Roche/454 pyrosequencing technology
The first high-parallel sequencing system was developed with an emulsion PCR method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized on the individual well of a PicoTiterPlate (PTP) [1]. The DNA sequencing protocol, including sample preparation, is supplied for a user to follow the method, which is developed by 454 Life Science (now part of Roche Diagnostics, Mannheim, Germany). Even following the manufacturer's protocol, to obtain high-quality DNA sequence with maximal total DNA sequence length as possible, one should make a library with both adapters on the sheared DNA fragment and mix the optical ratio of library DNA versus bead for emulsion PCR. Now, the system is upgraded to through-put a total of an average of 700 Mbp with an average 600 bp/read in one PTP run. The sequencer is available for single reads and paired-end read sequencing for the application of eukaryotic and prokaryotic whole-genome sequencing [2, 3], metagenomics and microbial diversity [4, 5], and genetic variation detection for comparative genomics [6].