Classic and Next-Gen Sequencing Technologies. David Miller Australian Genome Research Facility

Transcription

1 Classic and Next-Gen Sequencing Technologies David Miller Australian Genome Research Facility 6 th July 2009

2 Overview of Presentation

3 Milestones 1953 Discovery of the structure of the DNA double helix Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA The first complete DNA genome to be sequenced is that of bacteriophage φx Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation". [3] Frederick Sanger, independently, publishes "DNA sequencing by enzymatic synthesis" Frederick Sanger and Walter Gilbert receive the Nobel Prize in Chemistry 1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the first semi-automated DNA sequencing machine Applied Biosystems markets first automated sequencing machine, the model ABI The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (at 75 cents (US)/base) Richard Mathies et al. publish dye-based sequencing Phil Green and Brent Ewing of the University of Washington publish phred for sequencer data analysis.

4 History of DNA Sequencing Frederick Sanger Only person to receive two Noble prizes in Chemistry. One of only four people ever to receive two Nobel prizes. Only living person to receive two Nobel prizes.

5 Human Genome Project Brief Overview 15 year project began in 1990 US $3 Billion dollar project 2000 first draft genome was announced 2003 complete genome announced 2006 final chromosome published in nature

6 Human Genome Project Project Goals identify all the approximately 20,000-25,000 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project.

7 Human Genome Project Goals and Completion Dates Area HGP Goal Standard Achieved Date Achieved Genetic Map 2- to 5-cM resolution map 1-cM resolution map (3,000 September 1994 (600 1,500 markers) markers) Physical Map 30,000 STSs 52,000 STSs October 1998 DNA Sequence 95% of gene-containing part of human sequence finished to 99.99% accuracy 99% of gene-containing part of human sequence finished to 99.99% accuracy April 2003 Capacity and Cost of Finished Sequence Human Sequence Variation Sequence 500 Mb/year at < $0.25 per finished base 100,000 mapped human SNPs Sequence >1,400 Mb/year at <$0.09 per finished base November million mapped human SNPs February 2003 Gene Identification Full-length human cdnas 15,000 full-length human cdnas March 2003 Model Organisms Complete genome sequences of E. coli, S. cerevisiae, C. eleg ans,d. melanogaster Finished genome sequences of E. coli,s. cerevisiae, C. elegans, D. melanogaster, plus wholegenome drafts of several others, including C. briggsae, D. pseudoobscura, mouse and rat April 2003

8 Shotgun Sequencing Approach

9 1977 Classic Sequencing Technologies Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation". [3] Frederick Sanger, independently, publishes "DNA sequencing by enzymatic synthesis". Both methods generate fragment pools which are resolved via electrophoresis with a resolution of 1 BP.

10 Reactions to 1 Lane Sequencing Reaction Products Progression of Sequencing Reaction

11 Classic Sequencing Technologies

12 ABI377

13 Capillary Based Electrophoresis

14 Raw Data

15 Electropherograms

16 ABI3700

17 MegaBACE 1000

18 ABI3730xl

19 Sanger Sequencing Maximum yield / days <3,000,000bp <0.1% of the human genome 1000 days of sequencing for a 1 fold coverage...

20 Next-Gen Sequencing Technologies

21 Next-Gen Sequencing Technologies Three platforms, three technologies All massively parallel sequencing Read lengths vary from ~36bp to >400bp Read numbers vary from ~ 1 Million to >150 Million per run

22 Next-Gen Sequencing Technologies 4.50E E+06 Raw Data Storage (Mb) 3.50E E E E E E E E GS- 20 GS- FLX GS- FLX TI GAII 1x 18 GAII 1x 26 GAII 1x 35 GAII GAIIx 2x 35 2x 35 GAII GAIIx 2x 50 2x 50 GAII GAIIx 2x 75 2x 75 Instrument Run Type

23 Next-Gen Sequencing Technologies Roche GS-FLX Illumina GAII ABI SOLiD

24 Roche GS-Flx

25 Workflow Sample Fragmentation Library Preparation empcr Setup empcr Amplification Pyrosequencing Data Analysis

26 Sample Fragmentation and Library Generation

27 empcr Emulsion PCR is a method of clonal amplification which allows for millions of unique PCRs to be performed at once through the generation of micro-reactors.

28 empcr The Water-in-Oil-Emulsion

29 Pyrosequencing

30 Data Analysis Raw Image Files

31 Platform Updates Maximum yield / days 1,000,000,000bp 30% of the human genome ~3 days of sequencing for a 1 fold coverage...

32 Illumina GAII

33 Illumina Library Preparation Stylised graphic of the Paired-End library preparation taken from the Paired-End Protocol

34 Illumina Sequencing Technology Robust Reversible Terminator Chemistry Foundation 3 5 DNA ( ug) Sample preparation Cluster growth A C T C T G C T G A A G 5 T G C T A C G A T A C C C G A T C G A T Sequencing T G C T A C G A T Image acquisition Base calling

35 Illumina GAII Development 2009 Roadmap

36 SCS 2.4 and Pipeline 1.4 Two recent software updates from Illumina Huge impact on data yields by not only increasing the number of clusters detectable, but also the number of clusters that pass quality filtering Originally aimed for ~50 Million reads / run Now aiming for ~150 Million reads / run

37 Platform Updates Maximum yield / days 2,000,000,000bp 60% of the human genome ~1.5 days of sequencing for a 1 fold coverage...

38 ABI SOLiD

39 ABI ColorSpace

40 ABI SOLiD

41 empcr and Enrichment

42 Bead Deposition 3 Modification allows covalent bonding to the slide surface

43 Sequencing by Ligation

44 Base Interrogations

45

46 Genome Web

47 Summary Both SOLiD and GS-FLX use empcr Both GS-FLX and GAII sequence by synthesis GAII uses cluster generation similar to the polony approach SOLiD sequencing by ligation GS-FLX provides ~100x decrease in costs compared to Sanger Sequencing GAII and SOLiD ~10x decrease in costs over GS- FLX (though this is probably increasing given the huge increase in output)

48 Applications

49 Targeted Enrichment Sequencing costs have shifted significantly from sequencing to up-front sample preparation, libraries, amplification Recent applications from Roche Nimblegen and Agilent Technologies have allowed for sequence capture through the use of microarrays Resulted in two products, Nimblgen Sequence Capture Arrays and Agilent SureSelect

50 SureSelect In-Solution Capture

51 Nimblegen Sequence Capture Arrays

52 PET-SEQ

53 PET-SEQ Mate-Pair P1 Tag1 Internal Adapter Tag2 P2 Tag Reads Reference Inversions Tag Reads Reference Insertions Deletions

54 Next-Next Next-Gen Sequencing Technologies

55

56 Websites of Note emsequencing/index.htm

57 THANKS David Miller Australian Genome Research Facility 6 th July 2009