1. High Throughput Sequencing Technologies at YCGA
Shrikant Mane
Director, YCGA
Director, Keck Laboratory

2. First-generation sequencing technology
Outline
First-generation sequencing technology
Sanger sequencing
Current massively parallel sequencing strategies
“Second Generation”
454
Illumina
Ion Torrent & Ion Proton
“Third Generation”
Pacific Biosciences
Oxford Nanopore
YCGA

3. Goals of biomedical investigation
Understand normal, healthy and disease biology
Enable prevention and early diagnosis of disease
Enable new effective treatments
Utility of Next Generation Sequencing/genetics in medicine
Unbiased approach to identify new pathways underlying basic physiology, health and disease

4. Evolution of genomic technologies
Genetic mapping studies: Discovery of genes for well characterized Mendelian diseases.
Dense SNP genotyping using microarray technology: GWAS for discovery of common variants in common disease.
High throughput sequencing: Discovery of rare variants in not previously recognized Mendelian diseases and common diseases.
Constant and rapid changes in genomic technologies have driven successive eras of discovery of loci underlying human traits. The development of complete genetic maps of the human genome in the 1980’s fueled the mapping of Mendelian loci in extended kindreds for dominant traits and predominantly in consanguineous kindreds for recessive traits. Further accelerated by the acquisition of the sequence of the human genome in 2001, this first Mendelian era identified over 2800 disease loci and profoundly changed our understanding of the biology and pathophysiology of every organ system. Labor intensive and slow process.
A second era, was defined by the development of microarray technology and identification more that 10 million common variants in human genome. The microarrays were developed to genotype 500K to 5 Million SNPs in order to identify common variants associated with human disorders This era led to the identification of more than 1000 loci that shows robust association with human disease that have changed the understanding of disease biology..
We have recently entered a third era of discovery, this one driven by spectacular reductions in the cost of DNA sequencing from ~$100,000 per million bases in 1998 to ~$0.10 today on the HiSeq instrument. Coupled with our development of robust methods for selectively sequencing complete coding regions of the genome, which harbor the overwhelming majority of Mendelian loci, and analytic methods to rapidly and with high sensitivity and specificity identify variations from the reference sequence, one can now sequence ostensibly all the genes in the human genome (the exome) to high levels of completion for ~$1000 (direct cost). This has provided fundamental new opportunities for identifying Mendelian loci that were previously elusive.

5. Why High-Throughput DNA Sequencing Number of PubMed Articles
DNA sequencing can provide a deeper understanding about DNA/RNA than any other technology
Microarray Technology revolutionized biomedical research, but has several limitations, which DNA sequencing may overcome
As the cost of sequencing is rapidly decreasing, it is becoming affordable to perform sequencing at a genome level
Why do we need highthroughput DNA sequencing Center at Yale? There is no doubt that microarray technology has revolutionalized the biomedical research but has several limitations such as indirect observation based on hybridization signals which can non specific due to cross hybridization and also is not sensitive enough to identify low levels of chages. Also microarrays can provide the information about what is representated on the chips . DNA sequencing may be able to over come some of these limitations to provide
Number of PubMed Articles
In recent years there has been an explosion of research articles using next generation sequencing technologies

6. Applications of Next-gen Sequencing
DNA Sequencing Applications
Re-Sequencing
Exome sequencing Mutation/SNP discovery and profiling
Interactome
DNA Protein Interactions
ChIP Seq
Transcriptome Analysis
Alternative splicing and allele specific expression
microRNA
Expression and Discovery
Diagnostic Services
CLIA certification
Epigenomics
DNA Methylation
De Novo Sequencing
Population Metagenomics
Copy Number Variation

7. First Generation: Sanger sequencing
( )
1980 Nobel Prize in chemistry
phi X 174
~5300 bp
gels read by hand
radiolabeled dideoxyNTPs
one lane per nucleotide
800 bp reads
low throughput (several kb/gel)

8. Second-generation sequencing
Massively parallel sequencing of millions of template
454/Roche
Illumina
Ion Torrent-Proton

9. Second Generation: Massively Parallel Sequencing.
Throughput (24 hours): Mb (Sanger)
60,000 Mb (HiSeq)
Cost: $1500/Mb (Sanger)
$0.06 /Mb (HiSeq)
Read Lengths: ~800 bp (Sanger)
~ 100 – 600 ( HiSeq- 454)
Error rates: < 0.5 % (Sanger)
~ %% (HiSeq)

10. Illumina next generation sequencing platform

11. HiSeq 2500 Sequencing System Fast turnaround and highest output in a single instrument
1 Instrument – 2 Run Modes
High Output Mode
600 Gb in ~10.5 days
Current v3 flow cell
Current v3 reagents
cBot required
Rapid Run Mode
120Gb in ~1 day
New 2-lane flow cell
New reagents
No cBot required
User configurable
6 human genomes
in 10.5 days
1 human genome
in a day
Highest Output
Fastest turnaround

12. New sequencing platforms by Illumina
HiSeq X Ten and HiSeq X Five:
Production-scale human whole genome sequencing: 18,000 genomes/year at $ 1,500 cost/genome
HiSeq 3000/HiSeq 4000:
Up to 1.5 Tb/run.
Whole genome as well as other applications including exome sequencing

13. Overall Illumina Sequencing Workflow
Sample Preparation
Sequencing Library Preparation
Adapter1
Adapter2
Sequencing
Primer
Insert
Cluster Generation
Hybridizing Library to Flow Cell
Creating clusters from
individual molecules
Introductory workflow--- good to start with the basics and go from here
Explain that these 3 steps are 3 separate kits that one purchases. They can work with their salesperson to determine which kits and in what amounts they want to purchase. Emphasize that for any of our products (genomic, expression, chip, etc) that you follow these 3 basic steps: Sample Prep (library prep); Cluster Generation on a Flowcell, and Sequencing on the Genome Analzyer.
For Sample Prep--- the processes used in the kits end up with a construct illustrated for all sequencing types--- 2 different adaptors, a sequencing primer, and an insert. If the group will do paired end, can mention it’ll be slightly different adapters, and different sequencing primers on both ends of the insert (will be confusing for a new group--- can come back to this slide later if someone asks).
Cluster generation-- Show them the flowcell picture--- 8 lanes for 8 different samples. Library hybridizes to flowcell with individual molecules forming clusters that will be sequenced. The different molecules of the library are physically separated from one another so the sequence of each one can be determined.
Sequencing by Synthesis--- describe the general process with the reversible terminators. Can introduce the concept that the GA has a “chemistry cycle” where you are removing the last block and then adding the next particular base, then an imaging cycle.
Sequencing by Synthesis
Add all 4 bases with Reversible Terminators
Image 4 colors
Remove Terminator, repeat

14. Genomic Sample Prep Workflow
Purified genomic DNA
1. Genomic DNA fragmentation
Fragments of less than 800 bp
2. End-repair
Blunt ended fragments with 5’-Phosphorylated ends
3. Klenow exo- with dATP
3’-dA overhang
4. Adapter ligation
Adapter modified ends
5. Gel purification/bead
Removal of unligated adapter
6. PCR
Genomic DNA Library
We’re using Genomic Sample Prep Workflow as an example of the basic sample prep protocol, each being different. All sample prep methods come with their own protocol which follow standard molecular biology cloning techniques.
Adapter1
Adapter2
Sequencing
Primer
Insert

15. What is a Flow Cell?
A flow cell is a thick glass slide with 8 channels or lanes
Each lane is randomly coated with a lawn of oligos that are complementary to library adapters
P5 oligo
P7 Oligo
Adapter1
Adapter2
Insert
Sequencing Primer
Index

16. Reversible Terminator Seq Chemistry
All 4 labeled nucleotides in 1 reaction (green, orange, red and blue)
Advantages of reversible terminators:
Only one base is added at a time
Fluor can be cleaved off after the imaging. Thus, it does not emit color at the next cycle allowing only newly added base (with attached fluor) to emit the light
Next cycle
Incorporation
Detection
Deblock; fluor removal O
DNA HN N 3’ 5’
free 3’ end X OH O
PPP HN N
cleavage
site
fluor 3’
block

17. Illumina sequencing

18. Sequencing By Synthesis (SBS)5’ 3’ 5’
Cycle 1: Add sequencing reagents
First base incorporated
Remove unincorporated bases G T C A
Detect signal/Imaging T G
Cleave off fluor and Deblock C A G T
Cycle 2-n: Add sequencing reagents and repeat
All four labeled nucleotides in one reaction
High accuracy
Base-by-base sequencing
No problems with homopolymer repeats
HCS:1.8.6

19. 4 Ion Protons: coming soon
Ion Torrent PGM and Proton
Ion PGM™ Sequencer
4 Ion Protons: coming soon
First PostLight sequencing technology: Instead of using light as an intermediary, PGM creates a direct connection between the chemical and the digital worlds.

20. The Chip is the Machine Uses semiconductor chips for sequencing.
Ion PI chip: >165 million wells per chip: 8 to 10 Gb data per run
Ion PII chips: ~100 Gb of data in ~4 hours

21. Base Calling
When a nucleotide is incorporated into a strand of DNA, a Hydrogen ion is released as a by product. The H ion carries a charge which the PGM’s ion sensor can detect as a base.
Ion Torrent technology video.

22. Advantages and Current Limitations
Low equipment cost
Rapid run times: 3 to 4 hours
Simple Chemistry
Limitations
Homopolymers detection
Error rates
Slow on introducing newer chips: Overpromise
PGM and Proton: two separate sequencing equipment
Library prep: Emulsion PCR/ New protocols

23. Third generation sequencing
PacBio RS

24. The Third Generation Sequencing Platform: PacBio RS
Pacific Biosciences has developed Single Molecule Real Time (SMRT™) DNA sequencing technology: PacBio RS.
This technology enables, for the first time, the observation of natural DNA synthesis by a DNA polymerase as it occurs.
This technology delivers long reads at single molecule level and fast time to result, enabling a new paradigm in genomic analysis.
Most people here are familiar with the Sanger sequencing which is the so callled first generation sequencing; and second generation sequencing technology such as illumina hiseq system. It starts with library prep with PCR amplification and cluster building. After sequencing, it generate tens of millions of short reads. Today I am going to introduce you the third generation sequencing platform pacbio RS, developed by Pacific Biosciences that can do single molecule real time sequencing. the technology is called SMRT for single molecule real time. This technology enables, for the first time, the observation of natural DNA synthesis by a DNA polymerase as it occurs. The major advantages of this new sequencing technology is that it can delivers long reads at single molecule level and fast time to results.

25. Pacific Biosciences SMRT® Technology
Technology Video

26. Key Applications for PacBio RS
Targeted sequencing
SNP and structure variants detection
Repetitive region
Full length transcript profiling
De novo assembly and genome finishing
Bacteria genome
Fungal genome
Gap-captured sequencing
Targeted captured sequencing
Base modifications detection
Methylations
DNA damages
First I want to show you a paper published in nature last week using pacbio sequencing to identifiy mutations in a kinase FLT3, which is associated with AML % of the aml patient would have this ITD mutation. This is an activating mutation and there are drugs can effectively inhibits the kinase. But the problem is that the drug develops resistance after certain time. And the drug resistance is likely caused by few mutations in the kinase domain. So to find out whether the ITD mutation and the drug resistance mutation are really the disease causing mutations, they have to determine whether any resistant mutations found were from the same strand as the FLT3-ITD.
**Projects at YCGA
YCGA PacBio RS

27. Comparisons Between PacBio RS and Illumina HiSeq
PacBio RS (Third generation)
Illumina HiSeq (Second generation)
Sequencing Chemistry
Sequencing by synthesis (SBS)
Single Molecule Real Time (SMRT)
Sequencing substrate
Smart Cell made up of
150,000 ZMWs
Flow cell has made of
8 separate lanes
Data output per day
1 to 2 billion/ day. $1.5/ Mb
60 billion/day at a cost of $.06 per Mb
Read Length
Average up to 5 Kb
50bp to 150bp
Error rates
Raw: %. With 30x coverage: Q50 (< 0.01)
0.5 to 1 %
Sample Library
SMRT Bell template
(Single-strand circular DNA) 250 bp to 10 Kb insert
dsDNA with adaptors (175 bp to 1 Kb)
As shown in this table. Bothe technologies are using the sequencing by synthesis chemistry. The difference for pacbio is that it performs the sequencing at the single molecule level and in real time. the sequencing comsumables are also different. In hiseq, the sequencing is carried on the flowcell, that has 8 lanes, each lane has millions of DNA clusters. For pacbio, the sequencing is carried on a SMRT cell, which is comprised of 150k microscopic holes called ZMWs which stands for Zero Mode Wavelenghth. Each Zmw is a can hold one dna molecule with primer and polymerase. For the base calling, illumina is using the images taken during the sequencing run. And pacbio is using the movies that is collected in real time while the dna synthesis is happening.

28. Upcoming Technologies

29. Oxford Nanopore Technology
Exonuclease
Protein nanopore (Alpha Hemolysin)
Cyclodextrin
Electrically resistant Lipid bilayer
Silicon nitride or graphene. This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the nanopore by setting a voltage across this membrane.   If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. Measurement of that current makes it possible to identify the molecule in question. For example, this system can be used to distinguish between the four standard DNA bases G, A, T and C, and also modified bases. It can be used to identify target proteins and small molecules, or to gain rich molecular information, for example to distinguish between the enantiomers of ibuprofen or study molecular binding dynamics.

30. PromethION

31. Recent advances in nanopore sequencing
Two types of nanopores: Protein and synthetic (silicon nitride). Protein nanopores appear to be better in recognizing nucleotides.
The rapid speed at which DNA strands pass through the tiny hole makes distinguishing bases more difficult.
Currently an enzyme is used to control the rate.
By shining low power green laser on synthetic nanopore immersed in salt water it is possible to manipulate DNA speed at will. As the current increases, positive ions drag water molecules in
Meller A. et al, Nat Biotech 2013
the opposite direction of incoming DNA, acting as a brake and slowing its passage through the pore. As a result, nanoscale sensors in the pore would be more accurately able to read each nucleotide going into the pore. Using nanopores, long stretches of DNA can be zipped back and forth through the pore and can be read several times
Protein nanopoers can also identify epigenetic changes.
The rapid speed at which DNA strands pass through the tiny holes makes distinguishing bases more difficult. They showed that shining a certain wavelength of light could slow the flow of DNA through synthetic nanopores, potentially making it easier to read the four bases that make up each molecule.
Reporting in the November 2013 issue of  Nature Nanotechnology, Dr. Meller's group found that by shining a low-power green laser on a synthetic nanopore made of a thin layer of silicon nitride, it was possible to increase the electric charge near the walls of the pore, which is immersed in salt water. As the current increases, positive ions drag water molecules in the opposite direction of incoming DNA, acting as a brake and slowing its passage through the pore. As a result, nanoscale sensors in the pore would be more accurately able to read each nucleotide going into the pore.

32. Performance/Limitations…..?
Advantages
Nanopores offer a label-free, electrical, single-molecule
DNA sequencing method
No costly fluorescent labeling reagents
No need for expensive optical hardware and sophisticated
instrumentation to detect DNA bases
Performance/Limitations…..?
First data was released in Feb Since then slow to release new data
Very little data available for the evaluation: High Error Rates - >5%

33. The YCGA Laboratory at West Campus

34. Located in a newly renovated building.
YCGA was established in January 2009 through generous funding support and the strong commitment from the Yale University and School of Medicine
Portion of the laboratory showing sequencing systems through the glass wall partition that separates laboratory from the rest of office and administrative area.
Located in a newly renovated building.
Approximately 7,000 Sq Ft laboratory and ~4,000 Sq Ft office
space
23 staff

35. Sequencing Platforms at YCGA
11 Illumina HiSeqs (2000 and 2500)
One MiSeq
Ion PGM™ Sequencer
One PacBio RS
YCGA is well equipped with cutting edge technologies . Since the technology keeps improving at a very fast pace, it has been a challenge to keep up with it. New technologies are expensive and some times we have to change the platform before we have recovered the investments. Despite numerous challenges YCGA has been very successful in keeping up with the change while maintaining data production and balancing operating budget..
YCGA has kept pace with cutting-edge sequencing technologies

36. Computer Infrastructure
BulldogN: Dell Cluster with 200 Nodes/2,500 Cores
Hitachi/BlueArc Scalable Storage: ~2.5 Petabytes

38. Types of samples processed and runs of sequence read lengths carried out at YCGA in a typical month

39. Need for strong R&D efforts for Next-Generation sequencing operation
Optimization of sample preparation protocols for exome capture that have decreased the cost of a single human exome from $8,000 in 2009 to the current price of ~$500, while improving the quality of the data.
Development of a highly efficient protocol to extract and repair DNA from formalin-fixed paraffin embedded blocks for exome analysis.
Improved protocols for gDNA-seq, RNA-seq, and ChIP-seq that show higher data complexity than traditional protocols, allow users to start with less material, and cost less.
Continuous improvements of various analysis pipelines
This point is extremely significant because >90% of our sequencing is human exomes. The improvements we have made have increased our data quality, decreased our costs, and allowed us to dramatically increase our throughput.
There are likely billions of formalin-fixed paraffin embedded (FFPE) samples around the world. The fixation/storage process destroys the DNA and it was thought these samples would be unusable for genetic analysis. Our protocol allows us to use these samples for exome analysis and makes many new and interesting experiments possible that would otherwise be impossible to perform.
By spending the time and money to improve all of our protocols – not just human exomes – we are able to offer the Yale community a variety of sample preparation options that produce the most complex data possible at some of the lowest costs in the country.

40. Whole- Genome VS. Whole Exome Sequencing
Protein coding genes (exome) constitute 1% of the human genome but harbor 85 % of disease causing mutations
Significantly cheaper than sequencing entire genome
Maq,
2.1M probes cover ~300,000
exons of 19,000 genes
Total covered bases: 44.1Mb

41. Scientific and economic impact of high throughput sequencing at Yale

42. Spatio-temporal transcriptome of the human brain. Kang and Sestan
List of select publications resulting form the next-generation sequencing at YCGA
Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations. Bilguvar
Nature, v467, 2010
A Novel miRNA Processing Pathway Independent of Dicer Requires Argonaute2 Activity. Cifuentes
Science, v328, 2010
Mitotic recombination in ichthyosis causes reversion of dominant mutations in KRT10. Choate K
Science, v330, 2010
Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds. Wang s.
Nature, v477, 2011
Transposom-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals. Lynch and Wagner
Nature, Genet. v43, 2011
K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension. Choi M
Science, v331, 2011
Recessive LAMC3 mutations cause malformations of occipital cortical development. Barak and Gunel.
Nat Genet., V43, 2011
Spatio-temporal transcriptome of the human brain. Kang and Sestan
Nature, v478, 2011
Langerhans cells facilitate epithelial DNA damage and squamous cell carcinoma. Modi and Girardi
Science, v335, 2012
Mutations in kelch-like 3 and cullin 3 causes hypertension and electrolyte abnormalities. Boyden et al
Nature, v482, 2012
De novo point mutations, revealed by whole-exome sequencing, are strongly associated with Autism Spectrum Disorders. Sanders and State
Nature, v485, 2012
Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Krauthammer
Nat Genet., V44, 2012
Genomic Analysis of Non-NF2 Meningiomas Reveals Mutations in TRAF7, KLF4, AKT1,& SMO. Clark V et al
Science, v339, 2013
De novo mutations in histone-modifying genes in congenital heart disease. Zaidi and Lifton
Nature, v498, 2013
Recessive mutations in DGKE cause atypical hemolytic-uremic syndrome. Lemaire and Lifton
Nat Genet., V45, 2013
Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism. Scholl and Lifton
The evolution of lineage-specific regulatory activities in the human embryonic limb. Cotney and Noonan
Cell, v154, 2013
Mutations in DSTYK and dominant urinary tract malformations. Sanna-Cherchi and Gharavi
N Eng J Med., 2013
Nanog, and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Lee et al
Nature, 2013
Co-expression networks implicate human mid-fetal deep cortical projection neurons in the pathogenesis of autism. Willsey and State
Cell, 2013
CLP1 Founder Mutation Links tRNA Splicing and Maturation to Cerebellar Development and Neurodegeneration. Schaffer AE and Gleeson JG.
Cell, V157, 2014
Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders. Novarino G and Gleeson JG.
Science, V363, 2014

43. Impact of High Throughput Sequencing: Grant Funding (partial list)
Mendelian center grant, NIH $12M (3y)
Gilead cancer grant $40M (4y)
Brain tumor gift $12M (4y)
ARRA brain development (NIH) $ 3M (2y)
ARRA kidney disease (NIH) $ 2M (2y)
Simons autism sequencing $ 4M (3y)
Brain transcriptome (NIH) $10M (2y)
Congenital heart disease (NIH) $ 5M (4y)
Pediatric Cardiac Genomic Consortium $ 2M (2Y)
Melanoma Spore (NIH) $12M (5y)
Biogen Inc. (PPMS) $ 2 M
VA- Schizophrenia/Bipolar disorder $12 M
Yale Comprehensive Cancer Center $14 M
Total $ 128 M

44. NGS and Personalized Medicine
Use of genomics to tailor medical care to individuals based on their genetic makeup.
Which treatment?
What are my
chances?
Which class of
cancer?
Is it benign?
Therapeutic
Choice
Prognosis
Diagnosis
Classification
How and why
Discovery
Elucidation of mechanism of cause
Identification of cancer biomarkers
Therapeutic targets
The use of microarrays can tell us a lot about specific disease such as cancer.
They can help us to
(1) Diagnose the specific cancer in a quick and accurate way
(2) Classify the specific subtype of cancer to allow for the best treatment
(3) Allow the most accurate prognosis of recovery based on the genetics of the tumor
(4) Identify the specific treatment for each type of specific genetic profile. The arrays can be used to study the genetics behind how a certain type of cancer reacts to a specific treatment

45. CLIA: The New Paradigm in Molecular Diagnostics
Conventional molecular testing- gene by gene
Genomic testing using Exome analysis
YCGA is carrying out clinical diagnostic work in collaboration with Dr. Allen Bale
Over 1,000 exomes are analyzed for various disorders

46. Challenges Equipment, reagents, protocols, analysis
What is valid and what is significant?
Individual judgment versus consensus guidelines

47. Sequencing a genome is simple finding a cause of a disease is not
First clinical use of whole genome sequencing shows just how challenging it can be.
Study of fraternal twins with monogenic disorder
Genome was sequenced of fraternal twins diagnosed with a movement disorder
Sci Transl Med Jun 15;3(87):87re3. doi: /scitranslmed
Whole-genome sequencing for optimized patient management.
Bainbridge MN1, Wiszniewski W, Murdock DR, Friedman J, Gonzaga-Jauregui C, Newsham I, Reid JG, Fink JK, Morgan MB, Gingras MC, Muzny DM, Hoang LD, Yousaf S, Lupski JR, Gibbs RA.
Abstract
Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)-responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.
Genomes on prescription: Nature 2011
Bainbridge M, Sci Transl Med 2011

48. Acknowledgement Jim Noonan
Yale University, School of Medicine and west Campus
NHGRI: CMG
YCGA staff

50. Questions?

51. Data Analysis Overview
Primary Analysis
Secondary Analysis
Data Visualization

52. Primary and Secondary Analysis Overview
Analysis Type
Software
Outputs
Images/TIFF files
Sequencing
ICS/RTA
Base Calling
Intensities
Primary Analysis
ICS/RTA
Alignments and Variant Detection
Secondary Analysis

53. Cluster Generation: Amplification
Template hybridization and Initial Extension
Original template is washed away
Template
hybridization
Initial extension
Denaturation
3' extension OH OH
P P5
Grafted flowcell
Initials steps for the PE chemistry are the same as the Single Read chemistry.
single molecules bound to flow cell in a random pattern
> million single molecules hybridize to the lawn of primers

54. Cluster Generation: Amplification
Result: two copies of covalently bound single-stranded templates
Single-strand flips over to hybridize to adjacent oligos to form a bridge
Hybridized primer is extended by polymerases
Double-stranded bridge is denatured
2nd cycle
denaturation
1st cycle
extension
1st cycle
annealing
1st cycle
denaturation
2nd cycle
annealing
n=35
total
2nd cycle
extension
Amplification steps are also the same except that 28 cycles is recommended for any samples where the insert is greater than 200 bp. More cycles for samples with insets greater then 200 bp will cause the clusters to get too large after P5 resynthesis (see slide 6)

55. Cluster Generation: Linearization, Blocking and sequencing
Cluster Generation: Linearization, Blocking and sequencing primer hybridization
dsDNA bridges are denatured
complement strands are cleaved and washed away
sequencing
primer
P5 Linearization
Block with
ddNTPS
Denaturation and
Sequencing Primer
Hybridization
Cluster
Amplification
The first linearization step uses the Linearization 1 Enzyme instead of Periodate. The blocking step still uses ddNTPs but uses Blocking Enzyme 1 and 2 in the PE protocol instead of terminal transferase for the Single Read protocol. Read 1 primer hybridization uses Read 1 PE Sequencing Primer. Enzymatic cleavage, uracyl incorporation enzyme
Free 3’ ends are blocked to prevent unwanted DNA priming

56. Sequencing Resynthesis of P5 Strand (15Cycles) Sequencing First ReadOH
Sequencing
First Read
Denaturation and
De-Protection OH
Denaturation and
Hybridization
P7 Linearization OH
Sequencing
Second Read
Denaturation and
Hybridization
Block with
ddNTPs
The steps up to and including the first read sequencing are pretty much the same as for a single read. The first read sequencing is where the single read protocol would stop. For the PE protocol, it continues with deprotecting the P5 primer using deprotection enzyme. Resynthesis of the P5 strand occurs over 15 cycles. P7 linearization uses Linearization 2 Enzyme. Blocking again occurs with ddNTPS and Blocking enzyme 1 and 2. Sequencing read 2 uses Read 2 PE Sequencing Primer.