GS FLX/Junior Titanium Technology

www.454.com

GS FLX/Junior Titanium Technology

GS – FLX and GS Junior

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

GS FLX/Junior TechnologyExample of a Library Sample Run on a Bioanalyzer High Sensitivity Chip

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


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


• 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.


• 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


DNA Capture Beads


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


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!

23

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

24

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.

25

Each chamber is filled with- DNA beads - sequencing beads- packing beads

GS FLX/Junior TechnologyLoad Beads into PicoTiterPlate

26

GS FLX/Junior TechnologyDepositing beads into the PicoTiterPlate

Load enzyme beadsLoad paking beads

DNA beads packed into wells with surrounding beads and sequencing

enzymes.

34 µm pitch, 29 µm diameter wells

GS FLX/Junior TechnologyDepositing beads into the PicoTiterPlate

29

GS FLX/Junior TechnologySequencing instrument

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.

34

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.

40www.roche-applied-science.com

GS FLX/Junior System FlexibilityMultiplex Identifiers (MIDs)

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

GS FLX/Junior Titanium Technology

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