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Process Steps Overview

3. Sequencing. 2. emPCR. 1. DNA Library Construction *. 4.5 h. 8 h. 7.5 h. emPCR sstDNA with adaptors attached to bead Clonally amplified sstDNA in emulsion sstDNA ready to sequence. Sequencing Quality filtered bases. Process Steps Overview. gDNA. Data output.

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Process Steps Overview

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  1. 3. Sequencing 2. emPCR 1. DNA Library Construction * 4.5 h 8 h 7.5 h • emPCR • sstDNA with adaptors attached to bead • Clonally amplified sstDNA in emulsion • sstDNA ready to sequence • Sequencing • Quality filtered bases Process StepsOverview gDNA Data output DNA Library Preparation • Prepare single-stranded DNA library with adapters • Ready for titration sequencing run** *One library provides enough DNA for thousands of sequencing runs. ** Only one titration is required for each sample.

  2. 3. Sequencing 2. emPCR 1. DNA Library Construction * 4.5 h 8 h 7.5 h Process Steps1. DNA library Construction Overview gDNA Data output • Library is created from any dsDNA • Genome fragmentation by nebulization • Ligation of adapters A & B • A/B fragments selected using streptavidin-biotin purification • Denaturation to select for sstDNA library with A/B adaptors • No cloning; no colony picking gDNA sstDNA library

  3. Nebulization Snap cap Condenser tube • Nebulization shears double-stranded DNA into fragments ranging from 50 to 900 base pairs. • High-pressure nitrogen gas is used to force the sample into small droplets of liquid which shears the DNA.

  4. Fragment Distribution Post Nebulization • AMPure bead purification used to remove small fragments (<250 bp) • Nebulized, purified sample run on Agilent 2100 DNA 1000 or 7500 LabChip • Mean size between 400 bp and 800 bp • < 10% of material smaller than 300 bp

  5. End Repair Reaction DNA ends are made blunt and phosphorylated. • 3’ overhanging ends are removed (exonuclease). • 3’ recessed ends are extended (polymerase). • 5’ phosphates are added (kinase). Dr. Gary Kaiser, PHD

  6. DNA Ends Adapted with Specific Sequences Left (A) and right (B) adaptor oligonucleotides are ligated onto the pool of nebulized polished genomic DNA. Polished insert DNA Ligase + left and right adaptors Right adaptor Left adaptor

  7. GS Adaptors • “A” adaptor • 44 bases long • 20 base PCR primer component • 20 base sequencing primer component • 4 base key • “B” adaptor • 44 bases long • 20 base PCR primer component • 20 base sequencing primer component • 4 base key • Biotin on 5’ end (green dot) • Both adaptors are blunt on one end and recessed on the other to ensure only the blunt ends ligate to the polished genomic fragments. “A” adaptor “B” adaptor

  8. Ligation Products • 4 types of products are generated during ligation. • AB and BA products are equivalent (50%). • Products are bound to streptavidin-coated magnetic particles. • AB fragments • AA fragments • BB fragments • BA fragments

  9. Library is Rendered Single-stranded Adapted fragments are purified on a solid support and single-stranded material is eluted as the final product. B Isolate AB fragments only. A

  10. AB Strands Purified as the Final Library 2. Strands are filled. 1. AB and BB strands bind to magnetic particles. 3. Non-biotinylated strands are melted off and recovered. (only the AB strand will be captured) AA products (no biotin) are washed away, BA and BB strands remain attached to magentic beads.

  11. Final Library Distribution • Typical single-stranded profile on Agilent 2100 RNA Pico 6000 LabChip. • Average size is 400-800 bp. • Quantitate using Ribogreen Assay and dilute for emPCR • Titration of DNA fragments is suggested to optimize the input copy number for sequencing.

  12. Final Library Quantitation • Avogadro’s number is 6.022 x 1023 (molecules/mole) • 328.3 x 109 (grams/mole) is the ave. molecular weight of nucleotides • Sample Concentration obtained from: • Agilent Bioanlayzer or via flourometry using a RiboGreen Assay • Average fragment length obtained from • Agilent Bioanalyzer ONLY

  13. Summary Genomic DNA library construction • Nebulization – Shear DNA into appropriate size fragments • Small fragment removal – SPRI based removal of fragments smaller than 300 bp. • DNA end repair – Make ends of DNA blunt and phosphorylated. • Adaptor ligation – Add specific ends for amplification and sequencing. • Fragment immobilization – Bind fragment to solid support. • Nick repair – Strand displacement to make fragments double-stranded. • Single-strand DNA isolation (library) – Isolate sstDNA fragments. • Quantitation – Estimate the number of molecules recovered.

  14. 3. Sequencing 2. emPCR 1. DNA Library Construction * 4.5 h 8 h 7.5 h Break microreactors and enrich for DNA- positive beads Anneal sstDNA to an excess of 28 µmDNA Capture beads Emulsify DNA Capture beads and PCR reagents in water-in-oil microreactors Clonal amplification occurs inside microreactors sstDNA library Clonally-amplified sstDNA attached to bead Process Steps2. Emulsion PCR Data output gDNA

  15. From DNA quantitation, calculate a single DNA molecule to bead ratio for each microreactor Wash Capture Beads Anneal one DNA molecule to each Capture bead Add PCR reagents to DNA+Capture bead Transfer sample to oil tube Shake to emulsify GS FLX TechnologyEmulsionPCR

  16. GS FLX TechnologyEmulsion Formation Emulsion Oil and PCR mix containing Capture Beads are mixed using a Qiagen Tissue lyser as a high speed shaker

  17. GS FLX TechnologyEmulsion PCR • Emulsion oil – Before and After • After emulsions are created, dispense into PCR tubes/plates

  18. GS FLX TechnologyEmulsion PCR • All samples processed in parallel • “B” primer is attached to capture bead. • “A” primer (in solution) is biotinylated. • Microreactors are amplified simultaneously. • Amplified products are driven to solid support (Capture Bead). • Each capture bead will contain ~10 million clonal copies. DNA Capture Beads

  19. GS FLX TechnologyEmulsion PCR • Anneal Single-stranded template to DNA Capture Beads • Emulsify millions of beads in PCR reagents to form water-in-oil microreactors • Microreactor contains complete amplification mix • Thermocycle • Break Microreactors • Enrich for DNA positive beads Before PCR After PCR

  20. GS FLX TechnologyBreaking the Emulsion • Load Emulsion into Syringe • Pass Emulsion through Filter (beads are retained) • Wash Beads using filter • Recover beads from filter

  21. GS FLX TechnologyEnrichment Bead with Amplified DNA Bead without Amplified DNA Enrichment Bead • Beads with amplified DNA have the biotinylated ”A” primer • Beads with DNA product are extracted using streptavidin coated, magnetic Enrichment Beads • Approximately 10% of beads have bound product Magnet Add Enrichment Beads Purify Beads with Product

  22. gDNA 3. Sequencing 2. emPCR 1. DNA Library Construction * 4.5 h 8 h 7.5 h Process Steps3a. Bead Deposition into PicoTiterPlate ™ Data output • Well diameter average for PicoTiterPlate is 44 µm • A single clonally amplified sstDNA bead is deposited per well. • A layer of packing and enzyme beads are deposited • Plate is loaded into instrument for sequencing Amplified sstDNA library beads Packed PTP

  23. GS FLX TechnologyAssembling the Bead Deposition Device The PTP is placed into the bead deposition device (BDD) bottom, a gasket is applied, the BDD top is placed over top and clamped securely in place.

  24. GS FLX TechnologyLoading Gaskets for 70X75 PTP ~420K reads ~280K reads ~192K reads

  25. GS FLX TechnologyLoading Gaskets for 25X75 PTP ~48K reads ~70K reads

  26. 10 min Pack + BIM + Recycled Pol (spin) Enzyme (spin) 10 min Recover supernatant 10 min DNA (no spin) 5 min Prewet with BB2 (spin) GS FLX TechnologyBead Deposition Procedure • Each chamber is filled with DNA beads, packing beads and enzyme beads in 3 separate layers

  27. GS FLX TechnologyBead Deposition Empty PicoTiterPlate ™ DNA beads are loaded into the wells of the PTP. DNA beads packed into wells with surrounding beads and sequencing enzymes. *A well diameter of 44 µm allows for only 1 bead per well*

  28. gDNA 3. Sequencing 2. emPCR 1. DNA Library Construction * 4.5 h 8 h 7.5 h Process Steps3b. Sequencing Data output DNA capture bead containing millions of copies of a single clonal fragment • 4 nucleotides (TACG) flowed for >100 cycles • Chemiluminescent signal generation • Signal processing to determine base sequence and quality score • 300 - 400,000 reads obtained in parallel on a large format PicoTiterPlate Amplified sstDNA library beads High Quality reads

  29. GS FLX TechnologySequencing Instrument

  30. GS FLX TechnologySequencing-by-synthesis • Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells. • Pyrophosphate signal generation upon complimentary 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 DNA capture bead containing millions of copies of a single clonal fragment

  31. Sequencing By Synthesis GS FLX TechnologySequencing-by-synthesis Repeated dNTP flow sequence: G T C A Process continues until user-defined number of nucleotide flow cycles are completed. 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 C A T T T T G A G T C T C A G A C G T Anneal Primer

  32. T C G A T SoftwareImage-processing Overview • Raw data is processed from a series of individual images. • Each well’s data is extracted, quantified, and normalized. • Read data is converted into flowgrams.

  33. Signal output from a single well (flowgram) Metric and image viewing software SoftwareSignal Processing

  34. TACG TTCTGCGAA Base flow Signal strength SoftwareFlowgrams and Base calling Key sequence = TCAG for identifying wells and calibration Flow of individual bases (TCAG) is 100 times.

  35. Flow Order TACG 4-mer 3-mer 2-mer 1-mer SoftwareFlowgrams and Base calling Signal strength is determined by homopolymer length.

  36. Image processing Signal processing GS Amplicon Variant Analyzier (amplicons) GS denovo Assembler (denovo) GS runMapper (re-sequencing) Process StepsData Output Image capture

  37. Process StepsOverview of Kits • Library Preparation • GS DNA Library Preparation Kit • Emulsion PCR (emPCR) • GS emPCR Kits I, II, and/or III • > 1 emPCR kit for 70X75 sequencing run • Sequencing • GSFLX Sequencing Kits and PicoTiterPlate Kit • Data Analysis/Interpretation • Signal Processing, Basecalling, Assembly and Mapping

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