The pyrosequencing method" Instrumentation" Applications" ""SNP Analysis (SNP)" ""Allele Quantification (AQ)" ""Sequence Analysis (SQA)"

Transcription

1 The pyrosequencing method" Instrumentation" Applications" ""SNP Analysis (SNP)" ""Allele Quantification (AQ)" ""Sequence Analysis (SQA)" 1

2 The pyrosequencing method" PPi ATP 2

3 The pyrosequencing method" - detection of the light" light time 3

4 Sequencing By Synthesis Sequencing- By- Synthesis Pyrophosphate signal generation DNA Capture Bead A A T C G G C A T G C T A A A A G T C A Sulfurylase APS PP i T Anneal Primer Luciferase ATP luciferin Light + oxy luciferin

5 The pyrosequencing method" - solution for applied DNA analysis" Sequence based technology" Accurate" Simple and robust" No labels or gels" Real-time results" 5

6 The pyrosequencing method" nucleotides dispensed sequentially" A! GG! C! A! G! A" G" C" T" A" G" the sequence in this pyrogram is AGGCAG! 6

7 Instrumentation" - PSQ 96" Automatic dispensation of reagents" 96 well format" CCD camera" Processes" " "500 samples per hour" " "4500 samples per day" 7

8 Instrumentation" The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. - working with the PSQ 96" 1. Prepare samples " 2. Insert samples in PSQ 96 " 3. Insert reagent cartridge (enzymes, substrate, nucleotides)" 4. Start run" sequence automatically scored 8

9 Instrumentation" - PSQ HS 96A Automatic dispensation of reagents" 96 well format (It will be possible to upgrade to a 384- format in the future)" CCD camera" Processes" " "10000 samples per day" samples per day" (Triplex analysis)" 9

10 Instrumentation" The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. - working with the PSQ HS 96A 1. Prepare samples " 2. Insert samples in PSQ HS 96A (up to 10 plates and two hours of unattended operation) " 3. Insert reagent cartridge (enzymes, substrate, nucleotides)" 4. Start run" sequence automatically scored 10

11 Vacuum Prep Tool 11

12 Applications" - one technology, many applications" Genetic variability (SNP, insertions, deletions) Haplotyping" Allele quantification / frequency" Expression profiling, clone identification etc." Bacterial and viral typing" Resistance typing" Mutation detection" Forensic study"..and more" 12

13 applications" - software modules for PSQ96" SNP Analysis " "(SNP)" Allele Quantification "(AQ)" Sequence Analysis "(SQA)" 13

14 applications" - SNP Analysis" - SNPs as genetic markers" Single Nucleotide Polymorphisms are isolated single base variations in the genome" Occur every bases along the 3 billion bases of the human genome" The most common form of genetic interindividual variation" The major source of phenotypic variability between individuals" 14

15 Applications" - SNP Analysis" - Pyrosequencing for SNP analysis" SNP! Discovery" SNP! Allele! Confirmation" Frequency! SNP! Relevance" SNP! Diagnostics! New SNPs" Sanger " In silico" etc..." Verify " SNP as " true SNP" Frequency" of SNP in" populations" Validate SNP" as marker " for phenotype" Utilization of " SNP markers" 15

16 Pyrogram Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001"

17 PSQ96 Instrument

18 Primer pool 1 Primer pool 2 Primer pool 3 HPV-16 HPV-18 HPV-45 C C C A C G T A C G T A C G T A C G T A C G T A C G T HPV-31 HPV-33 HPV-35 G T G A C G T A C G T A C G T A C G T A C G T A C G T HPV-59 HPV-52 HPV-58 T G T A C G T A C G T A C G T A C G T A C G T A C G T HPV-39 HPV-56 HPV-51 C C G A C G T A C G T A C G T A C G T A C G T A C G T

19 Pyrosequencing, L1 MSPs Figure from Gharizadeh, B. et al. Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses. Molecular and Cellular Probes, H. Watson HPV Detection

20 HPV quadruple co- infec?on a) Primer pool 1 d) Primer pool 2 HPV- 16 HPV- 52 HPV- 31 HPV- 33 C G G T b) Specific primer HPV-16 3A e) Specific primer HPV-33 2G 2G 2A C A G C T A C T A T T G A T A c) Specific primer HPV-31 G A T A C T A C A 3T 4A f) Specific primer HPV-52 3A G G A C A C A T A

21 a HPV-16 HPV-18 Uon-specific amp. General primer General primer General primer General primer Mul?ple infec?ons and Non- specific amplifica?on products! b HPV-16 primer HPV-18 primer HPV-31 primer HPV-33 primer HPV-45 primer HPV-6 primer HPV-11 primer HPV-16 HPV-16 site General primer site HPV-18 Non-specific amp. HPV-18 site General primer site Sequencing and genotyping by pattern recognition Gharizadeh et al. Mol Cell Probes Gharizadeh et al. J Mol Diagn Gharizadeh et al. Mol Cell Probes. 2006

22 Sequencing of a low-yield PCR product containing Unspecific amplification products! a Sequenced by general primer b Sequenced and genotyped by multiple sequencing primers T A C A T A T A 5 T HPV-33 Sequence Pyrogram of HPV-33 c T A C A T A T A 5 T T

23 a General primer Pyrosequencing for Iden?fica?on of Fungi S. chartarum Irrelevant type Non-specific amp. General primer General primer General primer b S. chartarum primer S. elegans primer S. bisbyi primer S. species primer S. subsimplex primer S. kampalensis primer S. dichora primer S. chartarum Irrelevant type Non-specific amp. S. chartarum primer General primer site General primer site Sequencing and genotyping Kaller et al. Submitted 2007"

24 General Primer! T 2 C 2 T 2 C 3 G T 4 C 3 T G Specific Sequencing Primer Pool! G 2 C G C 3 G C 2 G 2 A G A C 4 A 3 C T C T 2!

25 G A T C G C A G T A C G A C A C Pyrosequencing for An?bio?c resistance a) Wild Type: GATTCCGCAGTTTACGACACC b) S91P and D95A: GATTTCGCAGTTTACGGCACC 3T 2T 2C 2C G A G C A G A C G A C A 3T 3T 2G 2C G A C G C A G A C C A c) S91P and D95G: GATTTCGCAGTTTACGCCACC 3T 3T 2C 2C G A C G C A G A C G A d) S91P: GATTTCGCAGTTTACGACACC 3T 3T 2C G A C G C A G A C G A C A e) D95N: GATTCCGCAGTTTACAACACC 3T 2T 2C 2A 2C G A G C A G A C C A

26 a) Group 1, Wild Type d) Group 4, S91P 3T 2T 2C 2C 3T 3T 2C G A G C A G A C G A C A G A C G C A G A C G A C A b) Group 2, S91P and D95A d) Group 5, D95N 3T 3T 3T G A C G C A G A C C A 2G 2C 2T 2C 2A 2C G A G C A G A C C A c) Group 3, S91P and D95G 3T 3T 2C 2C G A C G C A G A C G A Unemo et al. In Press APMIS 2007 Lindback et al. Mol Cell Probes Gharizadeh et al. Int J AnSmicrob Agents 2005

27 Pattern-recognition for improved genotyping! Multiple sequencing primer method and pattern recognition improves rapid genotyping!! The first few basecallings are enough for correct genotyping! Every sequence-pattern has its own characteristics!

28 Applications" - SNP Analysis" - Clearly distinguish heterozygotes and homozygotes" Homozygotes" ACTGCCT! Heterozygote" A/GCTGCCT! GCTGCCT! 28

29 Applications" - SNP Analysis" - SNP Analysis of HPV" 29

30 Applications" - SNP Analysis" - SNP Analysis" 5 ACTGCCT 3! 5 A/GCTGCCT 3! 5 GCTGCCT 3! 30

31 Applications" - SNP Analysis" - Insertions/deletions" Sequence to analyze: (C)ACGTGT... Theoretical Pyrosequencing output 4G/5G peak heights 1,2 1 0,8 0,6 0,4 0,2 0 T C G T A C G T G T dispensations Theoretical Pyrosequencing output 4G/4G peak heights 1,2 1 0,8 0,6 0,4 0,2 0 T C G T A C G T G T dispensations Theoretical Pyrosequencing output 5G/5G peak heights 1,2 1 0,8 0,6 0,4 0,2 0 T C G T A C G T G T dispensations 31

32 Applications" - SNP Analysis" - multiplex genotyping by pyrosequencing "..analysis of more than one SNP per well!! Reduces cost per genotype" Increases efficiency " Increases speed...of genotyping studies" 32

33 Applications" - SNP Analysis" - principle of multiplex genotyping by pyrosequencing " SNPs located on different fragments.! 1." 2." 3."...or on the same! 33

34 Applications" - SNP Analysis" - analysis of several SNPs" 5 -C/TGGCCGGGTCACGAT/GGCCC-3! 34

35 Applications" - allele quantification" association/linkage studies" "genome-wide scans of SNPs!!!large sample populations! SNP confirmation" "potential SNPs identified in silico!!!snp confirmation in different ethnic populations! mutations associated with cancer"! presence of normal cells in tumor samples give mixed genotypes! Analysis of SNPs in polyploid genoms" " "" 35

36 Applications" - allele quantification" - pooling DNA samples"..influence efficiency and cost of SNP studies! Reduction in number of analyses" Reduced costs (reagents and labor)" Less genomic material required" 36

37 Applications" - allele quantification" - result from Karolinska Institute" 85.3%" 14.7%" SNP 1:!!!1126 individuals! SNP Software AQ:!!G: 14.7% T: 85.3%! Expected:!!!G: 15% T: 85%! 37

38 Applications" - sequence analysis" SQA - whenever a specific DNA sequence! needs to be analyzed! 38

39 Applications" - sequence analysis"...short and medium length DNA fragments! 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 base" 39

40 Applications" - sequence analysis" - Helicobacter pylori, a clinical case study" Associated with gastric cancer" Treatment: Clarithromycin, but combined antibiotic treatments often required due to resistant strains" Prof Lars Engstrand et al SMI, Uppsala Univ." 40

41 Applications" - sequence analysis" Identify H. Pylori! Analysis of 20 bases in the 16S rrna gene" Determine resistance/ sensitivity to clarithromycin " Analysis of 2 point mutations in the 23S rrna gene " Patient genotyping" - Helicobacter pylori" Interleukin 1 beta SNPs appear to be 41 linked to a susceptibility to gastric cancer"

42 Applications" - sequence analysis" - Identification of 16S rrna gene from H. pylori" Conserved region Conserved region H. pylori-specific region CGCGCAATCA GCGTCAGTAA = H. pylori! 131 isolates sequenced 126 correctly base called (99.87%) 42

43 Applications" - sequence analysis" - Determination of Clarithromycin resistance in H. pylori! 23S rrna gene AAA ( wt ) Sensitive GAA Resistant AGA Resistant 154 isolates genotyped 154 correctly typed (100%) 43

44 Applications" - sequence analysis" and Helicobacter pylori! -511! -31! Il-1 beta! A/A " G/A! G/G! 44

45 Applications" - sequence analysis" - typical time course! Isolate DNA PCR - pure culture -gastric biopsies (H. pylori) 10 min-1 h 2.5 h Preparation of PCR-products manual preparation of 96 samples <1 h Pyrosequencing <1 h <5 h 45

46 Summary: The strength of Pyrosequencing" Many applications" Accurate" Sequence confirmation (compared to yes/no)" Fast, 96 genotypes in 10 minutes" Easy, little hands-on and short optimization time" 96-well format easy to automate" Automatic scoring of the results " 46

47 Template Tailoring for DNA FingerPrin?ng Seq. primer Bio TCAC. TCAC. TCAC. TCAC. TCAC. TCAC. ACGT. ACGTU TCAC. TCAC. TCAC. TCAC. TCAC. TCAC. ACGT. Seq. primer ACGTU TCAC. TCAC. TCAC. TCAC. TCAC. TCAC. TCAC. TCAC. TCAC. ACGT. TCAC. ACGT. Bio

48 DNA Finger Prin?ng Sample D29629, locus D7S820 (9/10 repeats): Sample D29630, locus D7S820 (8/11 repeats): Sample D29624, locus D16S539 (9/12 repeats):

49 ~16 ~39 ~56 ~68 ~73 ~34 ~42 ~45 ~52 ~59 ~69 HPV MIP- assay ~6b ~31 ~35 ~11 ~51 ~33 ~40 ~43 ~58 ~82 ~18 ~44 ~ bp E6 E2 E5 L1 E7 E1 E4 L2 UTR U 1 /U 2 Pyrosequencing by barcodes β-globin + β-globin + HPV-16 + HPV-18 + Inverted Probes Validation Microarray chip with barcodes β-globin + β-globin + HPV-16 + HPV-18 + Chip1 Chip2 β-globin HPV-6 HPV-11 HPV-16 HPV-18 HPV-31 HPV-33 HPV-34 HPV-35 HPV-39 HPV-40 HPV-42 PGMY MY09/11 GP5+/6+ SPF 10 ~ 65 bp Conventional genotyping region, ~ 450 bp ~150 bp Validation Cloning or hybridization based screening MULTIPLEX- MULTIPLEXING! conventional method HPV Detection

50 Rapid, Low- Cost, Accurate Diagnos?c Test for Poten?ally Pandemic Influenza

51 Alignment of 362 Strains of H5N1

52

53 Ini?al Results of Proof of Principle Experiment with CDC (using PCR, mulsplex pyrosequencing & mulsple sequencing primers) ChrarcterizaSon of the hemagglusnin from the H5N1 influenza viruses tested. G l y c o s y l a t i o n M o t i f ( N X T / S, X P ) a t a a 154 R e c e p t o r B i n d i n g P o c k e t ( ) C l e a v a g e M o t i f H o s t / O u t c o m e H A V i r u s N a m e C l a d e G o o s e / G u a n g d o n g / 1 / 96 - P r e s e n t G Q S G R R R K K R G o o s e H o n g K o n g / 156 / 97 3 A b s e n t G Q S G R R R K K R H u m a n / D i e d H o n g K o n g / 483 / 97 3 P r e s e n t G Q S G R R R K K R H u m a n / D i e d H o n g K o n g / 213 / 03 1 P r e s e n t G Q N G R R R K K R H u m a n / D i e d V i e t n a m / 1203 / 04 1 P r e s e n t G Q S G R R R K K R H u m a n / D i e d V i e t n a m / J P 14 / 05 1 P r e s e n t G Q S G R R R K K R H u m a n / D i e d V i e t n a m / H N / 05 1 P r e s e n t G Q S G R R K K R H u m a n / S u r v i v e d C h i c k e n / K o r e a / E S / 03 I n d o n e s i a / 5 / A b s e n t P r e s e n t G Q S G G Q S G K R K K R S R R K K R C h i c k e n H u m a n / D i e d Pourmand et al. Rapid and highly Informative Diagnostic Assay for H5N1 Influenza Viruses. PLoS One 2006"

54 Pyrosequencing Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001"

55 Pyrosequencing - Solid Phase Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001"

56 Pyrosequencing - Liquid Phase Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001"

57 Bioluminescence Regenera?ve Cycle (BRC) The Principle US Patent B &" Hassibi et al. Biophys Chem 2005"

58 BRC Performance Characteris?cs

59 BRC Prototype Performance over 4 orders of magnitude 200 picom 20 picom 2 picom 200 femtom 20 femtom

60 Samples in Human Serum Clean sample Sample in 10% serum 200 picom sample

61 Applications" - SNP Analysis" - SNP Analysis of HPV" 61

62 Applications" - SNP Analysis" "-Human papilloma virus (HPV)" "-Angiotensin converting enzyme (ACE)" "-Melanocortin receptor 1 (MC1R)" "-Leukocyte adhesion deficiency (LAD) "" "-Obesity" "-Helicobactor pylori (IL-1B gastric cancer)" "-Apolipoprotein E (Alzheimer s disease)" " 62

63 gyra Pyro results Codon 87 Codon 83

64 Applications" - sequence analysis" - SQA Software: Evaluation" Automatic base calling by dedicated algorithm" Editing of sequence" Color coding shows quality for each well" Basic alignment function" 64

65 mexr pyro results