Roche 454 was the first commercially successful next generation system. This sequencer uses pyrosequencing technology [5]. Instead of using dide — oxynucleotides to terminate the chain amplification, pyrosequencing tech­nology relies on the detection of pyrophosphate released during nucleotide incorporation. The library DNAs with 454-specific adaptors are denatured into single strand and captured by amplification beads followed by emul­sion PCR [6]. Then on a picotiter plate, one of dNTP (dATP, dGTP, dCTP, dTTP) will complement to the bases of the template strand with the help of ATP sulfurylase, luciferase, luciferin, DNA polymerase, and adenosine 5′ phosphosulfate (APS) and release pyrophosphate (PPi) which equals the amount of incorporated nucleotide. The ATP transformed from PPi drives the luciferin into oxyluciferin and generates visible light [7]. At the same time, the unmatched bases are degraded by apyrase [8]. Then another dNTP is added into the reaction system and the pyrosequencing reaction is repeated.

The read length of Roche 454 was initially 100-150 bp in 2005, 200000+ reads, and could output 20 Mb per run [9, 10]. In 2008 454 GS FLX Titanium system was launched; through upgrading, its read length could reach 700 bp with accuracy 99.9% after filter and output 0.7 G data per run within 24 hours. In late 2009 Roche combined the GS Junior a bench top system into the 454 sequencing system which simplified the library preparation and data processing, and output was also upgraded to 14 G per run [11, 12]. The most outstanding advantage of Roche is its speed: it takes only 10 hours from sequencing start till completion. The read length is also a distinguished character compared with other NGS systems (described in the later part of this paper). But the high cost of reagents remains a challenge for Roche 454. It is about $12.56 * 10-6 per base (counting reagent use only). One of the shortcomings is that it has relatively high error rate in terms of poly-bases longer than 6 bp. But its library construction can be automated, and the emulsion PCR can be semiautomated which could reduce the manpower in a great extent. Other informatics infrastructure and sequencing advantages are listed and com­pared with HiSeq 2000 and SOLiD systems in Tables 1(a), 1(b), and 1(c).