Overview scheme of 454 sequencing. Double-stranded sequencing templates are blunt end repaired, and two universal adapters, A and B, are ligated to their ends. The B-adapter carries a 5′ biotin (I). Streptavidin beads are used to isolate only molecules carrying an A and a B adapter (II). The single-stranded sequencing library is melted from the beads through alkaline treatment (III). A PCR reaction mix containing 600 000 oligonucleotide-coated sepharose beads and an appropriate number of library molecules is emulsified to produce physically separated droplets as reaction vessels (IV). After amplification, the emulsion is broken while the PCR products remain attached to the beads. Since most beads remain empty in emPCR, amplified beads are isolated through a bead enrichment procedure (V). A total of 50 000 beads are required for loading onto the wells of a 16th 454 FLX picotitre plate region, and the sequencing reaction is performed by flowing nucleotides over the plate and measuring light emissions. Beads carrying multiple amplicons produce mixed signals, which are recognized and filtered out by the run-processing software.