerase ( large fragment ) because it incorporates tplfns efficiently , has good strand displacement activity , a very temperature - sensitive incorporation rate ( high above 50 c but low below 5 c ) , and no exonuclease activity . after the microreactors are sealed , we trigger primer extension by raising the temperature to 55c , driving tplfn incorporation and release of 3-o - methyl-5(6)-carboxyfluorescein triphosphate to completion within two minutes . we introduce calf intestinal alkaline phosphatase ( ciap ) along with the polymerase to rapidly digest the released 3-o - methyl-5(6)-carboxyfluorescein triphosphate , generating fluorescent product . fluorogenic pyrosequencing occurs within a flow cell that facilitates rapid fluid exchange while minimizing sample volume . the instrumentation required for fluorogenic pyrosequencing consists of three modules : imaging , fluidics , and temperature control ( fig . we generated arrays of micron - sized pillars on a silicon wafer using conventional photolithography ( see online methods ) , and used this silicon master to generate pdms microreactor arrays with soft lithography . the regular array format has the potential for greatly simplified data acquisition and analysis while providing a straightforward means of increasing sample density and throughput . pdms is largely transparent to visible light and has low autofluorescence relative to other polymers used in microfluidics , making it compatible with fluorescence microscopy . although pdms is largely inert , it can be treated with oxygen plasma to form reactive groups for functionalization . this feature allows biotinylation of pdms microreactors , facilitating capture of streptavidin - coated beads to which dna templates are bound ( see online methods ) . similar platforms involving silica microreactors sealed to a silicone gasket have also been applied to single molecule enzymology . we constructed pdms microreactor flow cells consisting of a microreactor array - containing slab , a spacer , and a glass coverslip coated with a thin layer of pdms ( fig . sealing is accomplished by applying vacuum to the outlet of the flow cell , causing the elastomeric pdms slab that forms the top of the flow cell to deform and seal to the pdms coating on the glass surface ( see online methods , supplementary fig . this process is repeated in every sequencing cycle to facilitate microreactor loading , fluorescent product trapping , and washing . a subset of the fluorescence images obtained during a 60-cycle fluorogenic pyrosequencing run are shown in fig . 3a , with each image corresponding to the measured fluorescence generated after one tplfn reaction mixture was introduced to the sample . a fraction of the microreactors were loaded with 1 m polystyrene beads ( not visible in the fluorescence images ) each with 5,000 copies of immobilized dna template . each of the beads was decorated with one of four different dna oligo test sequences ( supplementary table 1 ) . the first test sequence is a  homopolymer ladder  ( hl ) oligo ( fig . 3b ) , and the other three are randomers with very different sequences ( r1 , r2 , and r3 ) ( fig . 4 , supplementary table 1 ) . data presented in figs the hl oligo sequence consists of a series of poly - t homopolymers ( up to five bases long ) interrupted by single g or c bases.unlike reversible terminator chemistry , in which primer extension can not proceed beyond the first base in a homopolymer within a single cycle , primer extension continues through the whole length of a homopolymer in pyrosequencing . the fidelity of fluorogenic pyrosequencing depends critically on the ability to quantitatively differentiate the levels of signal generated at homopolymers of different lengths . ideally , the signal is directly proportional to homopolymer length . homopolymer sequencing accuracy is one of the major challenges of methods with a pyrosequencing - type workflow such as fluorogenic pyrosequencing . the system achieved a single read raw accuracy of > 99% , and 80% of these hl sequencing traces are error - free . the origins of this accuracy can be visualized from the average and standard deviations ( fig . 3b , grey points ) of the signal generated in each cycle for all hl oligo - containing microreactors analyzed from this run . in addition , a histogram of the signal distribution for all analyzed hl oligo - containing microreactors across all sequencing cycles shows that the signal levels corresponding to zero , one , two , three , four , and five bases are well - separated ( fig . , we designed three quasi - random dna templates with very different sequence characteristics , and generated individual sequencing traces from the three oligos ( r1 in fig . 4a , r2 in fig . 4b , and r3 in fig 4c ) . a global analysis of all analyzed traces for all three random oligos sequenced in this experiment ( 36 traces , 12 for each sequence , fig . 4d ) demonstrates that our current system maintains a mean single read raw accuracy of at least 99% ( that is an error rate of 1% or less ) to a read length of 30 bases . 4d ) is due to signal decay and dephasing which occur in many clonal sequencing systems . 3 and 4 were background subtracted and corrected for signal decay ( see online methods , supplementary fig . however , we can reasonably expect that substantial improvements in read length and accuracy could be achieved by more sophisticated means of data analysis . fluorogenic pyrosequencing has the potential for dramatically improved performance over this proof - of - principle demonstration . the current cycle times of < 10 minutes can be reduced by increasing the speed of nucleotide incorporation and the rate of fluidic exchange ( for example by using positive pressure and optimized flow - cell geometries ) . tplns with longer phosphate chains have been shown to impart better catalytic efficiency compared to the -labeled nucleotides used here , making incorporation more rapid . the time required for washing the system can be further reduced with more sophisticated fluidics and enzymes that thoroughly digest excess nucleotides between cycles . finally , more advanced data processing algorithms to correct for de - phasing will increase the tolerance of our system to faster cycle times . the observed signal decay can occur for a number of reasons including dna loss resulting from misincorporation , primer - template melting , digestion by enzymatic impurities , or even dna dissociation . de - phasing occurs as a result of either incomplete extension , in which primer extension for a sub - population of dna templates falls behind , or carry - forward , in which incomplete washing causes a sub - population of dna templates to advance ahead of the main population . commercial sequencing platforms employ computer algorithms to model and correct for dephasing in combination with sophisticated image processing , leading to significant increases in read length . we expect these improvements to our sequencing chemistry , wash cycle efficiency , and data analysis to yield increases in read length and accuracy . because real - time monitoring is unnecessary , dna samples in two flow cells may be sequenced simultaneously with one sample undergoing wash and tplfn incorporation cycles while the other is imaged . although imaging a very large array will inevitably lead to increased cycle times , further increases in throughput can be realized by fabrication of smaller microreactors and the implementation of high - speed fluorescence imaging schemes such as time delay integration ( tdi ) line - scanning . these microreactors are 8 smaller in volume than those shown in figs . 2 and 3 , and higher density arrays may be combined with more efficient sample loading methods that are not limited by poisson statistics to increase the fraction of dna - containing microreactors ( supplementary fig . increases in array density and loading efficiency will lead to decreases in reagent consumption per base ( see supplementary note 2 for discussion ) . the off - the - shelf polymerase and phosphatase enzymes are currently the dominant reagent cost . fabrication and material costs of the pdms microreactor flow cells are extremely low and highly scalable . despite having similar chemistry , fluorogenic pyrosequencing has the potential for much lower reagent costs than conventional pyrosequencing due to the relatively small dna copy number ( thousands vs. 10 million for pyrosequencing ) and feature size required in fluorescence - based approaches . at the same time , rapid reduction in the reagent costs per sequenced base in commercial sequencers has made up - front instrumentation cost increasingly important . for sequencing systems that employ an optical readout , the illumination and detection equipment , including lasers , optics , and cameras , contribute substantially to instrument cost . furthermore , dna colonies in fluorogenic pyrosequencing are arranged in a regular array allowing more efficient use of the detector than in most commercial platforms , where colonies are immobilized randomly . the realization of high - speed , scanning - based fluorescence detection is substantially simplified if spectral separation is unnecessary and the sample can be spatially registered with the detector . sample preparation costs are also becoming more critical , particularly as diagnostic applications are contemplated . fluorogenic pyrosequencing is , in principle , compatible with all commercially available methods for library preparation and clonal amplification . we have demonstrated the sequencing of dna oligos immobilized on polymer beads , making clonal amplification by emulsion pcr a natural extension of our current technique . however , because the number of template dnas required for sequencing is small ( < 10 ) , less efficient but simpler on - chip amplification methods such as bridge pcr or solid - state rolling circle amplification ( rca ) are also applicable . additionally , our pdms microreactor platform can be readily integrated into pdms microfluidic devices for processing complex samples , single cell manipulation , extraction of genetic material , whole genome amplification , and targeted amplification on - chip ( see supplementary note 3 ) . this potential for seamless microfluidic integration opens the door to sample - to - sequence analysis of raw biomaterials on a unitary microfluidic device . supplementary figure 1 : synthesis of 3-o - methyl-5(6)-carboxyfluorescein - monophosphate supplementary figure 2 : synthesis of dg4p--3-o - methyl-5(6)-carboxyfluorescein supplementary figure 3 : vacuum - based sealing of pdms microreactor array supplementary figure 4 : sample sequencing data processing supplementary figure 5 : demonstration of fluorogenic pyrosequencing in high density microreactor arrays . supplementary figure 6 : super - poissonian loading of pdms microreactors supplementary table 1 : dna sequences used in fluorogenic pyrosequencing experiments supplementary note 1 : synthesis of terminal phosphate - labeled fluorogenic nucleotides supplementary note 2 : the scalability of fluorogenic pyrosequencing supplementary note 3 : potential for microfluidic integration of fluorogenic pyrosequencing
we have developed a multiplex sequencing - by - synthesis method combining terminal - phosphate labeled fluorogenic nucleotides ( tplfns ) and resealable microreactors . in the presence of phosphatase , 
 the incorporation of a non - fluorescent tplfn into a dna primer by dna polymerase results in a fluorophore . 
 we immobilize dna templates within polydimethylsiloxane ( pdms ) microreactors , sequentially introduce one of the four identically labeled tplfns , seal the microreactors , allow template - directed tplfn incorporation , and measure the signal from the fluorophores trapped in the microreactors . 
 this workflow allows sequencing in a manner akin to pyrosequencing but without constant monitoring of each microreactor . with cycle times of 
 < 10 minutes , we demonstrate 30 base reads with 99% raw accuracy . 
 fluorogenic pyrosequencing  combines benefits of pyrosequencing , such as rapid turn - around , native dna generation , and single - color detection , with benefits of fluorescence - based approaches , such as highly sensitive detection and simple parallelization .