Top Heavy

Raw data view of sequence. Raw data is only accessible through ABI Prism Sequencing Analysis.

Top heavy refers to data that starts out strong, but eventually dies out. Like most sequencing problems, top heavy results can be lie on a spectrum from being a nuisance to no significant problems. This particular example is bad as it dies out to background noise by base 435. It needed to be repurified by the client. The cause is twofold. A contaminant could be affecting the processivity of Taq such that it has a hard time elongating the primer. The contaminant would have to be prevalent enough to disturb the sequencing buffer. Also, a contaminant that affects the electrokinetic injection of the samples into the capillaries could be present. Ionic contamination disturbs the conductivity of the loading solution and is preferentially injected over dye-terminated fragments. Gel sequencing is not subject to this type of contamination; it does not use electrokinetic injection. Samples need to be free from salt (especially EDTA-sodium), protein, lipid, genomic DNA, carbohydrate, and alcohol contamination.