emulsion pcr - one template-fragment per bead?

[Triticum]

hi!
how is it assured, that only one fragment per bead is contained in an emulsion-droplet? why can´t there be more different(!) template-fragemts per bead, which would lead to nonsense-sequences?

triticum

[454Sequencing]

Hi Triticum,

In fact, there is a certain percentage of droplets that contain more than one template fragment. We minimize the occurrence of this via appropriate library dilution. A good reason for functional characterization (i.e. enrichment titration) is that you can ascertain the percent enrichment at a given copy-per-bead and thereby have some expectations about the quality of sequencing expected when you scale up the emPCR reactions. Very high enrichment could be an indicator that your library copy-per-bead is too high.

In any case, PicoTiterPlate wells containing beads with multiple fragments will not yield productive sequence and will be removed as Mixed Reads during quality filtering.

Best regards,
Jason

hi!
how is it assured, that only one fragment per bead is contained in an emulsion-droplet? why can´t there be more different(!) template-fragemts per bead, which would lead to nonsense-sequences?

triticum