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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.

KitLanesReads / RegionThroughput per  RegionThroughput per Plate

FLX+

FLX

(Amplicons)

2

450,000–650,000

450,000–650,000

(360,000-520,000)

250-400 Mb

180-280 Mb

500-800 Mb

360-560 Mb

FLX+

FLX

(Amplicons)

4

160,000–250,000

160,000–250,000

(130,000-200,000)

160-250 Mb

60-110 Mb

400-640 Mb

240-440 Mb

FLX+

FLX

(Amplicons)

8

80,000–120,000

80,000–120,000

(65,000-100,000)

80-120 Mb

30-55 Mb

320-560 Mb

160-320 Mb

FLX+

FLX

(Amplicons)

16

25,000–40,000

25,000–40,000

(20,000-30,000)

25-40 Mb

10-20 Mb

240-400 Mb

160-320 Mb

Junior+

Junior

(Amplicons)

1

100,000

100,000

(70,000)

70 Mb

35-50 Mb

70 Mb

35-50 Mb

 

For more information take a look at Roche 454.