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2. Comparing sequencing methods

(a) The length of sequence read from any one template for both 454 and Illumina sequencing depends on maintaining synchronous extension of all templates (i.e. that nucleotide is added to almost all copies of a template at each cycle that the correct nucleotide is supplied).

Read lengths are characteristically shorter for Illumina than for 454 sequencing. Why is that? Try to think of at least TWO differences in the sequencing protocols that contribute to this difference.

The length of the sequence reads in Illumina method is less than that in 454 sequencing, because in the Illumina method we have to unblock the 3’OH end of the bound dNTP and also to delink the fluorophor in each step.

The length of the sequence read is also limited by the template that fails to be extended in each cycle. In 454 sequencing method we need not perform these unblocking steps and delinking of flurophor. Also in this procedure the PCR is performed in a well with a bead. The bead prior to sequencing contains millions of copies of an identical DNA fragment and hence during sequencing reaction (pyrosequencing), we can get higher efficiency even if some fragments (templates) fail to extend....

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