In traditional Sanger Paired End sequencing, the ends of a fragment are sequenced using Forward and Reverse Primers (see Figure 95). In the 454 Sequencing System, by contrast, Paired End sequencing is performed by first circularizing the fragment using a linker to join the 2 ends of the fragment of sample DNA. The circularized fragment is then nebulized, and the nebulized fragments containing the linker are then sequenced (see Figure 96).

The GS De Novo Assembler or GS Reference Mapper application takes Paired End reads that have been basecalled, removes the linker sequence, and creates two “half” reads for computation and reporting purposes. The half of the read corresponding to the reverse read in the Sanger method (coming from the 3’-end of the original linear fragment) is reverse-complemented to match the orientation one would obtain by extending the Reverse primer in the Sanger method; this half read is labeled with the original read’s accno and given the extension ‘_left’. The half of the read corresponding to the forward read in the Sanger method is left in its original orientation and labeled with the original read’s accno and given the extension ‘_right’.