Pyrosequencing分析仪

1、1Pyrosequencing分析儀分析儀的原理及應用的原理及應用博微生物科技股份有限公司博微生物科技股份有限公司詹淳勝詹淳勝(A New Light on High-throughput SNP Analysis)2Presentation overview The pyrosequencing method Instrumentation ApplicationsSNP Analysis (SNP)Allele Quantification (AQ)Sequence Analysis (SQA)3The pyrosequencing method Sequence based techno

2、logy Accurate Simple and robust No labels or gels Real-time results- solution for applied DNA analysis4The pyrosequencing methodPPiATP5lighttimeThe pyrosequencing method- detection of the light612345678910110-1AAGCTGthe sequence in this pyrogram is AGGCAGAGGCAG- nucleotides dispensed sequentiallyThe

3、 pyrosequencing method7Pyro animation8Automatic dispensation of reagents96 well formatCCD cameraProcesses500 samples per hour4500 samples per dayInstrumentation- PSQ 9691. Prepare samples 2. Insert samples in PSQ 96 3. Insert reagent cartridge (enzymes, substrate, nucleotides)4. Start run- working w

4、ith the PSQ 96Instrumentation10Automatic dispensation of reagents96 well format (It will be possible to upgrade to a 384-format in the future)CCD cameraProcesses10000 samples per day 30000 samples per day (Triplex analysis)Instrumentation- PSQ HS 96A 111. Prepare samples 2. Insert samples in PSQ HS

5、96A (up to 10 plates and two hours of unattended operation) 3. Insert reagent cartridge (enzymes, substrate, nucleotides)4. Start run- working with the PSQ HS 96A Instrumentation12 Vacuum Prep Tool13 Genetic variability (SNP, insertions, deletions) Haplotyping Allele quantification / frequency Expre

6、ssion profiling, clone identification etc. Bacterial and viral typing Resistance typing Mutation detection Forensic study- one technology, many applicationsApplications.and more14 SNP Analysis(SNP) Allele Quantification (AQ) Sequence Analysis(SQA) - software modules for PSQ96applications15- SNPs as

7、genetic markersapplications - SNP Analysis Single Nucleotide Polymorphisms are isolated single base variations in the genome Occur every 500-1000 bases along the 3 billion bases of the human genome The most common form of genetic inter- individual variation The major source of phenotypic variability

8、 between individuals16SNPDiscoverySNPConfirmationAlleleFrequencySNPRelevanceSNPDiagnosticsNew SNPs Sanger In silico etc.Verify SNP as true SNPFrequencyof SNP inpopulationsValidate SNPas marker for phenotypeUtilization of SNP markers- Pyrosequencing for SNP analysisApplications - SNP Analysis17Homozy

9、gotesHeterozygoteACTGCCTGCTGCCTA/GCTGCCT- Clearly distinguish heterozygotes and homozygotesApplications - SNP Analysis18-Human papilloma virus (HPV)-Angiotensin converting enzyme (ACE)-Melanocortin receptor 1 (MC1R)-Leukocyte adhesion deficiency (LAD)-Obesity-Helicobactor pylori (IL-1B gastric cance

10、r)-Apolipoprotein E (Alzheimers disease)Applications - SNP Analysis19- SNP Analysis of HPVApplications - SNP Analysis20- SNP Analysis of HPVApplications - SNP Analysis215ACTGCCT35A/GCTGCCT35GCTGCCT3- SNP Analysis of ACE exon 15Applications - SNP Analysis22- Insertions/deletionsApplications - SNP Ana

11、lysisTheoretical Pyrosequencing output 5G/5G00,20,40,60,811,2TCGTACGTGTdispensationspeak heightsTheoretical Pyrosequencing output 4G/4G 00,20,40,60,811,2TCGTACGTGTdispensationspeak heightsTheoretical Pyrosequencing output 4G/5G00,20,40,60,811,2TCGTACGTGTdispensationspeak heightsSequence to analyze:

12、(C)ACGTGT.23.analysis of more than one SNP per well Reduces cost per genotype Increases efficiency Increases speed .of genotyping studies- multiplex genotyping by pyrosequencingApplications - SNP Analysis24SNPs located on different fragments.or on the same1.2.3.- principle of multiplex genotyping by

13、 pyrosequencingApplications - SNP Analysis255-C/TGGCCGGGTCACGAT/GGCCC-3- analysis of several SNPsApplications - SNP Analysis26The CYP2D6 gene - Triplex genotyping C/CA/delAA/AC/CA/AA/AC/CdelA/delAA/AC/TA/AA/A27association/linkage studiesgenome-wide scans of SNPslarge sample populationsSNP confirmati

14、onpotential SNPs identified in silicoSNP confirmation in different ethnic populationsmutations associated with cancer presence of normal cells in tumor samples give mixed genotypesAnalysis of SNPs in polyploid genomsApplications - allele quantification28.influence efficiency and cost of SNP studies

15、Reduction in number of analyses Reduced costs (reagents and labor) Less genomic material required- pooling DNA samplesApplications - allele quantification29SNP 1:1126 individualsSNP Software AQ: G: 14.7% T: 85.3%Expected: G: 15% T: 85%14.7%85.3%- result from Karolinska InstituteApplications - allele

16、 quantification30 Allele frequencies automatically calculated Statistics of replicate reactions Mean allele frequencies SD Max, min frequencies Peak heights for all dispensationsApplications - allele quantification31SQA - whenever a specific DNA sequence needs to be analyzedApplications - sequence a

17、nalysis32 96 well format Fast and cost-effective Gel free, no dyes or labels Automatic scoring Easy - installation & training within 1 1/2 day Real-time sequence information from first baseApplications - sequence analysis.short and medium length DNA fragments33- SQA Software: EvaluationnAutomati

18、c base calling by dedicated algorithmnEditing of sequencenColor coding shows quality for each wellnBasic alignment functionApplications - sequence analysis34 Associated with gastric cancer Treatment: Clarithromycin, but combined antibiotic treatments often required due to resistant strainsProf Lars

19、Engstrand et al SMI, Uppsala Univ.- Helicobacter pylori, a clinical case studyApplications - sequence analysis35 Identify H. PyloriAnalysis of 20 bases in the 16S rRNA gene Determine resistance/ sensitivity to clarithromycin Analysis of 2 point mutations in the 23S rRNA gene Patient genotypingInterl

20、eukin 1 beta SNPs appear to be linked to a susceptibility to gastric cancer- Helicobacter pyloriApplications - sequence analysis36Conserved regionConserved regionH. pylori-specific regionCGCGCAATCA GCGTCAGTAA = H. pylori131 isolates sequenced126 correctly base called (99.87%)- Identification of 16S rRNA gene from H. pyloriApplications - sequence analysis3723S rRNA gene2142 2143GAA ResistantAGA ResistantAAA (”wt”) Sensitive154 isolates genotyped154 correctly typed (100%)- Determination of Clarithromycin resistance in H. p