I'm new and need help

[Shamol]

Greetings everyone,

I'm a graduate student from Bangladesh (Department of Microbiology in the University of Dhaka). My work focuses on sequencing complete genomes of local high-risk HPV isolates. I've been using a more conventional PCR-based Sanger sequencing technique for the past few months, with limited success. My supervisor has recently suggested considering Next Generation Sequencing as an alternative.

The technology is rather exotic around here, so there's virtually no knowledgeable local distributor to seek help or consultation from. I've been poring through dozens of papers with themes of NGS and virology. While I do have something of a skeletal idea, I don't think I can progress without more direct advice from experts. I've been looking for an online forum where I could get some specific answers for the past week. This search led me to a webinar on NGS by Dr. Nandita Mullapudi on Bite Size Bio, where at the end she referenced this forum as a good resource.

I've really been having an awful time trying to figure out some nuances of NGS as it applies to my work, so I'd be incredibly grateful for any help I can get. I'll post my specific questions in appropriate sub-forums, in the meantime any general suggestion would be terrific.

Thanks again!

[GenoMax]

Don't want to sound discouraging but if you have not been able to get sanger sequencing to work right then NGS may not be the right choice.

NGS is useful when you have a lot of samples to screen (and a defined prep method for them). It sounds like you are having some experimental problems getting samples of good quality in the first place. Even though NGS prices have come down over the years that technology is still expensive compared to RT-PCR/Sanger. If you expect to send your samples to a provider then you will need to figure in the cost of making libraries in addition to sequencing.

First thing you should probably work on is getting good quality DNA preps that sequence well with conventional sanger.

[Shamol]

Thanks for your answer. This is news for me (and presumably will be for my supervisor as well!). Am I then right in saying that NGS offers no additional benefit in sequencing a limited number of samples over Sanger, since both processes involve enrichment (with PCR) and are equally cumbersome and both demand good quality of input DNA?

[DNA_Dan]

The best you'll do on an Illumina platform is ~600 bp targets on a MiSeq paired-end 2x300 bp kit. It takes time to develop primers for your targets, but once they are made the advantage of NGS is you can run 384 patient samples in a single run. So GenoMax has it correct. NGS offers incredible scaleability. Sanger can cover larger targets >600 bp but is a lower throughput platform. For newbies it's actually easier to work with the data too. Lots of easy to use tools out there for Sanger data.

[Shamol]

The main problem in the project for which my supervisor and I are considering NGS to begin with is the fact that it's difficult to amplify the samples with PCR, probably because of low DNA concentration in the samples. Wouldn't I have the same problem on an NGS platform as well? This is the basic question I have.

[GenoMax]

Unless you have clean/good starting material (DNA) there is no advantage to using NGS over sanger. If you are having trouble due to bad sample quality with Sanger sequencing then you will have the same problems with NGS (albeit at a greater expense).

[DNA_Dan]

The only possibility I think NGS can help is if perhaps you treat the PCR product as a genome and use a low-input library kit to get the adapters on the fragments. If you can do this, then it will amplify and generate libraries that can be sequenced. I don't recall the input amounts for a typical Sanger reaction, but NGS library prep may have more sensitive offerings for this. Also you may have to shear your product down to get the size to be appropriate. But as GenoMax eluded to, "garbage in, garbage out" and NGS will give the same or similar results.