Roche 454 sequencing technology
The Roche 454 sequencing technology is a large-scale parallel pyrosequencing system using bead-bound DNA templates, and is capable of generating more than 1,000,000 individual reads with improved Q20 read length of 400 bases per 10-hour instrument run. Basic steps include:
- Generation of a single-stranded template DNA library
- Emulsion-based clonal amplification of the library
- Data generation via sequencing-by-synthesis
A single-stranded DNA library is created from adaptor-ligated sheared genomic DNA, and is bound to beads.
The bead-bound library is emulsified with the amplification reagents and is contained within its own microreactor where PCR amplification occurs.
The DNA-containing beads are loaded into a PicoTitrePlate device for sequencing. Only one bead is contained within each well. The loaded PicoTiterPlate device is placed into the Genome Sequencer FLX Instrument. The four DNA nucleotides are added sequentially across the PicoTiterPlate device during a sequencing run. As a result, millions of copies of bead-bound DNA are sequenced in parallel. Addition of one (or more) nucleotide(s) generates a light signal that is recorded by the CCD camera in the instrument.
Read Lengths: Modal length = 500 bases, Average length = 400 bases
Reads per run / Throughput : >1 million high-quality reads
System Accuracy: 99% at 400 bases (and higher for prior bases)
Quality Score: Q20 read length of 400 bases
Data Format: SFF
Information taken from http://454.com/products-solutions/system-features.asp , accurate as of June 2010.