Our Approach
Rx Biosciences offers 454 sequencing using the Genome Sequencer FLX Instrument, powered by GS FLX Titanium and Standard series reagents. The high throughput sequencing technology features a ground breaking combination of quick, long readouts with exceptional precision in a high throughput manner.
Steps involved in 454 sequencing
- Preparation of a single-stranded template DNA library
- Emulsion-based amplification of individual clones of the library
- High throughput sequence data generation via sequencing-by-synthesis
- Data analysis using different bioinformatics tools, contig generation and assembly into long sequences
Wide variety of starting materials including genomic DNA, PCR products, BAC clones, and cDNA can be used for 454 FLX sequencing. Samples such as large genomic DNA and BAC clones are fragmented into small, 300 to 800 base-pair fragments where as for smaller templates, such as small non-coding RNA or PCR amplicons, fragmentation is not needed. Instead, short PCR products amplified using Genome Sequencer fusion primers are used for immobilization onto DNA capture beads.
300-800 base-pair fragments are blunted and ligated with 5’ and 3’ specific unique adapters which are used further in down stream purification, amplification, and sequencing steps. Single-stranded fragments with 5’ and 3’ specific adapters constitute the sample library and used for subsequent immobilization and sequencing steps.
The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads in such a way that each bead carries a unique single-stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture resulting in micro-reactors (vesicles) containing just one bead with one unique sample-library fragment.
Each unique sample library fragment is amplified within its own micro-reactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this result in a copy number of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads.
The clonally amplified fragments are enriched and loaded onto a PicoTiterPlate device for sequencing. The diameter of the PicoTiterPlate wells allows for only one bead per well. After addition of sequencing enzymes, the fluidics subsystem of the Genome Sequencer FLX Instrument flows individual nucleotides in a fixed order across the hundreds of thousands of wells containing one bead each. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera of the Genome Sequencer FLX Instrument.
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