1. ★Primer ID
   

2. ★Library prep

Primer ID tips

1. ★cDNA synthesis primers should not be purified by size selection, as degeneracy can become truncated (see further reading).
   

1. ★A population of Primer IDs should be under-sampled, minimizing the probability that two randomly generated identical Primer IDs tag two different templates in the cDNA synthesis reaction. For example, we sampled ~10,000 viral templates with a population of 65,536 Primer IDs.
   

1. ★It is critical that the long, tagging primer is removed after cDNA synthesis, as residual primer could potentially tag new templates during subsequent PCR reactions.
   

Library preparation tips

1. ★Amplicon populations should always be quantified by fluorescence, not absorbance.
   

1. ★Lib-L quantification can be done directly off of the adaptors using the Rapid Library Standard. Do not extrapolate outside the linear range (dilute your library stock instead).
   

Assessment of Primer IDs

1. ★Illumina sequencing can be done on a pooled library prior to 454 sequencing to check the Primer ID distribution per sample.
   

© 2011 by C. Jabara