When sequencing papers refer to single molecule sequencing, what is their definition of a "molecule". Are they saying base by base? The entire DNA chain in a chromosome can also be though of as a "molecule". In that case, that's a much larger read. What's the default interpretation?

For instance, is the Illumina sequencing "single molecule" as they fluoresce one base at a time?



For the classic Sanger sequencing you need quite large amounts of DNA to be able to run the sequence reaction. This is a problem when you want to sequence whole genome since the DNA needs to be amplified then, which for some sequences can cause problems, introduce errors or cause bias.

Single molecule sequencing is a huge step forward here, since only one molecule of DNA is used for sequencing. There are different sequencing technologies based on this principle. The system of pacific biosciences uses a polymerase bound to a small well which binds a singlestranded DNA and begins to make the opposite strand, when nucleotides are present. See the image below or this video, which explains the technique quite nicely.

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Illumina binds singlestranded DNA molecules to the surface of the reaction chamber and starts amplifying them there. The use of fluorescently labelled nucleotide allows to monitor the reaction. This looks like the following (left side of the figure from the review mentioned below):

enter image description here

An illustration from Illumina which is more detailed, can be found here. Although it is a bit older (and the technology develops fast), this review is a nice introduction: "Sequencing technologies - the next generation."

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  • $\begingroup$ Nanopores typically use an unlabelled oligo. Your figure is Pac Bio's immobilized DNA polymerase approach that uses the wonderfully named Zero Mode Waveguide to visualize a 20 zeptoliter volume and observe each labelled oligo being incorporated by the polymerase. $\endgroup$ – PlaysDice Apr 19 '14 at 11:17
  • $\begingroup$ Yep, thats right. Have a look at the video, it is about the same method. $\endgroup$ – Chris Apr 19 '14 at 11:54
  • $\begingroup$ But it doesn't use a pore. Its a well. My comment was that a 'true' nanopore approach like Oxford Nano doesn't use label. Both are examples of single molecule analysis. $\endgroup$ – PlaysDice Apr 19 '14 at 12:30
  • $\begingroup$ I'd +1 if you add a sentence about nanopores or other single molecule approaches and answer OP re Illumina. $\endgroup$ – PlaysDice Apr 19 '14 at 12:38
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    $\begingroup$ @biogirl Yes, as far as I know this is one of the other names for it. $\endgroup$ – Chris Apr 20 '14 at 11:14

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