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About 454 Sequencing

Information courtesy of Roche 454 Life Sciences

Genome Sequencing

454-FLXThe Genome Sequencer System provides a unique technology for sequencing DNA samples in an efficient, massively parallel and cost–effective manner. The Genome Sequencer FLX System has a number of applications, including de novo sequencing, resequencing of whole genomes and target DNA regions, metagenomics and RNA analysis.

 454 Sequencing is based on sequencing–by–synthesis where nucleotides are flowed sequentially in a fixed order across the Pico Titer Plate device during a sequencing run. During the nucleotide flow, hundreds of thousands of beads each carrying millions of copies of a unique single–stranded DNA molecule are sequenced in parallel. If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding nucleotides. Addition of one or more nucleotides results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides incorporated in a single nucleotide flow.454-Junior

 

 

The Pico Titer Plate (PTP)can be divided into a number of different regions allowing the sequencing of a number of different samples on a single PTP. Multiplex Identifiers (MIDs) expand multiplexing options beyond physical separation of samples by introducing a unique 10–bp tag, enabling pooling for simultaneous amplification and sequencing. The sizes of the different regions will dictate the number of reads per region that can be expected.

Sequencing KitNumber of RegionsReads per RegionRun Throughput per RegionRun Throughput per PicoTiterPlate
FLX+ 2 450,000–650,000 250-400 Mb 500-800 Mb
FLX 2 450,000–650,000 180–280 Mb 360–560 Mb
FLX+ 4 160,000–250,000 160-250 Mb 400-640 Mb
FLX 4 160,000–250,000 60-110 Mb 240-440 Mb
FLX+ 8 80,000–120,000 80-120 Mb 320-560 Mb
FLX 8 80,000–120,000 30-55 Mb 240-440 Mb
FLX+ 16 25,000–40,000 25-40 Mb 240-400 Mb
FLX 16 25,000–40,000 10-20 Mb 160-320 Mb
Ti Amplicon 2 360,000–520,000
Ti Amplicon 4 130,000–200,000
Ti Amplicon 8 65,000–100,000
Ti Amplicon 16 20,000–30,000
Junior 1 70,000–100,000 35-50 Mb 35-50 Mb

 

For more information take a look at Roche 454.