Post on 13-Jan-2016
description
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Process StepsOverview
Data output
DNA Library Preparation Prepare single-stranded DNA
library with adapters
Ready for titration sequencing run**
Sequencing
Quality filtered bases
emPCR
sstDNA with adaptors attached to bead
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.
8 h 7.5 h
3. Sequencing
2. emPCR
4.5 h
1. DNA Library Construction *gDNA
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Process Steps1. DNA library Construction Overview
sstDNA library
gDNA
A/B fragments selected using streptavidin-biotin purification
Denaturation to select for sstDNA library with A/B adaptors
No cloning; no colony picking
Library is created from any dsDNA
Genome fragmentation by nebulization
Ligation of adapters A & B
Data output8 h 7.5 h
3. Sequencing
2. emPCR
4.5 h
1. DNA Library Construction *gDNA
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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.
Nebulization
Snap cap
Condenser tube
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Fragment Distribution Post Nebulization
AMPure bead purification used to remove small fragments (<250 bp) Nebulized, purified sample run on Agilent 2100 DNA 1000 or 7500
LabChip Mean size between 400 bp and 800 bp < 10% of material smaller than 300 bp
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End Repair Reaction
Dr. Gary Kaiser, PHD
DNA ends are made blunt and phosphorylated. 3’ overhanging ends are removed (exonuclease). 3’ recessed ends are extended (polymerase). 5’ phosphates are added (kinase).
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DNA Ends Adapted with Specific Sequences
Ligase + left and right adaptors
Left adaptor Right adaptor
Polished insert DNA Left (A) and right (B) adaptor oligonucleotides are ligated onto the pool of nebulized polished genomic DNA.
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“A” adaptor 44 bases long 20 base PCR primer component 20 base sequencing primer component 4 base key
“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.
“A” adaptor
“B” adaptor
GS Adaptors
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AB fragments
AA fragments
BB fragments
BA fragments
4 types of products are generated during ligation.
AB and BA products are equivalent (50%).
Products are bound to streptavidin-coated magnetic particles.
Ligation Products
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Adapted fragments are purified on a solid support and single-stranded material is eluted as the final product.
A
B
Isolate AB fragments only.
Library is Rendered Single-stranded
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3. Non-biotinylated
strands are melted
off and recovered.
(only the AB strand
will be captured)
1. AB and BB strands bind to magnetic particles.
2. Strands are
filled.
AB Strands Purified as the Final Library
AA products (no biotin) are washed away, BA and BB strands remain attached to magentic beads.
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Final Library Distribution
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
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:
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
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 Steps2. Emulsion PCR
Clonally-amplified sstDNA attached to bead
sstDNA library
Anneal sstDNA to an excess of 28 µm DNA Capture beads
Emulsify DNA Capture beads and PCR reagents in water-in-oil microreactors
Break microreactors and enrich for DNA- positive beads
Clonal amplification occurs inside microreactors
8 h 7.5 h
3. Sequencing
2. emPCR
4.5 h
1. DNA Library Construction *
gDNAData output
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GS FLX Technology Emulsion PCR
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
Add PCR reagents to DNA+Capture bead
Transfer sample to oil tube
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
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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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
C) Thermocycle
D) Break Microreactors
E) Enrich for DNA positive beads
GS FLX Technology
Emulsion PCR
Before PCR
After PCR
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GS FLX Technology Breaking the Emulsion
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
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
Add Enrichment Beads
Ma
gn
et
Bead with Amplified DNA
Bead without Amplified DNA
Enrichment Bead
Purify Beads with Product
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Process Steps3a. Bead Deposition into PicoTiterPlate ™
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
Packed PTPAmplified sstDNA library beads
8 h 7.5 h
3. Sequencing
2. emPCR
4.5 h
1. DNA Library Construction *gDNA Data
output
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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 ~48K reads
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GS FLX Technology Bead Deposition Procedure
Each chamber is filled with DNA beads, packing beads and enzyme
beads in 3 separate layers
Prewet with BB2 (spin)
DNA (no spin)
Pack + BIM + Recycled Pol (spin)
Enzyme (spin)10 min
10 min
10 min
5 min
Recover
supernatant
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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 Steps3b. Sequencing
High Quality readsAmplified sstDNA library beads
DNA capture bead containing millions of copies of a single clonal fragment
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
8 h 7.5 h
3. Sequencing
2. emPCR
4.5 h
1. DNA Library Construction *gDNA Data
output
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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)
•Sulfurylase creates ATP from PPi
•Luciferase hydrolyses ATP and uses luciferin to make light
DNA capture bead containing millions of copies of a single clonal fragment
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Sequencing By Synthesis
A A T C G G C A T G C T A A A A G T C A
C TA
Repeated dNTP flow sequence:
GGTCAGTCAGTTTTCAG GAT CCCGATT
G CT A
Anneal Primer
Process continues until user-defined number of nucleotide flow cycles are completed.
GS FLX Technology Sequencing-by-synthesis
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SoftwareImage-processing Overview
• Raw data is processed from a series of individual images.
• Each well’s data is extracted, quantified, and normalized.
• Read data is converted into flowgrams.
T
AG
CT
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SoftwareSignal Processing
Metric and image viewing software
Signal output from a single well (flowgram)
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SoftwareFlowgrams and Base calling
Key sequence = TCAG for identifying wells and calibration Flow of individual bases (TCAG) is 100 times.
TTCTGCGAA
TACG
Base flowSignal strength
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SoftwareFlowgrams and Base calling
Signal strength is determined by homopolymer length.
Flow Order
1-mer
2-mer
3-mer
4-mer
TACG
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Image capture
Image processing
Signal processing
GS denovo Assembler (denovo)
GS runMapper
(re-sequencing)
Process StepsData Output
GS Amplicon Variant Analyzier
(amplicons)
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Process StepsOverview of KitsLibrary Preparation
GS DNA Library Preparation Kit
Emulsion PCR (emPCR) GS emPCR Kits I, II, and/or III
> 1 emPCR kit for 70X75 sequencing run
Sequencing GSFLX Sequencing Kits and PicoTiterPlate Kit
Data Analysis/Interpretation Signal Processing, Basecalling, Assembly and Mapping