I've been searching for a list of the types of DNA sequencing (e.g. Sanger, Next-Generation) and how prone they are to sequencing errors (None, Somewhat, Very), but I haven't been able to find anything. Here are a few scenarios:

  1. I have a bacterial culture I want to sequence to see if a plasmid was properly transformed.
  2. I want to verify a PCR product.
  3. I want to sequence a large organism's genome.
  4. I want to verify a mutation found in a human tumor (found using exome data)

Given these examples, and any other you use regularly in the lab, can you please give me information on overall platforms (not specific like Illumina, but general like Sanger sequencing) and their error prone-ness?

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    $\begingroup$ Don't you think your question is a bit broad? Did you check out reviews on this topic? There are many. See these: dx.doi.org/10.1101/gr.168393.113, dx.doi.org/10.1186/1471-2105-13-185, biostars.org/p/2015 $\endgroup$ – WYSIWYG Jul 7 '16 at 5:48
  • $\begingroup$ I'll be honest, this seems like an exam question reworded. $\endgroup$ – James Dec 16 '19 at 11:56
  • $\begingroup$ @James It's 3 years old, but definitely wasn't an exam question. I was a PhD student and was getting started in bioinformatics & my uni's resources to inform us on this tech was underwhelming. I was trying to figure out what tech I needed for different experiments based on their error rates. $\endgroup$ – Gaius Augustus Dec 17 '19 at 18:15
  • $\begingroup$ In that case, I think it suffers from needing to be more focussed like @WYSIWYG suggested originally. Essentially this would be more helpful if it was split into 4 questions. $\endgroup$ – James Dec 18 '19 at 10:25
  1. Sequencing does not seem necessary for this... most plasmids contain an antibiotic resistance gene (ex. kanamycin-resistance) that will allow cells that have been appropriately transformed to survive in the presence of kanamycin.
  2. If it's just a PCR product, Sanger sequencing seems the best way to go. It is quite specific and not prone to algorithmic errors as are next-gen sequencers.
  3. Next-gen sequencing would be ideal as you're dealing with the whole-genome. Sanger would prove laborious and low throughput. Any variants of interest you find call always be confirmed through sanger. Illumina sequencers are quite robust at sequence generation at the whole genome level.
  4. This mutation can be verified through sanger sequencing, but you could also conduct targeted re-sequencing of the particular exon (although this seems like over-kill). Since it is a single mutation, I believe the specificity of sanger would be your best bet.

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