Sequencing Long-Range Amplicons affordably?

[SDK]

Hi All,

I have a question regarding sequencing long-range amplicons affordably, as the title suggests! So, in short I have about 40 long range PCR amplicons from 40 different species (amplicons are ~10kbp). I have no internal sequencing primers. I'm wondering what's my best course of action to get these sequenced. I have a MiSeq in my lab, but the only way I can think to do this is to break up the samples using a Nextera kit which could get expensive. Sanger is out of the question because even if I took the time to develop internal primers, they wouldn't amplify across the 40 species...so I'd have to develop internal primers 40 times! Any other ideas? Please keep in mind that I can't pool the samples as I need to know "who is who".

I'm thinking there has to be a kit out there or a way to do it with restriction enzymes and MIDS?

Thanks for any suggestions!

[SNPsaurus]

You can scale down Nextera. I would pool the amplicons from each sample (it sounds like that is allowed, just not pooling the species), fragment a few ng worth from each sample and PCR with a unique dual index barcode, and then sequence by either slipping the samples in with a friend's run or finding the cheapest possible "lane" like a low NextSeq.

[SDK]

You can scale down Nextera. I would pool the amplicons from each sample (it sounds like that is allowed, just not pooling the species), fragment a few ng worth from each sample and PCR with a unique dual index barcode, and then sequence by either slipping the samples in with a friend's run or finding the cheapest possible "lane" like a low NextSeq.

Thanks for the reply SNPsaurus. So, how would you choose to fragment? I've never used restriction enzymes before but I assume that is the idea here? Would you choose a common cutter? Thanks for any additional details

[SNPsaurus]

I would pool the amplicons of each sample and fragment with Nextera using just a 2-3 ng reaction. Then PCR the pools, combine the samples and sequence. So the workflow is:
40 PCRs per sample -> pool the amplicons of a sample -> 40 Nextera reactions total, 1 per sample -> 40 PCRs total, 1 per sample -> pool -> sequence

[Simone78]

Thanks for the reply SNPsaurus. So, how would you choose to fragment? I've never used restriction enzymes before but I assume that is the idea here? Would you choose a common cutter? Thanks for any additional details

Tn5 transposase used in the Nextera kit is not a "traditional" enzyme. It´s a transposase that will cut (semi)-randomly your DNA in small fragments, usually 300-600 bp, depending on the amount you use.
The Tn5 transposase works as a dimer and cuts the DNA and ligate adaptors on both side of a DNA fragment in a single step (a 5-min reaction). Every Tn5 monomer carries "mosaic ends" (partly double-stranded oligos) that are used in the reaction. Once the Tn5 has attached the oligos it becomes inactive ("empty") and cannot work on other DNA molecules, that´s why it is important to titrate the amount of DNA and/or Tn5 you use in order to get the desired length.
As a general rule:
- more enzyme and/or less DNA --> shorter avg size of your final library
- less enzyme and/or more DNA --> longer avg size of your final library.

Pooling the amplicons, cutting and then pooling everything is, as said above, the best, cheaper and most efficient option you have. good luck!

[bilyl]

Hi All,

I have a question regarding sequencing long-range amplicons affordably, as the title suggests! So, in short I have about 40 long range PCR amplicons from 40 different species (amplicons are ~10kbp). I have no internal sequencing primers. I'm wondering what's my best course of action to get these sequenced. I have a MiSeq in my lab, but the only way I can think to do this is to break up the samples using a Nextera kit which could get expensive. Sanger is out of the question because even if I took the time to develop internal primers, they wouldn't amplify across the 40 species...so I'd have to develop internal primers 40 times! Any other ideas? Please keep in mind that I can't pool the samples as I need to know "who is who".

I'm thinking there has to be a kit out there or a way to do it with restriction enzymes and MIDS?

Thanks for any suggestions!

Why not just randomly fragment the amplicon with fragmentase and then make a library with barcodes corresponding to each one? You could assemble them afterward. You would get around using the Covaris. You could also use Kapa's new HyperPlus kit which has a fragmentation enzyme included. Because these are amplicons you can get away with using a lot less DNA than the recommended amounts.

[nucacidhunter]

I would pool the amplicons of each sample and fragment with Nextera using just a 2-3 ng reaction. Then PCR the pools, combine the samples and sequence. So the workflow is:
40 PCRs per sample -> pool the amplicons of a sample -> 40 Nextera reactions total, 1 per sample -> 40 PCRs total, 1 per sample -> pool -> sequence

This approach is the most convenient and less expensive option. Further saving can be made by reducing tagmentation reactions to 1/4 of Illumina recommendation for Nextera XT. In this case PCR volume still need to be 25 or 50 ul to have optimum yield. When using full contents of the kit, PCR can be done with KAPA HiFi polymerase in 25 or 50 ul volume.