Process Steps
Overview
gDNA
1. DNA Library Construction *
4.5 h
2. emPCR
3. Sequencing
8h
7.5 h
Data output
DNA Library Preparation
emPCR
Sequencing
 Prepare single-stranded DNA library with adapters
 sstDNA with adaptors
attached to bead
 Quality filtered
bases
 Ready for titration sequencing run**
 Clonally amplified sstDNA in
emulsion
 sstDNA ready to sequence
*One library provides enough DNA for thousands of sequencing runs.
** Only one titration is required for each sample.
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Process Steps
1. DNA library Construction Overview
gDNA
1. DNA Library Construction *
4.5 h
 Library is created from any dsDNA
 Genome fragmentation by nebulization
 Ligation of adapters A & B
gDNA
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2. emPCR
3. Sequencing
8h
7.5 h
Data output
 A/B fragments selected using streptavidin-biotin purification
 Denaturation to select for sstDNA library with A/B adaptors
 No cloning; no colony picking
sstDNA library
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Nebulization
Snap cap
Condenser tube
Nebulization shears double-stranded DNA into fragments ranging from 50 to 900 base pairs.
 High-pressure nitrogen gas is used to force the sample into small droplets of liquid which shears the DNA.
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Fragment Distribution
Post Nebulization
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AMPure bead purification used to remove small fragments (<250 bp)
Nebulized, purified sample run on Agilent 2100 DNA 1000 or 7500 LabChip
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Mean size between 400 bp and 800 bp
< 10% of material smaller than 300 bp
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End Repair Reaction
DNA ends are made blunt and phosphorylated.
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3’ overhanging ends are removed (exonuclease).
3’ recessed ends are extended (polymerase).
5’ phosphates are added (kinase).
Dr. Gary Kaiser, PHD
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DNA Ends Adapted
with Specific Sequences
Polished insert DNA
Left (A) and right (B) adaptor
oligonucleotides are ligated onto
the pool of nebulized polished
genomic DNA.
Ligase +
left and right adaptors
Left adaptor
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Right adaptor
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GS Adaptors
“A” adaptor
“A” adaptor
 44 bases long
 20 base PCR primer component
 20 base sequencing primer component
 4 base key
 “B” adaptor
“B” adaptor
 44 bases long
 20 base PCR primer component
 20 base sequencing primer component
 4 base key
 Biotin on 5’ end (green dot)
 Both adaptors are blunt on one end and recessed on the other to ensure only the blunt ends ligate to the polished
genomic fragments.
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Ligation Products
4 types of products are generated during
ligation.
 AB and BA products are equivalent (50%).
 Products are bound to streptavidincoated magnetic particles.
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AB fragments
AA fragments
BB fragments
BA fragments
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Library is Rendered Single-stranded
B
Isolate AB
fragments only.
A
Adapted fragments are purified on a solid support and single-stranded material is eluted as the final product.
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AB Strands Purified as the Final
Library
1. AB and BB strands bind to magnetic
particles.
2. Strands are filled.
AA products (no biotin) are washed away, BA and BB strands remain attached to magentic beads.
3. Non-biotinylated strands are
melted off and recovered.
(only the AB strand will be captured)
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Final Library Distribution
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Typical single-stranded profile on Agilent 2100 RNA Pico 6000 LabChip.
Average size is 400-800 bp.
Quantitate using Ribogreen Assay and dilute for emPCR
Titration of DNA fragments is suggested to optimize the input copy number for sequencing.
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Final Library Quantitation
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Avogadro’s number is 6.022 x 1023 (molecules/mole)
328.3 x 109 (grams/mole) is the ave. molecular weight of nucleotides
Sample Concentration obtained from:
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Agilent Bioanlayzer or via flourometry using a RiboGreen Assay
Average fragment length obtained from

Agilent Bioanalyzer ONLY
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Summary Genomic DNA library construction
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Nebulization – Shear DNA into appropriate size fragments
Small fragment removal – SPRI based removal of fragments smaller than 300 bp.
DNA end repair – Make ends of DNA blunt and phosphorylated.
Adaptor ligation – Add specific ends for amplification and sequencing.
Fragment immobilization – Bind fragment to solid support.
Nick repair – Strand displacement to make fragments double-stranded.
Single-strand DNA isolation (library) – Isolate sstDNA fragments.
Quantitation – Estimate the number of molecules recovered.
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Process Steps
2. Emulsion PCR
1. DNA Library Construction *
gDNA
4.5 h
Anneal sstDNA to an excess of 28 Emulsify DNA Capture beads and
µm DNA Capture beads
PCR reagents in water-in-oil
microreactors
sstDNA library
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2. emPCR
3. Sequencing
8h
7.5 h
Data
output
Clonal amplification occurs inside Break microreactors and
enrich for DNA- positive beads
microreactors
Clonally-amplified sstDNA attached to bead
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GS FLX Technology
Emulsion PCR
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
From DNA quantitation, calculate a single
DNA molecule to bead ratio for each
microreactor

Wash Capture Beads

Anneal one DNA molecule to each
Capture bead
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Add PCR reagents to DNA+Capture bead
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Transfer sample to oil tube
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Shake to emulsify
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GS FLX Technology
Emulsion Formation
Emulsion Oil and PCR mix containing Capture Beads are
mixed using a Qiagen Tissue lyser as a high speed shaker
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GS FLX Technology
Emulsion PCR
Emulsion oil – Before and After
 After emulsions are created, dispense into PCR tubes/plates
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GS FLX Technology
Emulsion PCR
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All samples processed in parallel
“B” primer is attached to capture bead.
“A” primer (in solution) is biotinylated.
Microreactors are amplified simultaneously.
Amplified products are driven to solid
support (Capture Bead).
Each capture bead will contain ~10 million
clonal copies.
DNA Capture Beads
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GS FLX Technology
Emulsion PCR
A) Anneal Single-stranded template to
DNA Capture Beads
B) Emulsify millions of beads in PCR
reagents to form water-in-oil
microreactors
• Microreactor contains complete
amplification mix
Before PCR
C) Thermocycle
D) Break Microreactors
After PCR
E) Enrich for DNA positive beads
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GS FLX Technology
Breaking the Emulsion
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Load Emulsion into Syringe
Pass Emulsion through Filter (beads are
retained)
Wash Beads using filter
Recover beads from filter
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GS FLX Technology
Enrichment
Bead with
Amplified DNA
Bead without
Amplified DNA
Purify Beads with Product

Beads with amplified DNA have the biotinylated ”A” primer

Beads with DNA product are extracted using streptavidin coated, magnetic Enrichment Beads

Approximately 10% of beads have bound product
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Magnet
Add Enrichment Beads
Enrichment Bead
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Process Steps
3a. Bead Deposition into PicoTiterPlate ™
gDNA
1. DNA Library Construction *
4.5 h
2. emPCR
3. Sequencing
8h
7.5 h
Data output
 Well diameter average for PicoTiterPlate is 44 µm
 A single clonally amplified sstDNA bead is deposited per
well.
 A layer of packing and enzyme beads are deposited
 Plate is loaded into instrument for sequencing
Amplified sstDNA library beads
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Packed PTP
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GS FLX Technology
Assembling the Bead Deposition Device
The PTP is placed into the bead deposition
device (BDD) bottom, a gasket is applied, the
BDD top is placed over top and clamped
securely in place.
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GS FLX Technology
Loading Gaskets for 70X75 PTP
~420K reads
~280K reads
~192K reads
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GS FLX Technology
Loading Gaskets for 25X75 PTP
~70K reads
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~48K reads
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GS FLX Technology
Bead Deposition Procedure
10 min
Enzyme (spin)
10 min
Pack + BIM + Recycled
Pol (spin)
Recover
supernatant
10 min
DNA (no spin)
5 min
Prewet with BB2 (spin)
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Each chamber is filled with DNA beads, packing beads and enzyme beads in 3 separate layers
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GS FLX Technology
Bead Deposition
Empty PicoTiterPlate ™
DNA beads are loaded into the wells
of the PTP.
DNA beads packed into wells
with surrounding beads and
sequencing enzymes.
*A well diameter of 44 µm allows for only 1 bead per well*
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Process Steps
3b. Sequencing
gDNA
1. DNA Library Construction *
4.5 h
DNA capture bead
containing millions of
copies of a single
clonal fragment
Amplified sstDNA library beads
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2. emPCR
3. Sequencing
8h
7.5 h
Data output
 4 nucleotides (TACG) flowed for >100 cycles
 Chemiluminescent signal generation
 Signal processing to determine base sequence
and quality score
 300 - 400,000 reads obtained in parallel on a
large format PicoTiterPlate
High Quality reads
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GS FLX Technology
Sequencing Instrument
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GS FLX Technology
Sequencing-by-synthesis
Simultaneous sequencing of the
entire genome in hundreds of
thousands of picoliter-size wells.
Pyrophosphate signal generation
upon complimentary nucleotide
incorporation — dark otherwise.
•Polymerase adds nucleotide (dATP)
•Pyrophosphate is released (PPi)
DNA capture bead containing
millions of copies of a single
clonal fragment
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•Sulfurylase creates ATP from PPi
•Luciferase hydrolyses ATP and uses
luciferin to make light
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Sequencing
GS
FLX TechnologyBy Synthesis
Sequencing-by-synthesis
Repeated dNTP flow sequence:
G
C
T
A
Process continues until user-defined
number of nucleotide flow cycles are
completed.
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A
A
T
C
G
G
C
A
T
G
C
T
A
A
A
A
G
T
C
A
T
T
A
G
A T T T G
A
T C
G
T C
A
T C
G
A G
T Anneal Primer
C C G T A C G C
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Software
Image-processing Overview
•
T
C
G
•
A
Raw data is processed from a series of individual
images.
Each well’s data is extracted, quantified, and
normalized.
T
•
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Read data is converted into flowgrams.
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Software
Signal Processing
Metric and image viewing software
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Signal output from a single well (flowgram)
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Software
Flowgrams and Base calling
Key sequence = TCAG for identifying wells and calibration
Flow of individual bases (TCAG) is 100 times.
T
A
C
G
TTCTGCGAA
Base flow
Signal strength
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Software
Flowgrams and Base calling
Signal strength is determined by homopolymer length.
4-mer
T
A
C
G
Flow Order
3-mer
2-mer
1-mer
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Process Steps
Data Output
Image capture
Image processing
Signal processing
GS Amplicon Variant Analyzier
(amplicons)
GS denovo Assembler
(denovo)
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GS runMapper
(re-sequencing)
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Process Steps
Overview of Kits
 Library Preparation
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GS DNA Library Preparation Kit
 Emulsion PCR (emPCR)
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GS emPCR Kits I, II, and/or III
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> 1 emPCR kit for 70X75 sequencing run
 Sequencing
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GSFLX Sequencing Kits and PicoTiterPlate Kit
 Data Analysis/Interpretation
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Signal Processing, Basecalling, Assembly and Mapping
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