454 Sequencing Technology | Rx Biosciences
Rx Biosciences offers advanced 454 sequencing technology using the Genome Sequencer FLX Instrument, powered by GS FLX Titanium and Standard series reagents. This 454 sequencing technology provides a groundbreaking combination of quick, long readouts with exceptional precision in a high throughput manner. Our 454 sequencing technology ensures fast, accurate, and high-throughput DNA sequencing to meet diverse research needs.
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
Each unique sample library fragment amplifies inside its own micro-reactor, which prevents competition or contamination from other sequences. Meanwhile, all fragments amplify in parallel, producing several million copies per bead. After amplification, the emulsion PCR breaks, but the amplified fragments stay attached to their specific beads.
Then, the process enriches the clonally amplified fragments, and technicians load them onto a PicoTiterPlate device for sequencing. Each well holds only one bead, ensuring isolated sequencing reactions.
After adding sequencing enzymes, the Genome Sequencer FLX Instrument flows individual nucleotides in a fixed order across hundreds of thousands of wells, each containing a single bead. When one or more nucleotides match the template strand, they bind and produce a chemiluminescent signal. Finally, the CCD camera of the Genome Sequencer FLX Instrument captures and records this signal.
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 amplifies inside its own micro-reactor, which prevents competing or contaminating sequences. The entire fragment collection amplifies in parallel. For each fragment, this creates several million copies per bead. After amplification, the emulsion PCR breaks, but the amplified fragments stay attached to their specific beads.
Next, the clonally amplified fragments are enriched and loaded onto a PicoTiterPlate device for sequencing. Each well in the PicoTiterPlate fits only one bead. After adding sequencing enzymes, the Genome Sequencer FLX Instrument’s fluidics system flows individual nucleotides in a fixed order across the hundreds of thousands of wells, each containing a single bead. When one or more nucleotides complement the template strand, they bind and create a chemiluminescent signal. The CCD camera of the Genome Sequencer FLX Instrument then records this signal.
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