1. Next Generation Sequencing Platforms
Sequencing by synthesis (SBS)
454/Pyrosequencing
Illumina/Solexa
Helicos
Pacbio
(Charge-based detection system, Now-sequencing)
Sequencing by hybridization
SOLiD

2. Pyrosequencing: Sequencing-By-Synthesis

4. UCSC Sequencing Center
454 Sequencing System Chemistry & Platform Sequencing-by-synthesis Library Preparation Emulsion Based Clonal Amplification SOLiD Bioanalyzer Chemistry & Platform Library Construction Emulsion PCR Applications Fragment Sequencing Transcriptome Studies Paired-End Sequencing Targeted sequencing Small RNA
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UCSC Sequencing Center

5. Overview of The 454 Sequencing System
1) Prepare Adapter Ligated ssDNA Library (A-[insert]-B)
2) EmPCR: Clonal Amplification on 28 µ beads followed by enrichment
3) Load beads and enzymes
in PicoTiter Plate™
4) Perform sequencing-by-synthesis
on the 454 Sequencer
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UCSC Sequencing Center

6. Emulsion Based Clonal Amplification
+ PCR Reagents
+ Emulsion Oil B
Micro-reactors
Adapter carrying
library DNA
Mix DNA Library
& capture beads
(limited dilution)
Create
“Water-in-oil”
emulsion
“Break micro-reactors”
Isolate DNA containing beads
Perform emulsion PCR
Generation of millions of clonally amplified sequencing templates on each bead
No cloning and colony picking
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UCSC Sequencing Center

7. Depositing DNA Beads into the PicoTiter™Plate
Load Enzyme Beads
Load beads into PicoTiter™Plate
Centrifuge Step
44 μm
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UCSC Sequencing Center

8. Sequencing-By-Synthesis
Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells
Pyrophosphate signal generation
DNA Capture Bead
Containing Millions of
Copies of a Single
Clonal Fragment
A A T C G G C A T G C T A A A A G T C A T
Anneal Primer
Sulfurylase
Luciferase PP i
APS
ATP
luciferin
Light + oxy luciferin
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UCSC Sequencing Center

9. Sequencing Workflow Overview
Generation of small DNA fragments via nebulization
Ligation of A/B-Adaptors flanking single-stranded DNA fragments
Emulsification of beads and fragments in water-in-oil microreactors
Sequencing and base calling
Sample input: Genomic DNA, BACs, amplicons, cDNA
One Fragment
One Bead
Clonal amplification of fragments bound to beads in microreactors
One Read ,000 reads per run
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UCSC Sequencing Center

10. Sequencing Workflow Library Preparation
Genome fragmented by nebulization
sstDNA library created with adaptors
A/B fragments selected using streptavidin-biotin purification
Library generation:
Nebulization of genomic DNA: 300 – 800 bp fragments
Low molecular weight DNA is used without fragmentation and sample preparation begins with adaptor ligation. For amplicon sequencing A and B sequences are added during PCR using FusionPrimers
Polishing, blunting and adaptor ligation (A and B): mixture of AA, BB and AB molecules; A adaptor is biotinylated on one strand
Adding of streptavidin coated magnetic beads: Only AA and AB molecules bind (A-adaptor biotin marker)
Denaturation of double strands: only single stranded fragments flanked by A and B sequences can be collected from supernatant after magnetic separation of beads.
Single stranded fragments are substrate for sequencing process.
8.0 h
7.5 h
4.5 h
and 10.5 h
DNA library preparation and titration
emPCR
Sequencing
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UCSC Sequencing Center

11. Sequencing Workflow Emulsion PCR
Emulsion-based clonal amplification
Anneal sstDNA to an excess of DNA Capture Beads
Emulsify beads and PCR reagents in water-in-oil microreactors
Clonal amplification occurs inside microreactors
Break microreactors, enrich for DNA-positive beads
emPCR:
Anneal to an excess of DNA capture beads: Making sure that statistically only one fragment is captured in a microreactor
Emulsion generation: Oil and water and PCR reagents are mixed and shaked in a tissue lyser
Clonal amplification results in beads carrying ca. 106 fragments on a bead
Subsequently, the emulsion is broken and enrichment using streptavidin coated magnetic beads is perfomrmed (the A-PCR primer is biotinylated),
8.0 h
7.5 h
4.5 h
and 10.5 h
DNA library preparation and titration
emPCR
Sequencing
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UCSC Sequencing Center

12. Sequencing Workflow Loading of PicoTiterPlate Device
Depositing DNA beads into the PicoTiterPlate device
Well diameter: average of 44 µm
> 400,000 reads obtained in parallel
A single clonally amplified sstDNA bead is deposited per well
Amplified sstDNA library beads
Quality filtered bases
Loading of beads on PicoTiterPlates
Current protocol:
Sample beads are loaded on PicoTiterPlate: Gravitation; mention size exclusion: Only one bead per well: sample bead has a diameter of around 30µ, diameter of a well: 44µ
Brown packing beads are loaded on top: Centrifugation 1
Enzyme beads are loaded on top: Centrifugation 2
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UCSC Sequencing Center

13. Sequencing Workflow Sequencing by Synthesis
Bases (TACG) are flowed sequentially and always in the same order (100 times for a large GS FLX run) across the PicoTiterPlate device during a sequencing run.
A nucleotide complementary to the template strand generates a light signal.
The light signal is recorded by the CCD camera.
The signal strength is proportional to the number of nucleotides incorporated.
Flowgram
Key sequence
Nucleotides are added sequentially: When the first of the four deoxynucleosid triphosphates is flowed over the plate and if it is complementary to the template strand, DNA polymerase catalyzes the incorporation of the nucleotide into the DNA strand. Pyrophasphate is released in a quantity equimolar to the amount of incorporated nucleotide and is converted by ATP.
Luciferase uses this ATP to convert luciferin to oxyluciferin and light, which is detected by a charge-coupled device (CCD) camera and recorded as a peak.
Each light signal is proportional to the number of nucleotides incorporated.
In parallel, apyrase continuously degrades unincorporated dNTPs and execc ATP. When the degradation is complete, the next dNTP is added.
8.0 h
7.5 h
4.5 h
and 10.5 h
DNA library preparation and titration
emPCR
Sequencing
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UCSC Sequencing Center

14. GS FLX Data Analysis Flowgram Generation
Key sequence = TCAG for signal calibration
Flow Order
1-mer
2-mer
3-mer
4-mer
TACG
Flowgram
TTCTGCGAA
Background information: Keypass and normalization (signal per base
& signal per flow)
a) Noise to signal calculation: Noise is calculated by averaging the well signals from negative flows of the key.
Signal flow pattern is checked against the known and expected key sequence pattern. If both match and signal to noise ratio exceeds a certain threshold, a signal per base value is generated for every well separately by averaging the signals of the 4 matching keys.
c) Signal normalization within a well: The normalized signal per flow (per base) is calculated by dividing each flow signal by the signal per base value.
d) Normalization across wells, i.e. calculation of final signal: Normalized signals (see c) from the first x T C, A and G flows of all wells are averaged. Thereafter the normalized signal from an individual flow in a well (see c) is divided by this “across well average”, thus resulting in the final normalized signal displayed in the flowgram.
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UCSC Sequencing Center

15. GS FLX Data Analysis Overview
GS De Novo Assembler
GS Reference Mapper
GS Amplicon Variant Analyzer
Image capture
Image processing
Signal processing
GS Run Browser
The Genome Sequencer FLX software package includes four unique software modules used for data analysis and results display:
GS De Novo Assembler Software
The De Novo Assembler software generates a consensus sequence of the whole DNA sample by assembly of de novo shotgun sequencing reads into contigs and subsequent ordering of these contigs into scaffolds. Contig ordering is done by generation and assembly of paired-end reads. The GS De Novo Assembler Software allows for de novo assembly of up to 120 Megabases of sequencing data.
GS Reference Mapper Software
The Reference Mapper software generates a consensus DNA sequence by mapping, or alignment, of the sequencing reads to a reference sequence. In addition, it generates a list of high-confidence mutations by identification of individual bases that differ between the generated consensus DNA sequence and the reference sequence. The GS Reference Mapper Software facilitates the re-sequencing of up to three Gigabases of sequencing data.
GS Amplicon Variant Analyzer Software
The Amplicon Variant Analyzer software compares amplicon sequencing reads to a reference sequence. Multiple alignment of several hundreds of clonal amplicon reads (ultra-deep amplicon sequencing) and comparison of these reads against a reference sequence allows for the detection of rare sequence variations even in heterogeneous specimens.
GS Run Browser Software
The Run Browser software is an interactive application that displays the results of a sequencing run as well as raw images and graphic representations of various metrics files. Run Browser software can be used to assess the general quality of a run, and therefore is a useful tool for troubleshooting if problems are observed. The application also facilitates evaluation of the results of titration experiments. Data generated by Run Browser software can be exported to an Excel spreadsheet.
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UCSC Sequencing Center

16. GS FLX System Performance Read Length
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UCSC Sequencing Center

17. The Genome is comprised of repeat regions
Depending upon the specific genome characteristics, microreads (~25 bp’s) cover only a portion of the genome
In human – 25 base pair reads can only be mapped uniquely to 80% of the genome
Short reads are limiting in known genomes – What about unknown genomes?
Mapping versus de novo assemblies
Mapping will miss genome rearrangements
Mapping is only as good as the reference
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UCSC Sequencing Center

18. UCSC Sequencing Center
Why Does Length Matter?
Longer sequencing reads mean more applications
Identify and characterize small and short RNA’s
Full length cDNA sequencing for expression levels and variations
Amplicon resequencing for genetic variation including somatic mutations
Sequencing of micro-organisms in a single instrument run
Sequencing of complex genomes – mammalian & plant
Sequencing of complex samples – Metagenomics, Ancient DNA
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UCSC Sequencing Center

19. Longer sequencing reads mean more applications
HIV Studies (3)
ChIP-Sequencing (8)
Boyle et al, Cell: Mixed technologies for mapping open chromatin
Metagenomics (12)
Palacios et al, New England Journal of Medicine, Pathogenic Virus Detection
Whole Genome Sequencing (30)
Velasco et al, PLoS: Pinot Noir Genome
Paired-End sequencing
Detecting Structural Variations across two human genomes
Technology and Bioinformatics (11)
Meyer et al, NAR,: Using Picogram quantities of sample
Transcriptome studies – cDNA (17)
Small RNA (32)
Amplicon and Methylation Studies (9)
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UCSC Sequencing Center

20. UCSC Sequencing Center
Applications of Whole Genome, Ultra Broad and Ultra Deep HT- Sequencing
HT-
Sequencing
Technology
Applications
Whole Genome
Ultra Broad
Ultra Deep
Sequencing
Sequencing
Sequencing
Virus
Small RNA
HIV
Bacteria
SAGE /
CAGE
Resistance
Tropism
Fungus
Expression
Higher Eukaryotes
Transcriptome
Amplicons
Human
Metagenomics
Population Biology
Novel strain ID
Bacterial 16S
HLA Typing
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UCSC Sequencing Center

21. The power of Metagenomics
How to Identify an environment based upon the microbial organisms that are present
Microbial Population Structures in the Deep Marine Biosphere
Huber et al., Science, 318, p97, 2007
Determining the state of an environment based upon the presence and mixture of microbial organisms
The interdependence of Coral and it’s microbial environment
Wegley et al., Environmental Microbiology, 9, p2707, 2007
Detecting viral pathogens – quickly and accurately
Less than 12 months from first identification of affected hives to possible pathogen
Cox-Foster et al., Science, 2007
Transplant victims from Australia
Palacios et al, New England Journal of Medicine, 2008
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UCSC Sequencing Center

22. Transcriptome Analysis Workflow Comparison
GS FLX (clonal sequencing ensured through emPCR)
Sanger (E. coli cloning, often concatemerization)
cDNA libraries
(short tag library,
EST library)
Concatemerization,
insert fragments into vectors
and clone into bacteria
Grow, pick colonies
Template Generation
Sequencing
Time: Weeks
emPCR
Time: Days
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UCSC Sequencing Center

23. UCSC Sequencing Center
Sequencing of approximately 400,000 small RNAs from C. elegans
Another 18 unknown miRNA genes were detected
Thousands of endogenous siRNAs acting preferentially on transcripts associated with spermatogenesis and transposons were identified
A new class of small RNAs was identified: 21U-RNAs. They all begin with an U and are precisely 21 nt long.
The Genome Sequencer System is perfectly suited for the discovery of small non coding RNAs on a genome wide level
Meanwhile > 15 papers dealing with the detection and characterization of sncRNAs based on GS have been published
This is a typical example: The researchers have found a new sncRNA class. This occurs frequently because not much is known about these regulatory elements
Advantages compared to Sanger:
No cloning
No bias
No concatenation
High throughput and low cost per clonal read allows for genome wide analysis
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UCSC Sequencing Center

24. Multiplex Identifier Basics
What is it?
Two new kits, each with 6 different library adapters (total of 12 adapters)
Each MID library adapter has an added, specially encoded 10-base region
Used to “bar-code” up to 12 different genomic library samples to be run in the same region of a single sequencing run
Standard Library
Seq. primer
Read
Primer A
Key
Library fragment
Primer B
#bases:
MID
Library
Seq. primer
Read
Primer A
Key
MID 1
Library fragment
Primer B
#bases:
Primer A
Key
MID 2
Library fragment
Primer B
Primer A
Key
MID n
Library fragment
Primer B
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UCSC Sequencing Center

25. Paired-End Applications
~100 bp sequencing tags separated by 3 kb spacing
Use for de novo assembly
Order contigs
Use for Structural Variation studies
Inversions, Deletions, Insertions…
High resolution detection – 3kb spacing vs 10 to 40 kb
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UCSC Sequencing Center

26. UCSC Sequencing Center
Paired-Ends workflow
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UCSC Sequencing Center

27. Targeted Enrichment of Human gDNA
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Fragment and hybridize to NimbleGen capture array
Elute
HT-Sequencing
Analyze
Exon
Sequences
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UCSC Sequencing Center

28. Sequencing all the known exons from the human genome
“Direct selection of human genomic loci by microarray hybridization,”
Albert et al., Nature Methods, (4) 11, , 2007
~6,700 gDNA loci selected
BRCA1 region
2 MB Region
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UCSC Sequencing Center

29. Another Sequence-Capture Example
19 Kb region from Chromosome 4
Targeted Exons
Seq-Cap Array Probes
GS FLX Seq Reads
Sequencing Coverage
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UCSC Sequencing Center

30. SOLiD Library Preparation
The SOLiD system uses either a fragment library or a mate-paired library depending on the user’s desired information or application.
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UCSC Sequencing Center

31. Emulsion PCR and Bead Enrichment
PCR takes place in oil in water microreactors. Post-PCR, templated beads are separated from non-templated beads, and modified at the 3’ end to allow covalent linkage to the SOLiD sequencing slide.
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UCSC Sequencing Center

32. Bead Deposition
Beads are deposited into 1,2,4, or 8 segmented chambers on a slide.
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UCSC Sequencing Center

33. Sequencing By Ligation and Data Analysis
Primers hybridize to adaptors and a set of 4 dye labeled probes competes for ligation to the primer with probe specificity determined by the 4th and 5th base interrogation during each ligation series, for 5-7 rounds.
After each round of ligation, a new primer offset by 1 base is hybridized for a new round of ligations bp are generated through 5 sequential primer reset and ligation rounds.
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UCSC Sequencing Center

34. UCSC Sequencing Center
Library Construction
2 different libraries can depending on the application and desired information.
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UCSC Sequencing Center

35. UCSC Sequencing Center
Fragment Library
DNA is fragmented and PCR primer adaptors are ligated to the DNA
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UCSC Sequencing Center

36. UCSC Sequencing Center
Mate-Pair Library
DNA is sheared, selected for a desired input size, and circularized around an internal adaptor.
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UCSC Sequencing Center

37. Mate-Pair Library (cont.)
The circularized DNA is enzymatically cleaved to yield 2 DNA fragments separated by an internal adaptor. PCR primer adaptors are ligated on to the end of this piece of DNA.
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UCSC Sequencing Center

38. UCSC Sequencing Center
Emulsion PCR (ePCR)
PCR takes place in oil in water microreactors containing P1-coupled beads, templates, primers, and all required PCR reaction components..
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UCSC Sequencing Center 38

39. UCSC Sequencing Center
ePCR
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UCSC Sequencing Center

40. UCSC Sequencing Center
Emulsion PCR yields both monoclonal (unique templates and polyclonal beads (multiple templates), as well as some non-templated beads.
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UCSC Sequencing Center

41. UCSC Sequencing Center
Clonal Amplification
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UCSC Sequencing Center

42. Post-Emulsion and ePCR
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UCSC Sequencing Center

43. UCSC Sequencing Center
Bead Enrichment
Templated beads are separated from non-templated beads via polystyrene beads
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UCSC Sequencing Center

44. Pre- and Post-Bead Enrichment P2-hybridization
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45. UCSC Sequencing Center
Bead Deposition
Templated beads are modified at their 3’-end and covalently attached to a glass slide.
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UCSC Sequencing Center

46. UCSC Sequencing Center
Slide Configurations
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UCSC Sequencing Center

47. UCSC Sequencing Center
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UCSC Sequencing Center

48. SOLiD Sequencing Chemistry
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UCSC Sequencing Center

49. 4-color Ligation Reaction
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UCSC Sequencing Center

50. UCSC Sequencing Center
A complementary dye-labeled probe hybridizes is ligated to the universal sequencing primer.
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UCSC Sequencing Center

51. UCSC Sequencing Center
De-phosphorylation
A phosphatase cleaves the 3’-phosphate from the universal sequencing primer preventing ligation to templates where no probe was ligated.
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UCSC Sequencing Center

52. UCSC Sequencing Center
Visualization
The fluorescently labeled dyes attached to each probe are visualized.
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UCSC Sequencing Center

53. UCSC Sequencing Center
Cleavage
Fluorescent dye labeled nucleotides are cleaved from hybridized probe.
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UCSC Sequencing Center

54. UCSC Sequencing Center
2nd Ligation Cycle
The next set of labeled probes hybridize and are ligated to the previously hybridized probe.
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UCSC Sequencing Center

55. 2nd Cycle Visualization
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56. UCSC Sequencing Center
2nd cycle cleavage
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UCSC Sequencing Center

57. Every 5th base is interrogated
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UCSC Sequencing Center

58. UCSC Sequencing Center
Reset
Template is stripped
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UCSC Sequencing Center

59. UCSC Sequencing Center
1st cycle after reset
A new set of universal sequencing primers is hybridized, offset by (n-1).
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UCSC Sequencing Center

60. UCSC Sequencing Center
1st cycle after reset
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UCSC Sequencing Center

61. UCSC Sequencing Center
2nd round
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UCSC Sequencing Center

62. Sequential Rounds of Sequencing with Fragment Library
In each round of sequencing ………….??????????????
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UCSC Sequencing Center

63. Sequential Rounds of Sequencing with Mate-Paired Library
In each round of sequencing ………….??????????????
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UCSC Sequencing Center

64. UCSC Sequencing Center
Di-Base Encoding
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UCSC Sequencing Center

65. Advantages of di-Base Encoding
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66. Advantages of di-Base Encoding Real SNPs
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67. Advantages of di-Base Encoding Miscall
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68. Only Certain Transitions are Allowed for Real SNPs
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69. Only Allowed Transitions
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70. Two color changes not allowed
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71. UCSC Sequencing Center
Benefits or solid
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UCSC Sequencing Center

72. Thank you for your attention
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UCSC Sequencing Center