3 What is Pyrosequencing? Sequencing by Synthesis:Ronaghi M., Uhlén M., Nyrén P. (1998)Real-Time Pyrophosphate Detection for DNA Sequencing.Science 281:Sequence based technology in real timeSimple and robustNo separation, gel, label or probesIn built controlsFlexiblein throughputin assay designin type of applicationsquantitationPyrosequencing is a sequencing chemistry based on sequencing by synthesis. It doesn’t use dyes or labels or gels so doesn’t suffer from all the usual artifacts associated with sequencing chemistries. This makes it very accurate and very fast. Sequencing takes place in solution “in front of your eyes” ie in real time, you don’t run a reaction then a gel then analyse your results…it all happens simultaneously.
4 Pyrosequencing Assays Amplification of Region of interest (PCR)Pyrosequencing AnalysisSequencingprimerPCR primerRegion ofinterestBiotinylatedPCR primer4
6 Pyrosequencing Workflow PCR and Sample preparation 1. PCR with one of the primers biotinylated2. Immobilize biotinylated PCR products onto streptavidin coated beads3. Separate strands by denaturation in NaOH4. Wash /neutralize the immobilized strand5. Anneal sequencing primer1-2 h10-15 minRun Pyrosequencing min
7 Pyrosequencing Workflow Sample Preparation Vacuum toolWaterWashing bufferDenaturation Solution(NaOH)EtOHPSQ plate with sequencing primerPCR product immobilized on Sepharose beads
8 Pyrosequencing Workflow Sample preparation 1-24 post-PCR samples prepared in parallel in less than 15 minutesMinimal pipettingActual hands-on time, less than 1 minutePlastic waste reduced to a minimum8
14 PyroMarkTM Q24 A Small Smart Affordable Pyrosequencing System with a 24 well plate formatA complete solution for Mutation and Methylation AnalysisCpG methylationAllele quantificationMutation analysis14
15 PyroMark™ Q24 Instrument Design X-Y drivePositioningACGTReagent cartridgeInk jet delivery24 well plateMixer and thermostatInstrument designThis is a technical drawing of the interior instrument design and shows the position of reagent cartridge, plate and CCD cameraKey things to describe:An X-Y drive moves the reagent cartridge over the plateDispensation of reagents by ink jet delivery in all wellsThe 96 well plate is placed in a thermostatted heating block housed on top of a shaker table for efficient mixing of reagents and beads in the wellsDetection of the light in all wells allows for simultaneous monitoring of the sequencing events in all wells, thus real time sequencing.24 individual CCD ChipsDetection
16 PyroMarkTM Q24 System Reagents Application and Assay Design software InstrumentVacuum Prep. Workstation
17 PyroMark™ Q24 BenefitsAn extremely easy to use system - Up to 24 samples in parallelSmall FootprintTakes very little bench spaceMeasuring only H420xW390xD525mmRobustCan be run at ambient temperatures between 15°C and 32°C.Runs can be set up and analyzed on any PC anywhere, running PyroMark™Q24 Software.Information is transferred between the instrument and computer via a USB memory stick.Conforms to EU IVDD so suitable for clinical use in Europe
18 PyroMark™ Q24 SoftwareThe software has two analysis modes:AQ - A variety of quantification studies and SNP analysis.CpG - Methylation analysis of multiple consecutive CpG sites.AQ assays and CpG assays can be performed on the same PyroMark™ Q24 Plate.5 multi licensesYou can toggle between the analysis modes in the analysis view of the software, by selecting AQ or CpG in the toolbar.Both modes offer detailed report information that can be exported to html, Excel, text and PDF formats.18
19 PyroMark™ Q24 software AQ mode Frequency calculations of variable positions in sequence context including;Single variable position AG/TCMultiple variable positions AG/TCAG/TCAG/T/ACDi- Tri- or Tetra-allelic mutations GA/C/G/TAHigh resolution of individual sitesQuality assessment of individual sites and sequence contextSNP analysis (Multiple positions, Di- Tri- or Tetra-allelic variants)Analysis of SNPs in the presence of CpG sites
20 Mutation example KRASMutations in the KRAS gene results in a constitutively active KRAS protein which leads to abnormal cell growth, proliferation and differentiation.KRAS is frequently mutated inColorectal cancerLung cancerThe most common KRAS mutations are found in codons 12,13 and 61In several types of cancer the K-Ras gene is mutated, resulting in a loss of activity regulation and subsequently increased invasion and metastasis, and decreased apoptosis.The highest frequency of mutated KRAS is found in pancreatic cancers (90%), but in colorectal and lung cancers up to 30% of the patients have mutations in KRAS. Characteristic for these cancers are that they are adeoncarcinoma (originate form adenovirus infections).The most common mutations in KRAS are found at residue G12 and G13 in the P-loop and the catalytic residue Q61.
21 Mutation example KRAS – mutation frequency Codon 12+13Codon 61= Guanine747770G= ThymineT= CytosineC= AdenineABasepair substitutionBasepair substitutionFlexible assay design facilitates analysis of contiguous, multivariable mutationsWt seqGGTGGCCAACodon 61FPFPBSeqRPBRPGGT GGC GTAGGTCCA GTT CTCCodon 12 & 13PyroMark Q96/Q24 KRAS v2.0 test is an assay for mutation analysis of KRAS; optimized for use on any Pyrosequencing instrument.The most frequent SNPs in KRAS codons 12, 13 and 61, resulting in amino acids exchange, is illustrated in the histogram. However, other rare mutations resulting in a aminoacid exhange is also reported, but the relevance of these are not known.PyroMark Q96/ Q24 KRAS v2.0 test assay is designed to amplify the two DNA regions in the KRAS gene where the most frequent mutations are found. The PCR results in a ≈120bp amplicon which is more than half the size compared to the previous assay. In paraffin embedded tissues the DNA is often degraded in pieces about 200 bp. The new primers improve the PCR and ensure high quality sequence data.The codons analysis is designed as an forward assay while the assay for codon 61 is designed in reverse order.Assays performed according to the ”Instructions for use” enables detection of nucleotide substitutions in position and 2 of codons 12 and 13 and position 3 in codon 61. In addition, post-run analysis of peaks in an Excel macro enables identification of rare mutations in the codons. Quantification of these mutations can be performed on MD and ID instruments by running a new sample with changed ”Sequence to Analyze”. PyroMark Q24 application software contains a function that post run enables quantification of rare mutations by just changing the sequence to analyze in the run-file.
22 Mutation example The KRAS 2.0 assay Codons121361Wt seqGCTCAACTAMulti-variable mutationsCTGCGCTGACTBNDFPSeqRPBNNt RVcCodon 12 & 13Codon 61FPBRP
23 Mutation example The KRAS 2.0 assay Efficient detection and quantification of mutations in codons 12, 13 and 61 with built in quality control1 well/sample for all Codon 12 & 13 mutations (9 reported mutations)1 well/sample for all Codon 61 mutations (7 reported mutations)Provides high quality data of DNA from fresh, frozen and paraffin-embedded tumor samplesSequence context provides built-in control and eliminates false positives/negatives
25 Mutation example The KRAS assay – codon 12 position 2 GGT>GCTGly12AlaGGT>GATGly12AspGGT>GTTGly12Val
26 Mutation example The KRAS assay – codon 12 position 1 GGT>TGTGly12CysGGT>AGTGly12Ser
27 Mutation example The KRAS assay – codons 13 and 61 GGC>GACGly13AspCAA>CACGln61HisTTG>GTG
28 Mutation example The KRAS assay – Conclusions 1 PCR to cover ALL mutations in codons 12 & 13Minimises amount of gDNA needed (10ng)Optional second PCR to cover all mutations in codon 61Provides results for rarer but important mutations not covered by other methodsFast resultsPCR product to quantitative result in ~ 30 minutes for 24 samples
29 CpG methylation analysis mCC1. Bisulfite conversion2. PCR amplification3. PyrosequencingCUCCUT25%75%Degree of methylation is automatically analyzed by the software.mCC
30 CpG methylation analysis Assay design CpG islandSequencing primerPCR primerCpG sitesPCR primerAll primers are located in non-variable regions, in between CpG sitesEnables analysis of several adjacent CpG sites with one sequencing primerFreedom in positioning of the sequencing primerdistance from CpG siteorientation of assayThe primer placement for Pyrosequencing analysis is flexible, unlike competitors like MSP, SnapMeth, COBRA
31 CpG methylation analysis A range of analysis possibilities Any single CpG siteMultiple consecutive CpG sitesOne gene at a timeSeveral genes in the same analysis (analyze up to 24 different assays in one run)
32 Benefits of Pyrosequencing for CpG methylation analysis Quantitative analysis of multiple consecutive sitesFlexible assay designForward – reverse/ Upper – lowerFlexible primer positioningBuilt-in Bisulfite treatment controlExcellent PerformanceAccuracyPrecisionReproducibility over timeConfidently discern even small changes in methylationFast results
33 Benefits of Pyrosequencing for CpG methylation analysis Built-in quality control of bisulfite treatmentRASSF1A geneBefore bisulfite treatment(RASSF1A)No separate reaction needs to be run to ensure complete bisulfite conversion. Every single well will contain a built-in control.CCGACATGGCCCGGTTGGGCCCGTGCTTCGCTGGCTTTGGGCGCTAGCAAGCGCGGGCCGGGCGGGGCAnalyzed sequenceTYGATATGGTTYGGTTGGGTTYGTGTTTYGTTGGTTTTGGGYGTTAGTAAGYGYGGGTYGGGYGGGGTAny C not followed by a G gives bisulfite QC
34 Benefits of Pyrosequencing for CpG methylation analysis Accuracy - Linear response in measured methylationNormal DNAColon cancer DNAThis is illustrating how accurate the analysis is – a perfectly linear relationship was observed in this experiment.Linear response of methylation measured by Pyrosequencing in PCR-amplified products generated from controlled dilutions of in-vitro methylated (IVM) genomic DNA with unmethylated DNA (IVM DNA is 80% methylated).Sequence to analyze:GGGTGGGGYGGATYGYGTGYGT
35 Methylation levels are consistent even when using different primers Benefits of Pyrosequencing for CpG methylation analysis Accuracy – using different sequencing primersMethylation levels are consistent even when using different primersSeq. Primer 3Seq. Primer 2Seq. Primer 1MLH1 geneA region of 73 bases, containing 12 CpG sites, was analyzed using 3 different sequencing primers. All values above are based on the average of 3 replicates. Note how well the methylation values correlate, regardless of primer.73 basesEach methylation value is the mean of 3 replicates
36 Benefits of Pyrosequencing for CpG methylation analysis Consistent precision among CpG sites Confidently measure the individual degree of methylation in adjacent CpG sites, even at long distances from the sequencing primerPos Pos Pos Pos. 4 Pos Pos Pos Pos Pos Pos. 10MGMT geneDoes the distance from the sequencing primer affect the precision of methylation results?The standard deviation of the 10th site is just as low as that of the first site from the sequencing primer.(Based on replicate Pyrosequencing, not replicate PCR.)
37 Benefits of Pyrosequencing for CpG methylation analysis Quantification of individual CpG sites Methylation levels may vary from site to sitePyrosequencing detects site variation reproduciblyThis experiment showsThe reproducibility of methylation analysis using PyrosequencingThe importance of analyzing several, consecutive CPG sites. Using other methods, that just look at one single CpG position, one might get a non-representative value (site number 2 in this experiment serves as a good example). The relevance of these variations are yet unknown. Shaw et al (2006) discusses this phenomena, and it might be due to steric hindrance of the methylating enzymes. Nevertheless, this shows the importance of analyzing multiple, consecutive sites.(Values based on repeated Pyrosequencing and repeated PCR.)Methylation pattern in RASSF1A in neighboring CpG sites in 4 tumor samples (duplicate runs)
38 Imprinting Gene expression dependent on the parent of origin Prader-Willi Syndrome (PWS) Angelman Syndrome (AS)Neuro developmental disorders caused by a deficiency with 15q parental contributionsmethylated on the maternal chromosomeremains unmethylated on paternal chromosomePWS: lack of paternal contributionpaternal deletion (70%)maternal uniparental disomy (25%)AS: lack of a maternal contributionmaternal deletion (70%)paternal uniparental disomy (5%)In particular they looked at two pediatric disease in neuro development, Prada Will Syndrome and Angelman syndrome.In PWS, there is a lack of paternal contribution and this can occur in two ways-paternal deletion or receiving two copies of the maternal allele (known as uniparental disomy).AS is the exact opposite-lack of maternal contribution(UPD-receive two copies of a chromosome from one parent)38
39 Benefits of Pyrosequencing for CpG methylation analysis Flexible assay design Analysis in either direction – Prader Willi/AngelmanForward assay: C/TNote how well the results achieved forward/reverse correlate to each other. The first site from left (upper pyrogram) corresponds to the first site from right (lower pyrogram).Reverse assay: G/A
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