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Hailed as the "gold standard" of sequencing, I was wondering to what Sanger sequencing owes its incredible accuracy to. If possible, I would like a quantification of the accuracy (because I doubt it is 100%, though possibly very close).

I understand ddNTP chain termination and the strategies involved in Sanger sequencing but I lack the connection to accuracy.

Thanks!

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  • $\begingroup$ * In searching, I have located something known as "phred programs" which had provided early confidence levels for Sanger sequencing. I do not directly see the correlation though to modern Sanger sequencing $\endgroup$ – Andy Apr 26 '15 at 3:59
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    $\begingroup$ I don't think it's referred to as the gold standard solely because of its accuracy but rather because it was virtually the only method used for some 20 odd years before second generation techniques were even talked about. Chain termination sequencing is accurate though and is theoretically only limited by the error rate of the polymerase. That said, next gen techniques are also accurate. $\endgroup$ – canadianer Apr 26 '15 at 6:24
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    $\begingroup$ Here's some accuracy comparisons: hindawi.com/journals/bmri/2012/251364/tab1 $\endgroup$ – canadianer Apr 26 '15 at 6:39
  • $\begingroup$ Thanks for the comparisons! Personally, I have heard that Sanger sequencing is sometimes even used to confirm next gen tests, and its 99.999% accuracy is as expected. But I do agree that next gen is improving, in both accuracy and convenience $\endgroup$ – Andy Apr 26 '15 at 16:02
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Reliability comes from the fact that in Sanger sequencing (AFAIK) the synthesis of the new strand to the template and the actual reading of the sequence is separated. As you may know Sanger seq uses capillary gel elfo. thus separating the newly produced DNA fragments on a single nucleotide difference, and the reading is done by fluorescence detectors (then base calling etc.) Now compared to this the other sequencing methods like Illumina and 454 detect each nucleotide incorporation that may produce less reliable signals. For eg. in Illumina colonies may overlap on the plate, or in 454 incorporation of multiple nucleotides at homopolymer regions cause a hard time deciding how many nucleotides have actually incorporated. Also it must be noted that quality of the singals / reads is not constant through out Sanger sequencing. The beginning and the end of the reads tend to have noisy and low quality signals, thus actually only the 100-500(-600)bp segment of the whole fragment can be read reliably. Also ABI's SOLID uses a 2 color color-space and each base is read twice and is extremely reliable (99.96% is said by the inventors). Also clever base calling and error correction algorithms may improve accuracy.

It is noteworthy to say that next gen sequencers also achieve high accuracy by taking advantage of their immerse throughput / output - due to clonal amplification of template sequences each original fragment is sequenced multiple times and errors in a single read can be corrected by the use of others.

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