Transcript
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..
8 h 10 h
3. Sequencing2. emPCR
4 h
1. DNA Library Construction *gDNA
Process Steps1. DNA library Construction Overview
sstDNAlibrarygDNA
Data output8 h 9 h
3. Sequencing
2. emPCR
5.5 h
1. DNA Library Construction *gDNA
• Nebulization shears double-stranded DNA into fragments ranging from 50 to 1000 base pairs
• High-pressure nitrogen gas is used to force the sample into small droplets of liquid which shears the DNA
GS FLX/Junior TechnologyNebulization
GS FLX/Junior TechnologyFragment End Polishing, A tailing
Filling of 3’ recessed ends
Removal of 3’ overhang ends
Blunting of frayed ends + A tailing for ligation of adaptors
AA
A tailing
12Number of Preps per Kit
YesAutomation Friendly
1 step(integrated)Library Quantification
YesOptional MID Adaptors Kit
1Columns Required
1Bioanalyzer Chips Required
500 ngDNA Input Requirement
7Protocol Steps
2 - 3 hoursProtocol Time
Rapid LibraryFeature
A
ALigase &
Adaptor
SPRI with
Sizing Solution
A
AT
T
TT
Quantify FAM
A
AT
T
GS FLX/Junior TechnologyRapid Library Adaptor
Clonally-amplified sstDNA attached to beadsstDNA library
Anneal sstDNA to an excess of DNA Capture beads
Emulsify DNA Capture beads and PCR reagents in water-in-oil microreactors
Break microreactorsand enrich for DNA-positive beads
Clonal amplification occurs inside microreactors
8 h 9 h
3. Sequencing2. emPCR
2.5 h
1. DNA Library Construction *gDNA Data output
GS FLX/Junior TechnologyEmulsion PCR
Annealing of single-stranded template to DNA capture beads
• from DNA quantitation:
calculate a DNA molecule to bead ratio
• Anneal:
one DNA moleculeone DNA moleculeto each Capture beadto each Capture bead
GS FLX/Junior TechnologyAnnealing of single-stranded to DNA capture beads
GS FLX/Junior TechnologyEmulsion PCR
• Add PCR reagents to DNA+Capture bead
• Transfer sample to tube or cup with oil
• Shake to emulsify
• 1 starting effective fragment per microreactor
• ~106 microreactors per ml
• All processed in parallel
• Microreactors contain complete amplification mix
,
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GS FLX/Junior TechnologyEmulsion formation
Emulsion oil and PCR mix containing capture
beads are mixed using a high-speed shaker.
GS FLX/Junior TechnologyEmulsion PCR
• Emulsion oil – Before and After for Small Volume Emulsions (SVE)
• After emulsions are created, dispense into PCR tubes/plates
Titanimu kits – 4x PCR plate
Junior - 1x PCR plate
GS FLX/Junior TechnologyEmulsion PCR
Before PCR After PCR
• All samples processed in parallel• “B” attached to capture bead• “A” primer is in solution• Microreactors are amplified simultaneously• Each capture bead will contain ~30 million clonal copies
GS FLX/Junior TechnologyBreaking the Emulsion
• SMALL VOLUME EMULSION BREAKING
• Load Emulsion into Syringe
• Pass Emulsion through Filter (beads are retained)
• Wash Beads using filterwith isopropanol
• Recover beads from filter
• LARGE VOLUME EMULSION BREAKING
• Large Volume Breaking kit will include:– Transfer pipette for aspirating emulsions from
plate– 50mL conical tube cap adaptors– Tubing shown in image
• Breaking apparatus is connected to a vacuum source supplied by the customer
• Emulsion is aspirated from plate using apparatus
• Plate is washed using isopropanol
• After collection samples undergo washes using centrifugation to complete breaking procedure
GS FLX/Junior TechnologyEnrichment (Titanium kits)
• Melt Solution added to create single stranded fragments bound to control beads• Biotinylated Enrichment primer is annealed to fragments on capture beads• Enrichment beads are added• Beads with DNA product are extracted using streptavidin coated, magnetic Enrichment
Beads
Approximately 10% of beads have bound product
+melt
solution
Process Steps3. Sequencing
Well diameter average for PicoTiterPlate is 29 µmA single clonally amplified sstDNAbead is deposited per well.Layers of packing, enzyme and PPiase Beads are depositedPlate is loaded into instrument for sequencing
Packed PTPAmplified sstDNA library beads
8 h 10 h
3. Sequencing2. emPCR
2.5 h
1. DNA Library Construction *gDNA Data output
GS FLX/Junior TechnologyAnneal Sequencing Primer
• Sequencing primer is annealed
• Excess primer is removed through a series of washes
• Beads are counted
Beads are ready to run!
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GS FLX/Junior TechnologyDepositing DNA beads into the PicoTiterPlate
Load beads into PicoTiterPlateTM
Titanium PicoTiter plate34 micron center to center29 micron diameter3,200,000 wells per 60 x 60mm
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GS FLX/Junior TechnologyAssembling the jig for bead deposition
The PTP is placed on the jig bottom, a gasket is applied, the jig top is placed over top and clamped securely in place.
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Each chamber is filled with- DNA beads - sequencing beads- packing beads
GS FLX/Junior TechnologyLoad Beads into PicoTiterPlate
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GS FLX/Junior TechnologyDepositing beads into the PicoTiterPlate
Load enzyme beadsLoad paking beads
DNA beads packed into wells with surrounding beads and sequencing
enzymes.
GS FLX/Junior TechnologySequencing-by-synthesis
Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells.
Pyrophosphate signal generation upon complementary 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
luciferin
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
GS FLX/Junior TechnologySequencing-by-synthesis
Simultaneous sequencing in hundreds of thousands of picoliter-size wells
Pyrophosphate signal generation upon complimentary nucleotide incorporation — dark otherwise.
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GS FLX/Junior TechnologyMassive parallelization
400 – 500 bases read lengthx
~ 1 000 000 reads
~ 450 Million Bases / run
FLX Titanium
400 – 500 bases read lengthx
~ 100 000 reads
~ 40 Million Bases / run
Junior
What is the GS Junior System?
5922160001
GS Junior Installation Kit
GS Junior Computer and Accessories
GS Junior Monitor and Accessories
GS Junior Sequencer
GS Junior Complete
Performance Summary
GS Junior System
Throughput > 35 million high-quality, filtered bases per run average
Read Length 400 bases average (GS FLX Titanium Series)
HQ Reads per Run 100,000 shotgun, 70,000 amplicon average
Accuracy Q20 read length of 400 bases (99% accuracy at 400 bases)
Run Time 10 hours sequencing, 2 hours data processing
Sample Input Purified gDNA, amplicons, cDNA, depending on application
Computing Linux-based OS on desktop PC, included. Point-and-click software
Physical Dimensions 40 cm high x 40 cm wide x 60 cm deep (size of a laser printer)
Robustness No complex optics or lasers; long-life reagents
*Per run specifications is for shotgun libraries, and can vary based on the organism and genomic content. Reference organism is E. coli.
Unique combination of read length & readsThe broadest applications portfolio
De Novo Sequencing Microorganisms (genome plasticity)Complex eukaryotic genomes (Plants, Animals)BACs, YACs, Fosmids, Viruses etc.Long and short paired-end sequencing available
ResequencingWhole GenomesDisease associated regionsStructural variations of the human genomeSomatic mutations (cancer research via amplicon sequencing)
Transcriptome AnalysisExpression profiling (e.g. SAGE-like, CAGE-like, GIS-PET)EST-sequencingFull length cDNA sequencing
Gene Regulation StudiesIdentification of transcription factor binding sites (ChIP-Sequencing)Identification and quantification of sncRNAs sequences
Epigenetic ChangesDNA-Methylation patterns
Metagenomes & Microbial DiversityShotgun sequencing of the metagenome16S amplicon sequencing
Ancient DNANeanderthals, Mammoths and many more
Over 1000 high-profile publications
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