Technology
System Workflow
One Fragment = One Bead = One Read
The complete sequencing workflow of the GS FLX and GS Junior Systems comprise of four main steps, leading from purified DNA to analyzed results. These basic steps include:
- Generation of a single-stranded template DNA library
- Emulsion-based clonal amplification of the library
- Data generation via sequencing-by-synthesis
- Data analysis using different bioinformatics tools
Watch the 454 Sequencing workflow animation
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Sample Input & Fragmentation
The GS FLX and GS Junior Systems support the sequencing of samples from a wide variety of starting materials, including genomic DNA, PCR products, BACs and cDNA. For shotgun, paired end or cDNA libraries, start with as little as 500 ng of sample DNA. Nebulize longer DNA samples to create shorter library fragments.
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Library Preparation
Ligate Rapid Library Adaptors to the fragments for use in subsequent purification, quantitation, amplification and sequencing steps. For amplicon libraries, create PCR products by amplifying with specific fusion primer containing 454 Sequencing adaptor sequences.
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One Fragment = One Bead
Attach library to DNA Capture Beads. Each bead carries a unique single-stranded library fragment. Emulsify beads with amplification reagents in a water-in-oil mixture to trap individual beads in amplification microreactors.
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emPCR: Emulsion PCR Amplification
Amplify the entire emulsion in parallel to create millions of clonally copies of each library fragment on each bead. Break the emulsion while the amplified fragments remain bound to their specific beads.
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Sequencing: One Bead = One Read
Load the beads onto the PicoTiterPlate device, where the surface design allows for only one bead per well. The PTP Device is then loaded in instrument for sequencing. Individual nucleotides are flowed in sequence across the wells. Each incorporation of a nucleotide complementary to the template strand results in a chemiluminescent light signal recorded by the camera.
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Pyrosequencing chemistry
Pyrosequencing reaction of 454 Sequencing Systems. Millions of copies of a single clonal fragment are contained on each DNA Capture Bead.
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Data Processing & Analysis
454 Sequencing Data Analysis software uses the signal intensity of each incorporation event at each well position to determine the sequence of all reads in parallel. Analyze the results in depth with powerful and user-friendly bioinformatics software for de novo assembly, mapping and amplicon variant detection.