GS FLX/Junior Titanium Technology

www.454.com 

GS FLX/Junior Titanium Technology

GS – FLX and GS Junior

Process Steps 

Overview 

gDNA 

1. DNA Library Construction * 

4h 

2. emPCR 

3. Sequencing 

8 h 10 h 

Data output 

DNA Library Preparation 

• Prepare single-stranded 

DNA library with adapters 

• Ready for titration 

sequencing run** 

emPCR 

• sstDNA with adaptors 

attached to bead 

• Clonally amplified 

sstDNA in emulsion 

• sstDNA ready to 

sequence 

Sequencing 

• Quality filtered 

bases 

*One library provides enough DNA for thousands of sequencing runs..

Process Steps 

1. DNA library Construction Overview 

gDNA 


5.5 h 

2. emPCR 

3. 

Sequencing 

8 h 9 h 

Data output 

gDNA 

sstDNA 

library

GS FLX/Junior Technology 

Nebulization 

• 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


Fragment End Polishing, A tailing 

Bluntingoffrayedends+ A tailingforligationofadaptors 

Filling of 3’ recessed ends 

Removal of 3’ overhang ends 

A 

A 

A tailing


Rapid Library Adaptor 

Quantify FAM 

T 

A 

A 

T 

A 

A 

Ligase & 

Adaptor 

SPRI with 

Sizing Solution 

T 

A 

A 

T 

T 

T 

Feature 

Protocol Time 

Protocol Steps 

DNA Input Requirement 

Bioanalyzer Chips Required 

Columns Required 

Optional MID Adaptors Kit 

Library Quantification 

Automation Friendly 

Number of Preps per Kit 

Rapid Library 

2 - 3 hours 

7 

500 ng 

1 

1 

Yes 

1 step 

(integrated) 

Yes 

12


Example of a Library Sample Run on a Bioanalyzer High 

Sensitivity Chip


Emulsion PCR 

gDNA 


2.5 h 

2. emPCR 

8 h 9 h 

3. Sequencing 

Data output 

Anneal sstDNA to an 

excess of DNA 

Capture beads 

Emulsify DNA Capture 

beads and PCR 

reagents in water-in-oil 

microreactors 

Clonal amplification 

occurs inside 

microreactors 

Break microreactors 

and enrich for DNApositive 

beads 

sstDNA library 

Clonally-amplified sstDNA attached to bead


Annealing of single-stranded to DNA capture beads 

• from DNA quantitation: 

calculate a DNA molecule to bead 

ratio 

• Anneal: 

one DNA molecule 

to each Capture bead 

Annealing of single-stranded 

template to DNA capture beads


Emulsion 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 

• ~10 6 microreactors per ml 

• All processed in parallel 

• Microreactors contain 

complete amplification mix


Emulsion formation 

Emulsion oil and PCR mix containing capture 

beads are mixed using a high-speed shaker. 

15


Emulsion 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


Emulsion PCR 

DNA Capture Beads


Emulsion 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


Breaking the Emulsion 

• 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 

• SMALL VOLUME EMULSION 

BREAKING 

• Load Emulsion into Syringe 

• Pass Emulsion through Filter (beads are 

retained) 

• Wash Beads using filterwith isopropanol 

• Recover beads from filter 

• Emulsion is aspirated from plate using apparatus 

• Plate is washed using isopropanol 

• After collection samples undergo washes using 

centrifugation to complete breaking procedure


Enrichment (Titanium kits) 

melt 

solution 

+ 

• 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

Process Steps 

3. Sequencing 

gDNA 


2.5 h 

2. emPCR 

3. Sequencing 

8 h 10 h 

Data output 

• Well diameter average for 

PicoTiterPlate is 29 µm 

• A single clonally amplified sstDNA 

bead is deposited per well. 

• Layers of packing, enzyme and PPiase 

Beads are deposited 

• Plate is loaded into instrument for 

sequencing 

Amplified sstDNA library beads 

Packed PTP


Anneal Sequencing Primer 

• Sequencing primer is annealed 

• Excess primer is removed through a series of washes 

• Beads are counted 

Beads are ready to run!


Depositing DNA beads into the PicoTiterPlate 

Load beads into 

PicoTiterPlate TM 

Titanium PicoTiter plate 

• 34 micron center to center 

• 29 micron diameter 

• 3,200,000 wells per 60 x 60mm 

23


Assembling 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. 

24


Load Beads into PicoTiterPlate 

Each chamber is filled with 

- DNA beads 

- sequencing beads 

- packing beads 

25


Depositing beads into the PicoTiterPlate 

Load enzyme beads 

Load paking beads 

DNA beads packed into 

wells with surrounding 

beads and sequencing 

enzymes. 

26


Depositing beads into the PicoTiterPlate 

34 µm pitch, 29 µm diameter wells


Sequencing instrument 

29


Sequencing-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) 

luciferin 

•Sulfurylase creates ATP from PPi 

•Luciferase hydrolyses ATP and 

uses luciferin to make light


Sequencing-by-synthesis 

Repeated dNTP flow sequence: 

G 

T 

C 

A 

A A T C G G C A T G C T A A A A G T C A 

T T A G C C G T A C G CA T T T AGT GTC TCA CA 

G TG 

Anneal Primer 

Simultaneous sequencing in hundreds of thousands of picoliter-size wells 

Pyrophosphate signal generation upon complimentary nucleotide incorporation 

— dark otherwise.


Massive parallelization 

FLX Titanium 

Junior 

400 – 500 bases read length 

x 

~ 1 000 000 reads 

400 – 500 bases read length 

x 

~ 100 000 reads 

~ 450 Million Bases / run 

~ 40 Million Bases / run 

34

What is the GS Junior System? 

5922160001 

GS Junior Complete 

GS Junior Sequencer 

GS Junior Monitor and Accessories 

GS Junior Computer and Accessories 

GS Junior Installation Kit

Performance Summary 

GS Junior System 

Throughput 

Read Length 

HQ Reads per Run 

Accuracy 

Run Time 

Sample Input 

Computing 

Physical Dimensions 

Robustness 

> 35 million high-quality, filtered bases per run average 

400 bases average (GS FLX Titanium Series) 

100,000 shotgun, 70,000 amplicon average 

Q20 read length of 400 bases (99% accuracy at 400 bases) 

10 hours sequencing, 2 hours data processing 

Purified gDNA, amplicons, cDNA, depending on application 

Linux-based OS on desktop PC, included. Point-and-click 

software 

40 cm high x 40 cm wide x 60 cm deep (size of a laser printer) 

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.

GS FLX/Junior System Flexibility 

Multiplex Identifiers (MIDs) 

www.roche-applied-science.com 

40

Unique combination of read length & reads 

The 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 

Resequencing 

• Whole Genomes 

• Disease associated regions 

•Structural variations of the human genome 

• Somatic mutations (cancer research via amplicon sequencing) 

Transcriptome Analysis 

• Expression profiling (e.g. SAGE-like, CAGE-like, GIS-PET) 

• EST-sequencing 

• Full length cDNA sequencing 

Gene Regulation Studies 

• Identification of transcription factor binding sites (ChIP-Sequencing) 

• Identification and quantification of sncRNAs sequences 

Epigenetic Changes 

• DNA-Methylation patterns 

Metagenomes & Microbial Diversity 

• Shotgun sequencing of the metagenome 

• 16S amplicon sequencing 

Ancient DNA 

• Neanderthals, Mammoths and many more 

Over 1000 high-profile publications

GS FLX/Junior Titanium Technology

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