while conducting a research with a small sample size (5-6) with a large exome size (21-25), should you go for Sanger sequencing or NGS? Keeping cost, labour and practicality in context.
The question does not make much sense. First the research focus need to be defined about what need to be analyzed. What method to be used is additional and secondary question that depends on the first.
I am looking for a descriptive answer and insights from someone who has been there already.
For the background, the gene in subject is ATP7B that spans 21 exomes, the study is done to explore novel mutations.
Now that my professor is going to employ NGS, and it is going to be expensive, though there are some studies where Sanger sequencing has been utilized only as a method of validation after RFLP, an exception to that was a study following the similar theme as my study which employed Sanger.
So, what benefit can Sanger have over NGS in this context besides cost-effectiveness? And if I employ NGS, what other molecular biology skills would I miss out on?
One limitation of Sanger is that it is hard to detect low-frequency changes. You can see SNPs, small InDels, etc if they are heterozygous or homozygous and use samples from individual organisms/bacterial colonies/etc.
If you use NGS, you would need to follow up and verify any potential changes with Sanger. So, you would not be missing out on any skills.
Talk with your advisor about the specific goals of the project. Also, unless this is just a "pilot study", I very much doubt that your total sample size will be 5-6, especially looking for novel mutations. Are you starting with cancerous tumors or something that is KNOWN to have a high mutation load?
Based on the gene & a quick Google search, you are studying Wilson's disease. You'll need to talk with your advisor - it's very difficult to show that a "novel variant" is associated with a specific disease. You should also consider sequencing the entire ATP7B gene (promotor, exons, introns, UTRs, etc). Not all disease-associated variations are in the coding region.
PS The term for all of the coding regions in a single gene is "exon". The "exome" is all of the protein-coding sequences in the entire genome.
Edited to add the OMIM link: https://omim.org/entry/606882
If my guess was correct, here's a link to the OMIM database for your gene. Great place to start diving into the literature.
Good luck!
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