I am familiar with Sanger sequencing, but at the level of an undergraduate. A le

Question

I am familiar with Sanger sequencing, but at the level of an undergraduate. A lecturer of mine tried to describe Sanger sequencing as losing the sequence information in noise when used to detect cancer. This paper also says "lacks suf?cient sensitivity for detecting mutant alleles in tumor biopsies,"(Thomas et al., 2006). What is it about Sanger that makes it too insensitive for SNP analysis?

Thomas, R.,K., et al. (2006) Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat. Med. 12, 852

Explanation / Answer

In the Sanger approach, DNA would be isolated from the biopsy and would contain both normal alleles and mutant alleles of genes associated with the development of the tumour. If, for example, PCR amplification was then used to derive a sample of a target template region, this material would end up being sequenced as a mixed population: the derived sequence would be an average of that population of sequences, and rare alleles would be masked.

The key difference in most next generation approaches is that the template DNA is "cloned" physically (e.g. by sequestering individual molecules into droplets, or by binding them to a surface) so that the sequences of these individual molecules can be determined in parallel. This approach would reveal tumour-associated polymorphisms when the sequences of the individual template molecules were compared.

Added later as supplementary information.

I have now looked at the paper cited in the question. This quotation from the Introduction bears out my main point about sequencing a mixture of templates differing at just a few key positions.

Although commonly used in many clinical settings, dideoxynucleotide chain termination (or

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