Fundamentals of Pyrosequencing: Principle, Analysis, and Limitations

e Fundamentals of Pyrosequencing Colleen T Harrington BA Elaine Lin BA Matthew T Olson MD James R Eshleman MD PhD ContextDNA sequencing is critical to identifying ers can input DNA sequences and other pyrosequencing many human genetic disorders caused by DNA mutations parameters to generate the expected pyrosequencing including cancer Pyrosequencing is less complex involves results fewer steps and has a superior limit of detection compared ConclusionsWe demonstrate how mutant and wild with Sanger sequencing The fundamental basis of pyrose type DNA sequences result in different pyrograms Using quencing is that pyrophosphate is released when a pyrograms of established mutations in tumors we explain deoxyribonucleotide triphosphate is added to the end of how to analyze the pyrogram peaks generated by different a nascent strand of DNA Because deoxyribonucleotide dispensation sequences Further we demonstrate some triphosphates are sequentially added to the reaction and limitations of pyrosequencing including how some com because the pyrophosphate concentration is continuously plex mutations can be indistinguishable from single base monitored the DNA sequence can be determined mutations Pyrosequencing is the basis of the Roche 454 ObjectiveTo demonstrate the fundamental principles nextgeneration sequencer and many of the same princi of pyrosequencing ples also apply to the Ion Torrent hydrogen ionbased next Data SourcesSalient features of pyrosequencing are generation sequencers demonstrated using the free software program Pyromaker Arch Pathol Lab Med 201313712961303 doi httppyromakerpathologyjhmiedu through which us 105858arpa20120463RA A variety of methods are available for sequencing DNA amplification and DNA sequencing reactions are most but Sanger and pyrosequencing are 2 of the most commonly run separately although they can be combined commonly used today Although both Sanger and Maxam into a single reaction Read lengths have increased for Gilbert DNA sequencing were invented in 1977 Sanger Sanger sequencing and 800 base reads can now be achieved sequencing has become the most used method of DNA routinely sequencing The Sanger method is also known as Pyrosequencing is designated as a sequencebysynthesis terminator sequencing because DNA fragments of varying technique because DNA synthesis is monitored in real time lengths are synthesized by incorporating both nucleotides It is based on the pioneering and elegant basic science work and dideoxy terminators deoxyribonucleotide triphosphates of Pal Nyrén PhD who first demonstrated in 1987 that dNTPs and dideoxynucleotide triphosphates ddNTPs DNA polymerization can be monitored by measuring respectively Random incorporation of the ddNTPs causes pyrophosphate production which can be detected by light chain termination that produces DNA fragments of every Edward Hyman PhD capitalized on Dr Nyréns work to possible length In a more recent adaptation each ddNTP invent pyrosequencing 1 year later although it took several A C T or G carries a unique fluorescent molecule such more years to be fully commercialized and morewidely that the extension products are both terminated and labeled implemented After an oligonucleotide is annealed to the with the appropriate fluorophore Terminated products template strand of DNA to be sequenced a DNA must be purified from unincorporated ddNTPs and the polymerase synthesizes DNA by extending the 3 end of fragments are subsequently separated by size usINS capillary the nascent strand using the information encoded in the electrophoresis in which the terminal nucleotide of each template strand During pyrosequencing dNTPs are se fragment is detected by fluorescence at wavelengths unique quentially dispensed into the chamber containing the to each of the terminators Polymerase chain reaction PCR template with the primer and DNA polymerase bound When the correct complementary dNTP is injected and Accepted for publication November 7 2012 added by the polymerase inorganic pyrophosphate PP is From the Sol Goldman Pancreatic Cancer Research Center all released during the condensation reaction as shown below authors and Departments of Pathology Mss Harrington and Lin and where n is the number of nucleotides in the nascent strand Drs Olson and Eshleman Oncology Dr Eshleman Johns Hopkins and H is a hydrogen ion University School of Medicine Baltimore Maryland The authors have no relevant financial interest in the products or companies described in this article P DNA dNTP DNA PPi H 1 Reprints James R Eshleman MD PhD the Sol Goldman Through a sequence of another 2 reactions the released Pancreatic Cancer Research Center Departments of Pathology and Oncology Johns Hopkins University School of Medicine CRB II pyrophosphate is converted into adenosine triphosphate Room 344 1550 Orleans St Baltimore MD 21231 email ATP a cofactor for the enzyme luciferase oxidizing jeshlemajhmiedu luciferin to oxyluciferin and light 1296 Arch Pathol Lab MedVol 137 September 2013 Fundamentals of PyrosequencingHarrington et al Biotin A 5AAGGGTCAGTTCGAACAGT3 3TTCCCAGTCAAGCTTGTCA f Biotin B 5AAGGGTCAGTTCGAACAGT3 v 3 TICCCAGTCAAGCTTIGICA Bead Streptavidin C Template strand 3TTCCCAGTCAAGCTTIGTCA Bead D x d Nascent strand vacdeey Template strand 3TTCCCAGTCAAGCTTGTCA Bead DNA Polymerase Figure 1 Steps of pyrosequencing A Polymerase chain reaction PCR amplification is performed using a reverseprimer biotin labeled on the 5 end filled squares to produce the PCR product shown B The biotinylated PCR product is bound to the streptavidin white open polygons coated onto the beads gray half circles C The bead is immobilized using a magnet not shown the DNA is denatured and the top strand is washed away D A complementary primer 5AAGGGT3 has been annealed and if the complementary deoxynucleotide triphosphate dNTP is added dCTP the DNA polymerase gray oval will incorporate it into the nascent elongating strand at the position of the open box Each bead is coated with innumerable PCR products represented here by a single molecule PP APS ATP SO ATP dNTP AMP dNMP 4P 4 catalyzed by ATPsulfurylase 2 where dNMP is deoxynucleoside monophosphate and P is inorganic phosphate ATP luciferin O2 AMP PPj oxyluciferin Before the actual pyrosequencing the region of interest is first amplified via PCR using a reverse primer that is CO2 v 3 biotinylated Figure 1 A This allows firm immobilization of where APS is adenosine 5phosphosulfate SO is sulfate the PCR products onto beads coated with streptavidin AMP is adenosine monophosphate COz is carbon dioxide through extremely tight avidinbiotin bonding Figure 1 and hv is light B Because the beads are magnetic they can be Thus light emission is proportional to the amount of immobilized against the wall of a tube by the application pyrophosphate produced which is directly proportional to of a magnetic field This allows purification of the template the number of nucleotides added Whether a given strand biotinylated bottom strand of the PCR product dispensed dNTP can be incorporated apyrase catalyzes after denaturation and washing Figure 1 C The strand the degradation of excess dNTPs via the following reaction attached to the bead becomes the template strand for primer and before the next dNTP is dispensed binding and undergoes the 4enzyme pyrosequencing Arch Pathol Lab MedVol 137 September 2013 Fundamentals of PyrosequencingHarrington et al 1297 Equation 3 Luciferin O ATP AMP PP Oxyluciferin CO Luciferase Equation 2 t PP APS ATP SO t ATP sulfurylase PPE dCTP Equation 1 Nascent strand 5AAGGG 3 Biotin Template strand 3T TCCCAGTCAAGCTTGTCA Bead DNA Polymerase Streptavidin Figure 2 Chemical reactions of pyrosequencing When the correct deoxynucleotide triphosphate dNTP can be incorporated into the strand of DNA being synthesized the released inorganic pyrophosphate PP from the condensation reaction initiates a chain of enzyme reactions ultimately producing light hv A hydrogen ion H is also released along with PP Equation numbers correspond to those listed in the Introduction in text The apyrase reaction equation 4 is not shown Abbreviations AMP adenosine monophosphate APS adenosine 5phosphosulfate ATP adenosine triphosphate CO2 carbon dioxide Ox oxygen SO4 sulfate reaction when the correct dNTP is added deoxycytidine versus 20 for Sanger of mutant alleles but the read triphosphate dCTP Figures 1 D and 2 Thus the length is shorter typically 100 to 400 bases71 A complementary nascent DNA strand is synthesized on the comparison of pyrosequencing Sanger sequencing and template strand attached to the bead An advantage of using nextgeneration sequencing is shown in the Table the antisense strand as the template strand is that the DNA In 2005 Rothberg and colleagues developed the first sequence produced is sense sequence The DNA polymerase massively parallel nextgeneration sequencer based on used in pyrosequencing is the Klenow fragment bacterial the pyrosequencing reaction Single DNA molecules are first exonuclease I with the exonuclease function deleted This painted onto the surface of individual beads They are enzyme is used as it was found to empirically decrease then clonally amplified in an emulsion PCR where each background spurious signals molecule is clonally amplified in its own aqueous bead Pyrosequencing is fundamentally different from Sanger containing droplet separated from its neighbors by oil The sequencing in that bioluminescence results from strand ampliconcoated beads are then deposited into picoliter elongation in real time whereas with Sanger sequencing sized wells and pyrosequenced in parallel In another fluorescence is detected as a separate step after chain method of nextgeneration sequencing produced by the termination Pyrosequencing with its low coefficient of company lon Torrent Systems Inc Guilford Connecticut variation is inherently more quantitative Different dNTPs the released hydrogen ion equation 1 is measured instead generate similar peak heights following single incorporation of the pyrophosphate When a nucleotide is incorporated events and the peak height generated from incorporating 3 the hydrogen ion released is detected by a corresponding dNTPs is 3 times the signal from incorporating one of the drop in pH in micronsized wells designated as the worlds same dNTPs It also has a superior limit of detection 5 smallest pH meters 1298 Arch Pathol Lab MedVol 137 September 2013 Fundamentals of PyrosequencingHarrington et al Given the widespread and growing use of pyrosequencing in the clinical molecular diagnostic laboratory there is a parallel need to train new userstechnologists residents fellows pathologists and physicians from other disci plinesin basic pyrosequencing and pyrogram interpreta tion In this article we demonstrate the fundamental principles of pyrosequencing specifically showing how 1 a peak is generated only when the correct nucleotide is dispensed and incorporated 2 peak heights are propor tional to the number of nucleotides incorporated 3 different dispensation sequences can produce different pyrograms 4 suboptimal dispensation sequences can mask mutations and 5 nonoptimized dispensation se quences can produce overly complex pyrograms We show these concepts via theoretical pyrograms generated by Pyromaker httppyromakerpathologyjhmiedu accessed October 1 2012 Johns Hopkins University School of Medicine Baltimore Maryland20 A tutorial demonstrating these concepts is posted on the Pyromaker Web site that also contains the free Pyromaker software Other reviews primarily focused on the biochemistry of pyrosequencing have been published elsewhere11132122 MATERIALS AND METHODS Pyromaker httppyromakerpathologyjhmiedu is an R script that accepts user inputs through a Webpage interface These inputs include the wildtype DNA sequence the mutant DNA sequence dispensation order the percentage of mutant bearing cells eg cancer cells and whether the mutation or single nucleotide polymorphism is present in the heterozygous or homozygous state It then determines the response as various dNTPs are dispensed and maintains the position of the polymerase on each respective strand of DNA It simulates real pyrograms in that the left edge of the peak is nearly straight vertical whereas the right edge of the peak contains a tail most likely indicating that the enzymes that convert pyrophosphate to light are not instantaneous In pyrosequencing the dispensation order is defined as the order in which individual dNTPs are sequentially injected into the chamber containing the pyrosequencing reaction and they are represented on the xaxis as single letters eg C for dCTP etc In pyrosequencing the natural deoxyadenosine triphosphate dATP results in false signals because like ribose adenosine 50 triphosphate rATP it is a substrate for luciferase13 Accordingly the dATP analog deoxyadenosine athio triphosphate dATPaS is used in lieu of dATP but that produces a higher peak than the other dNTPs which needs to be considered when comparing homopolymers of A that are equal in length to the other nucleotides23 This relative peak height A versus C G or T is incorporated into Pyromaker and can be appreciated in many of the pyrograms shown below eg Figure 3 RESULTS Presence or Absence of Peaks Following Dispensation of a dNTP Indicates the Sequence If we imagine the sequencing template and primer shown in Figure 3 shaded box left the extension of the primer will generate 50ACGT30 Because this is done in realtime where the xaxis of the graph is time the first 3 nucleotides dispensed deoxythymidine triphosphate dTTP deoxygua nosine triphosphate dGTP and dCTP do not elongate the growing strand because the first complementary nucleotide is dATP Thus there are no peaks on the pyrogram for these dispensed dNTPs abbreviated T G and C in the graph because they could not be incorporated into the growing strand However when dATP is dispensed it is incorporat ed pyrophosphate is released and light is emitted The light is detected by a chargecoupled device sensor and is represented by peaks in the pyrogram21 The subsequent injection of dCTP also results in incorporation and light producing the extended product shown shaded box right When the following 2 bases dTTP and dATP are dispensed they cannot be used to extend the nascent DNA strand whereas peaks are seen when dGTP and dTTP are dispensed Accordingly the sequence of DNA can be determined from the light pattern that results from serially dispensing dNTPs into a chamber containing polymerase bound to a primerbearing DNA template molecule Peak Heights Are Proportional to the Number of Nucleotides Incorporated The height of the peak is proportional to the number of identical bases of a homopolymeric run as they are simultaneously incorporated into the elongating DNA strand during a single dispensation13 The pyrogram of codons 12 13 and 14 of KRAS demonstrates the propor tionality between peak height and homopolymer length Figure 4 The first G peak of codon 12 is twice as high as the subsequent T peak because the G peak represents the light produced from the extension of 2 dGTPs 23 whereas the T peak represents the incorporation of only one dTTP 13 into the elongating strand This is also seen with codon 13 GGC but not for codon 14 GTA where all nucleotides are present as single incorporation events Pyrosequencing is more accurate at detecting a difference between low numbers of mononucleotide bases such as one dATP versus 2 as opposed to the difference between 8 and 9 dATPs This is an inherent limitation of pyrosequenc ing Comparison of Sequencing Methods Sequencing Method Strengths Weaknesses Common Applications Pyrosequencing Better limit of detection 5 Shorter read lengths 100400 bases homopolymers Oncogene detection methylation analyses resolving complex Sanger results Sanger sequencing Longer read lengths 800 bases Worse limit of detection 20 Somatic and germline mutation detection Nextgeneration sequencing Massively parallel sequencing or extremely high depth of coverage cost reduction for gene panels Cost Wholegenome sequencing wholeexome sequencing RNASeq amplicon sequencing rapid microbial identification gene panels early detection of cancer Arch Pathol Lab MedVol 137 September 2013 Fundamentals of PyrosequencingHarrington et al 1299 ai Nascent sequence 5ACGT3 on Codons iz 13 14 Templatesequence TGCA3 r Nascent sequence 5GGT GGC GTA3 za Template oe A CCG CAT5 S 2 2 x Zu es n fee oi ip as 5 2uwn a z 5CCCC 3CCCCAC 2 1x g 3GGGGTGCA5 3GGGGTGCA5 a a wn 2 L TTC l I I i Il T G A Cc A G Cc YF TAC GAC TC AGA TC GTAG 3 Dispensalion 4 Dispensation tn Wild type 5GGTGGCGTA3 2 Wild type 5GGTGGCGTA3 B Mutant 5GATGGCGTA3 os is Mutant 5GATGGCGTA3 oun é BB force fen eerste mane tec ctnennininnstcniaetins st zw 50 Se oN 8 eo a ahah Po Co TAC GA CT CAGAT CGTAG AGC T AGC TAGE TA GCOTA 5A Dispensation 5B Dispensation Figure 3 Incorporation or lack of incorporation of deoxynucleotide triphosphates dNTPs indicates the DNA sequence A virtual pyrogram for hypothetical DNA sequence ACGT where the polymerase chain reaction product bottomstrand with annealed topstrand sequencing primer is shown on the left shaded box and the extended nascent strand after addition of deoxyadenosine triphosphate dATP and deoxycytidine triphosphate are shown in the right shaded box The xaxis is the dispensation sequence time and the yaxis is the intensity of the bioluminescence The presence of a peak indicates the injected dNTP was incorporated whereas the absence of a peak indicates the polymerase was unable to incorporate the dispensed dNTP eg note the absence of light produced when the first T G and C dNTPs were dispensed Because of the nature of the dATP isomer see Materials and Methods the A peaks are always slightly higher than peaks from the other 3 nucleotides Figure 4 Peak height is proportional to the number of deoxynucleotide triphosphates incorporated A virtual pyrogram for wildtype KRAS codons 12 13 and 14 demonstrates that the height of the peaks is proportional to the number of nucleotides incorporated For example the first G peak is twice as tall as the first T peak indicating that 2 consecutive deoxyguanosine triphosphates underlined were incorporated whereas only one deoxythymidine triphosphate underlined was incorporated 1X and 2X lines represent peak heights because of single or double base incorporation events respectively Figure 5 Dispensation order should be optimized for a known gene target Pyrosequencing results for simple mutation in KRAS codon 12b GGTGAT Different dispensation sequences can produce different pyrograms A Pyrogram generated with a dispensation sequence optimized to KRAS and its known mutation spectrum Note that the G peak is higher than the A peak because the G peak is due to the combined activity from wild type and mutant sequences B Pyrogram generated with a cyclic dispensation for the same mutation Cyclic dispensation sequences generally produce pyrograms harder to analyze The down arrow indicates a reduction of the expected signal Up arrows indicate novel peaks from the mutant allele Dispensation Order Is Important unknown target sequencing such as whole genome The dispensation sequence for pyrosequencing can affect sequencing the only option is cyclic dispensation In comparing 2 pyrograms that detect the same mutation how the pyrogram will appear Different sequences will j 5 A hat j imized for KRAS duce nonidentical pyrograms for the same simple igure 9 Jy uses a sequence thal 1s optimized Tor produ defined Py 1 bstituti np whereas Figure 5 B uses a cyclic dispensation sequence A mutation defined as a single base substitution Two options G and T repeated in that order Note how the 2 different for dispensation sequences exist cyclic or optimized based dispensation sequences create different pyrograms for this on the unique order of the bases in the region of DNA being simple codon 12b KRAS mutation GGTGAT The sequenced A programmed sequence is more efficient at optimized dispensation sequence generates a cleaner pyro detecting mutations and facilitates a longer read length a gram with an easily identifiable mutant peak at the second faster readout and less out of phase shifts Of course for dATP dispensed Figure 5 A On the other hand the use of 1300 Arch Pathol Lab MedVol 137 September 2013 Fundamentals of PyrosequencingHarrington et al A B Wild type 5GGTGGCGTA3 Wild type 5GGTGGCGTA3 re Mulant 5 AATGGCGTA3 Mutant 5AGTGGCGTA3 Ww wy 2 2 2 5 Cc 2 1 1 TACGACTCAGATCGTAG TACGACTCAGATCGTAG Dispensation Dispensation Figure 6 Different mutations can be indistinguishable Some complex mutations may be indistinguishable from simple mutations In clinical pyrosequencing the percentage of mutant cancer cells in a tumor is not known with complete accuracy A Pyrogram demonstrating a complex mutation in codon 12a and 12b of KRAS GGTAAT B Pyrogram demonstrating a simple mutation in codon 12a GGTAGT Pyrograms A and B are identical The tumor percentage in A was 25 and in B it was 50 Thus a sample with a complex mutation and a sample with a related simple mutation in the same codon can produce identical pyrograms when the percentage of mutant molecules is not identical between the 2 samples a cyclic dispensation sequence results in a complex pyro base location on both the wildtype and the mutant gram with 6 novel peaks making the pyrogram extremely sequences and are out of phase when they are not aligned difficult to analyze Figure 5 B The presence of additional When the molecules are out of phase it suggests that 2 peaks indicates that the wildtype and mutant elongating dissimilar molecule species are being simultaneously se strands are out of phase with one another discussed quenced because of either a mutation or a polymorphism In below contrast to cyclic dispensation an optimized dispensation sequence is more effective at keeping the mutant such as Suboptimal Dispensation Can Mask Mutations codon 12b KRAS and wildtype molecules in phase at the When creating an optimized dispensation sequence it is base locations that are identical in both samples while important that the sequence does not mask any mutations allowing the base locations that are truly dissimilar to be out that might be present Pyrosequencing is unable to produce of phase Figure 5 A pyrograms that distinguish between some simple and For codons 12 13 and 14 of wildtype KRAS and 12b complex mutations if the dispensation order is not optimal KRAS mutant GA both molecules are elongated when A complex mutation consists of 2 or more base substitu dGTP is dispensed but the nascent strands are out of phase tions not necessarily consecutive In clinical pyrosequenc because the strand replicating the wildtype allele incorpo ing the percentage of mutant cells is not known with rates 2 dGTPs whereas the one replicating the mutant allele complete accuracy whereas with Pyromaker the user can only incorporates one dGTP Figures 5 A and 7 A The define a precise percentage of mutant cells Thus a sample subsequent addition of dATP brings the molecules back in with a complex mutation and a sample with a simple phase because the polymerase replicating the wildtype mutation in the same codon can produce identical pyro allele does not advance whereas the one for the mutant grams when the percentage of cancer cells is not identical advances by the one nucleotide Thereafter the polymerases between the 2 samples For example the complex mutation replicating the wildtype and mutant alleles remain in phase in codon 12 of KRAS GGT to AAT results in a pyrogram for the rest of the pyrogram Because the molecules are in Figure 6 A that is identical to the pyrogram for the simple phase for most of the pyrogram it is easy to identify the codon 12a KRAS mutation GGT to AGT Figure 6 B This simple mutation by the peak at the second dATP dispensed is because the percentage of tumor cells is 25 with the A detailed basebybase extension of this pyrogram shows AAT mutation and 50 with the AGT mutation An the location of inphase and outofphase molecules as alternative dispensation order could be designed to distin sequencing occurs Figure 7 A guish these mutations and that highlights the importance of In contrast using an AGCT cyclic dispensation sequence knowing all the mutations that may occur in a given region for the identification of the same simple codon 12b KRAS when optimizing the dispensation sequence mutation generates a morecomplex pyrogram Figures 5 B and 7 B The presence of more peaks at varying heights in Phase Affects Ease of Interpretation comparison to tthe pyrogram of the same simple mmitation Phase is defined as the relative positions of the DNA which uses an optimized dispensation sequence is due to polymerase molecules on 2 different DNA template strands the wildtype and mutant molecules continuously being out and is affected by dispensation order The polymerase of phase because the polymerase is incorporating nucleo molecules are in phase when they are aligned at the same tides in an unsynchronized manner many of the peaks Arch Pathol Lab MedVol 137 September 2013 Fundamentals of PyrosequencingHarrington et al 1301 represent nucleotides incorporated by only one allele Therefore outofphase sequencing products can lead to complex pyrograms with more ambiguous peaks A detailed analysis of the pyrogram in Figure 5 B depicting basebybase extension demonstrates how a cyclic dispensation sequence causes the polymerase to remain out of phase Figure 7 B The cyclic dispensation sequence employed is AGCT The first nucleotide dispensed dATP is not incorporated into either nascent strand whereas the next one dGTP is incorporated twice on the strand for the wildtype allele and only once on the strand for the mutant allele causing the polymerase molecules to go out of phase The dCTP produces no peak whereas the dTTP is incorporated by the elongating nascent strand from the wildtype allele moving the polymerase that is replicating the wildtype allele out of phase with the polymerase replicating the mutant allele by 2 nucleotides The second dATP dispensed is incorporated by the nascent strand for the mutant allele yet the polymerase molecules remain out of phase Because the dispensation is cyclic once the sequencing products get out of phase they are usually unable to get back in phase for the rest of the sequencing This shows how a cyclic dispensation sequence can generate Figure 7 Stepwise advancement of a DNA polymerase demonstrating phase A Virtual pyrogram for codons 12 13 and 14 of KRAS with optimized dispensation shows that the polymerase molecules on the wildtype top sequence and mutant bottom sequence DNA molecules can move back in phase after being out of phase at the singlemutant base location Both molecules are shown in the 50 to 30 direction B Virtual pyrogram for codons 12 13 and 14 of KRAS with cyclic dispensation demonstrating how polymerases on wildtype and mutant DNA sequences can advance out of phase relative to each other Boxes designate the incorporated nucleo tides during that dispensation and the right edge of the box indicates the position of the polymerase on each DNA molecule after the incorporation event If there is no incorpora tion in response to the dispensed deoxynu cleotide triphosphate the polymerase position is indicated by a vertical line 1302 Arch Pathol Lab MedVol 137 September 2013 Fundamentals of PyrosequencingHarrington et al suboptimal pyrograms for even a simple mutation espe cially one located at the beginning of the DNA sequence COMMENT In this article we demonstrate the unique features of pyrosequencing a sequencebysynthesis method of DNA sequencing with the advantage of realtime analysis Even though the pyrosequencing read length has an upper limit of approximately 400 bases it is more quantitative and has a superior limit of detection 5 compared with conventional Sanger sequencing It reports the incorporation of nucleo tides base by base by converting the production of pyrophosphate into light The amount of pyrophosphate produced is proportional to the number of identical bases incorporated in a homopolymer represented by the height of the peaks in pyrograms Pyromaker is a free online software tool that generates pyrograms from DNA sequenc es entered by users In this article we used Pyromaker to demonstrate the salient fundamentals of pyrosequencing Some principles include 1 the incorporation of a correct nucleotide to generate a peak 2 the proportionality between peak heights and the number of nucleotides incorporated 3 the importance of dispensation sequence and 4 the limitations of pyrosequencing such as the masking of mutations and the generation of overly complex pyrograms Despite its limitations pyrosequencing employs unique features that make it advantageous over other sequencing methods for certain situations and we support its continued and improved use in biomedicine The primary application of pyrosequencing is short reads where a superior limit of detection than can be provided by Sanger sequencing is preferred Pyrosequencing in a clinical molecular diagnostics laboratory requires technologists capable of performing highcomplexity testing and a pyrosequencing instrument commonly PyroMark Q24 and PyroMark Q96 Qiagen Germantown Maryland Roche 454 and GS Junior Roche Diagnostics Indianapolis Indiana One common application is sequencing onco genes in tumors which are DNA mixtures of malignant and normal stromal cells where the tumor cell percentage can be low for example only 10 Pyrosequencing is a valuable tool to solve ambiguous Sanger sequencing results such as differentiating between one dinucleotide substitution and 2 adjacent singlebase substitutions and between cis and trans configurations of closely juxtaposed mutations Additional applications include human genetics testing and promoter methylation analysis using the relative degree of methylation between different CpG cytosinephos phateguanine sites The Pyromaker software is useful for other applications We previously used it to demonstrate the expected pyro grams for all reported KRAS mutations and these can serve as reference patterns to compare with clinical results20 We also demonstrated its utility as a tool to help resolve complex uninterpretable clinical pyrograms using 2 methods In the hypothesis testing method virtual pyro grams are generated for different hypothesized mutations and those patterns are matched to the unknown actual pyrogram In the iterative mode the user starts with the wildtype sequence and titrates in the minimum mutation to create the first change seen in the pyrogram and then iteratively titrates in additional mutations to eventually reproduce the experimental result Pyrosequencing is an especially valuable tool for onco gene detection in tumors for methylation analysis for identifying ambiguous 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