Additional Sequencing Methods¶
This one is widely taught and known, but a little outdated.
1. Lyse the cells and extract DNA. The DNA itself is fragmented and copied many many times over.
2. Attach primers to the fragments and separate the experiment into 4 tubes.
3. Each tube receives plenty DNA polymerase, plenty deoxynucleosidetriphosphates (dNTPs) and about 1/100th the amount of di-deoxynucleotidetriphosphates (ddNTPs). The issue with ddNTPs is that they don’t have the 3’-OH group. Synthesis stops when it a ddNTP is attached, which happens at various points on various fragments.
4. DNA is negatively charged. Heat the DNA to separate it and put through a gel from pos->neg side, which separates fragments with a resolution of 1 nucleotide.
5. The trick is that the ddNTPs are fluorescent! You now have many many fragments whose lengths you know down to the nucleotide. Expose an x-ray film to the gel, and now you have 4 rows representing the 4 nucleotides and dark bands where each of the 4 nucleotides terminated a fragment. Here’s a picture to clarify:
image of sanger gel
1. DNA is immobilized at the bottom of a small well with a DNA polymerase.
2. Fluorescent nucleotides are introduced to into the well, each labeled with a unique fluorophore.
3. Light from the bottom of the well makes the bases fluoresce as they are added to the DNA template by the polymerase and the sequence of light colors is recorded.
|Pros |Cons| |—|—||- long reads - up to 15kpb |- expensive | |- high throughput| - high error rate |
Longer reads means that fewer reads are needed for the same coverage. Clearly, PacBio is best used for the reconstruction of longer genes or entire genomes.
|Pros |Cons| |—|—||- High precision - error rates of ~.001% |- low throughput | |- long read length |- expensive |
Sanger sequencing is currently only really for small sequence lengths in a small number of samples.
|Pros |Cons| |—|—||- low cost |- short reads | |- high throughput |- not cost effective for small number of targets| |- decent precision - error rate: 0.46% - 2.4% | |
Although the reads are short and the error rate is not as low as Sanger sequencing, Illumina sequencing produces so many sequences that it doesn’t matter. Getting the most out of Illumina means getting the most out of its high throughput and per base coverage, which requires the proper software.