GS FLX/Junior Titanium Technology

Transcription

1 GS FLX/Junior Titanium Technology

2

3 GS FLX and GS Junior

4 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..

5 Process Steps 1. DNA library Construction Overview gdna 1. DNA Library Construction * 5.5 h 2. empcr 3. Sequencing 8 h 9 h Data output gdna sstdna library

6 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

7 Fragment End Polishing, A tailing Bluntingoffrayedends+ A tailingforligationofadaptors Filling of 3 recessed ends Removal of 3 overhang ends A A A tailing

8 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 ng 1 1 Yes 1 step (integrated) Yes 12

9 Example of a Library Sample Run on a Bioanalyzer High Sensitivity Chip

10 Emulsion PCR gdna 1. DNA Library Construction * 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

11 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

12 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

13 Emulsion formation Emulsion oil and PCR mix containing capture beads are mixed using a high-speed shaker. 15

14 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

15 Emulsion PCR DNA Capture Beads

16 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

17 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

18 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

19 Process Steps 3. Sequencing gdna 1. DNA Library Construction * 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

20 Anneal Sequencing Primer Sequencing primer is annealed Excess primer is removed through a series of washes Beads are counted Beads are ready to run!

21 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

22 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

23 Load Beads into PicoTiterPlate Each chamber is filled with - DNA beads - sequencing beads - packing beads 25

24 Depositing beads into the PicoTiterPlate Load enzyme beads Load paking beads DNA beads packed into wells with surrounding beads and sequencing enzymes. 26

25 Depositing beads into the PicoTiterPlate 34 µm pitch, 29 µm diameter wells

26 Sequencing instrument 29

27 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

28 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.

29 Massive parallelization FLX Titanium Junior bases read length x ~ reads bases read length x ~ reads ~ 450 Million Bases / run ~ 40 Million Bases / run 34

30 What is the GS Junior System? GS Junior Complete GS Junior Sequencer GS Junior Monitor and Accessories GS Junior Computer and Accessories GS Junior Installation Kit

31 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.

32 GS FLX/Junior System Flexibility Multiplex Identifiers (MIDs) 40

33 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