GS FLX/Junior Titanium Technology
GS FLX/Junior Titanium Technology
GS FLX/Junior Titanium Technology
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<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Titanium</strong> <strong>Technology</strong>
<strong>GS</strong> – <strong>FLX</strong> and <strong>GS</strong> <strong>Junior</strong>
Process Steps<br />
Overview<br />
gDNA<br />
1. DNA Library Construction *<br />
4h<br />
2. emPCR<br />
3. Sequencing<br />
8 h 10 h<br />
Data output<br />
DNA Library Preparation<br />
• Prepare single-stranded<br />
DNA library with adapters<br />
• Ready for titration<br />
sequencing run**<br />
emPCR<br />
• sstDNA with adaptors<br />
attached to bead<br />
• Clonally amplified<br />
sstDNA in emulsion<br />
• sstDNA ready to<br />
sequence<br />
Sequencing<br />
• Quality filtered<br />
bases<br />
*One library provides enough DNA for thousands of sequencing runs..
Process Steps<br />
1. DNA library Construction Overview<br />
gDNA<br />
1. DNA Library Construction *<br />
5.5 h<br />
2. emPCR<br />
3.<br />
Sequencing<br />
8 h 9 h<br />
Data output<br />
gDNA<br />
sstDNA<br />
library
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Nebulization<br />
• Nebulization shears double-stranded DNA into fragments<br />
ranging from 50 to 1000 base pairs<br />
• High-pressure nitrogen gas is used to force the sample<br />
into small droplets of liquid which shears the DNA
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Fragment End Polishing, A tailing<br />
Bluntingoffrayedends+ A tailingforligationofadaptors<br />
Filling of 3’ recessed ends<br />
Removal of 3’ overhang ends<br />
A<br />
A<br />
A tailing
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Rapid Library Adaptor<br />
Quantify FAM<br />
T<br />
A<br />
A<br />
T<br />
A<br />
A<br />
Ligase &<br />
Adaptor<br />
SPRI with<br />
Sizing Solution<br />
T<br />
A<br />
A<br />
T<br />
T<br />
T<br />
Feature<br />
Protocol Time<br />
Protocol Steps<br />
DNA Input Requirement<br />
Bioanalyzer Chips Required<br />
Columns Required<br />
Optional MID Adaptors Kit<br />
Library Quantification<br />
Automation Friendly<br />
Number of Preps per Kit<br />
Rapid Library<br />
2 - 3 hours<br />
7<br />
500 ng<br />
1<br />
1<br />
Yes<br />
1 step<br />
(integrated)<br />
Yes<br />
12
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Example of a Library Sample Run on a Bioanalyzer High<br />
Sensitivity Chip
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Emulsion PCR<br />
gDNA<br />
1. DNA Library Construction *<br />
2.5 h<br />
2. emPCR<br />
8 h 9 h<br />
3. Sequencing<br />
Data output<br />
Anneal sstDNA to an<br />
excess of DNA<br />
Capture beads<br />
Emulsify DNA Capture<br />
beads and PCR<br />
reagents in water-in-oil<br />
microreactors<br />
Clonal amplification<br />
occurs inside<br />
microreactors<br />
Break microreactors<br />
and enrich for DNApositive<br />
beads<br />
sstDNA library<br />
Clonally-amplified sstDNA attached to bead
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Annealing of single-stranded to DNA capture beads<br />
• from DNA quantitation:<br />
calculate a DNA molecule to bead<br />
ratio<br />
• Anneal:<br />
one DNA molecule<br />
to each Capture bead<br />
Annealing of single-stranded<br />
template to DNA capture beads
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Emulsion PCR<br />
• Add PCR reagents to<br />
DNA+Capture bead<br />
,<br />
• Transfer sample to tube or<br />
cup with oil<br />
• Shake to emulsify<br />
• 1 starting effective fragment<br />
per microreactor<br />
• ~10 6 microreactors per ml<br />
• All processed in parallel<br />
• Microreactors contain<br />
complete amplification mix
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Emulsion formation<br />
Emulsion oil and PCR mix containing capture<br />
beads are mixed using a high-speed shaker.<br />
15
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Emulsion PCR<br />
• Emulsion oil – Before and After for<br />
Small Volume Emulsions (SVE)<br />
• After emulsions are created, dispense<br />
into PCR tubes/plates<br />
Titanimu kits – 4x PCR plate<br />
<strong>Junior</strong> - 1x PCR plate
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Emulsion PCR<br />
DNA Capture Beads
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Emulsion PCR<br />
Before PCR<br />
After PCR<br />
• All samples processed in parallel<br />
• “B” attached to capture bead<br />
• “A” primer is in solution<br />
• Microreactors are amplified simultaneously<br />
• Each capture bead will contain ~30 million clonal copies
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Breaking the Emulsion<br />
• LARGE VOLUME EMULSION BREAKING<br />
• Large Volume Breaking kit will include:<br />
– Transfer pipette for aspirating emulsions from<br />
plate<br />
– 50mL conical tube cap adaptors<br />
– Tubing shown in image<br />
• Breaking apparatus is connected to a vacuum<br />
source supplied by the customer<br />
• SMALL VOLUME EMULSION<br />
BREAKING<br />
• Load Emulsion into Syringe<br />
• Pass Emulsion through Filter (beads are<br />
retained)<br />
• Wash Beads using filterwith isopropanol<br />
• Recover beads from filter<br />
• Emulsion is aspirated from plate using apparatus<br />
• Plate is washed using isopropanol<br />
• After collection samples undergo washes using<br />
centrifugation to complete breaking procedure
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Enrichment (<strong>Titanium</strong> kits)<br />
melt<br />
solution<br />
+<br />
• Melt Solution added to create single stranded fragments bound to control beads<br />
• Biotinylated Enrichment primer is annealed to fragments on capture beads<br />
• Enrichment beads are added<br />
• Beads with DNA product are extracted using streptavidin coated, magnetic Enrichment<br />
Beads<br />
Approximately 10% of beads have bound product
Process Steps<br />
3. Sequencing<br />
gDNA<br />
1. DNA Library Construction *<br />
2.5 h<br />
2. emPCR<br />
3. Sequencing<br />
8 h 10 h<br />
Data output<br />
• Well diameter average for<br />
PicoTiterPlate is 29 µm<br />
• A single clonally amplified sstDNA<br />
bead is deposited per well.<br />
• Layers of packing, enzyme and PPiase<br />
Beads are deposited<br />
• Plate is loaded into instrument for<br />
sequencing<br />
Amplified sstDNA library beads<br />
Packed PTP
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Anneal Sequencing Primer<br />
• Sequencing primer is annealed<br />
• Excess primer is removed through a series of washes<br />
• Beads are counted<br />
Beads are ready to run!
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Depositing DNA beads into the PicoTiterPlate<br />
Load beads into<br />
PicoTiterPlate TM<br />
<strong>Titanium</strong> PicoTiter plate<br />
• 34 micron center to center<br />
• 29 micron diameter<br />
• 3,200,000 wells per 60 x 60mm<br />
23
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Assembling the jig for bead deposition<br />
The PTP is placed on the jig<br />
bottom, a gasket is applied, the<br />
jig top is placed over top and<br />
clamped securely in place.<br />
24
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Load Beads into PicoTiterPlate<br />
Each chamber is filled with<br />
- DNA beads<br />
- sequencing beads<br />
- packing beads<br />
25
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Depositing beads into the PicoTiterPlate<br />
Load enzyme beads<br />
Load paking beads<br />
DNA beads packed into<br />
wells with surrounding<br />
beads and sequencing<br />
enzymes.<br />
26
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Depositing beads into the PicoTiterPlate<br />
34 µm pitch, 29 µm diameter wells
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Sequencing instrument<br />
29
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Sequencing-by-synthesis<br />
Simultaneous sequencing of the entire<br />
genome in hundreds of thousands of<br />
picoliter-size wells.<br />
Pyrophosphate signal generation upon<br />
complementary nucleotide<br />
incorporation — dark otherwise.<br />
•Polymerase adds nucleotide (dATP)<br />
•Pyrophosphate is released (PPi)<br />
luciferin<br />
•Sulfurylase creates ATP from PPi<br />
•Luciferase hydrolyses ATP and<br />
uses luciferin to make light
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Sequencing-by-synthesis<br />
Repeated dNTP flow sequence:<br />
G<br />
T<br />
C<br />
A<br />
A A T C G G C A T G C T A A A A G T C A<br />
T T A G C C G T A C G CA T T T AGT GTC TCA CA<br />
G TG<br />
Anneal Primer<br />
Simultaneous sequencing in hundreds of thousands of picoliter-size wells<br />
Pyrophosphate signal generation upon complimentary nucleotide incorporation<br />
— dark otherwise.
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> <strong>Technology</strong><br />
Massive parallelization<br />
<strong>FLX</strong> <strong>Titanium</strong><br />
<strong>Junior</strong><br />
400 – 500 bases read length<br />
x<br />
~ 1 000 000 reads<br />
400 – 500 bases read length<br />
x<br />
~ 100 000 reads<br />
~ 450 Million Bases / run<br />
~ 40 Million Bases / run<br />
34
What is the <strong>GS</strong> <strong>Junior</strong> System?<br />
5922160001<br />
<strong>GS</strong> <strong>Junior</strong> Complete<br />
<strong>GS</strong> <strong>Junior</strong> Sequencer<br />
<strong>GS</strong> <strong>Junior</strong> Monitor and Accessories<br />
<strong>GS</strong> <strong>Junior</strong> Computer and Accessories<br />
<strong>GS</strong> <strong>Junior</strong> Installation Kit
Performance Summary<br />
<strong>GS</strong> <strong>Junior</strong> System<br />
Throughput<br />
Read Length<br />
HQ Reads per Run<br />
Accuracy<br />
Run Time<br />
Sample Input<br />
Computing<br />
Physical Dimensions<br />
Robustness<br />
> 35 million high-quality, filtered bases per run average<br />
400 bases average (<strong>GS</strong> <strong>FLX</strong> <strong>Titanium</strong> Series)<br />
100,000 shotgun, 70,000 amplicon average<br />
Q20 read length of 400 bases (99% accuracy at 400 bases)<br />
10 hours sequencing, 2 hours data processing<br />
Purified gDNA, amplicons, cDNA, depending on application<br />
Linux-based OS on desktop PC, included. Point-and-click<br />
software<br />
40 cm high x 40 cm wide x 60 cm deep (size of a laser printer)<br />
No complex optics or lasers; long-life reagents<br />
*Per run specifications is for shotgun libraries, and can vary based on the organism and genomic<br />
content. Reference organism is E. coli.
<strong>GS</strong> <strong>FLX</strong>/<strong>Junior</strong> System Flexibility<br />
Multiplex Identifiers (MIDs)<br />
www.roche-applied-science.com<br />
40
Unique combination of read length & reads<br />
The broadest applications portfolio<br />
De Novo Sequencing<br />
• Microorganisms (genome plasticity)<br />
• Complex eukaryotic genomes (Plants, Animals)<br />
• BACs, YACs, Fosmids, Viruses etc.<br />
• Long and short paired-end sequencing available<br />
Resequencing<br />
• Whole Genomes<br />
• Disease associated regions<br />
•Structural variations of the human genome<br />
• Somatic mutations (cancer research via amplicon sequencing)<br />
Transcriptome Analysis<br />
• Expression profiling (e.g. SAGE-like, CAGE-like, GIS-PET)<br />
• EST-sequencing<br />
• Full length cDNA sequencing<br />
Gene Regulation Studies<br />
• Identification of transcription factor binding sites (ChIP-Sequencing)<br />
• Identification and quantification of sncRNAs sequences<br />
Epigenetic Changes<br />
• DNA-Methylation patterns<br />
Metagenomes & Microbial Diversity<br />
• Shotgun sequencing of the metagenome<br />
• 16S amplicon sequencing<br />
Ancient DNA<br />
• Neanderthals, Mammoths and many more<br />
Over 1000 high-profile publications