TruSeq PCR free DNA with 16 amplicons

[maddo1ck]

New member and new to running my own MiSeq Sequencer!

We are trying to figure out how to modify TruSeq PCR free DNA library prep kit protocol to make a library with our pool of barcoded amplicons (16s and ITS) for sequencing on the MiSeq. Has anyone else done this or seen any published protocols combining these methods?

The problem I am running into is that we use SequalPrep Plates to normalized our PCR amplicons (~350bp) and then pool. This theoretically provides us with 1.9ml of pooled PCR product at ~1ng/ul (we ended up with about 0.7 bc of negtive controls and failed PCR reactions).

We would start the TruSeq protocol with the dAtailing step, which calls for 15ul of sample that I guess is around 800ng since you start the full protocol with 1100 (for a 350bp insert).

Speed vac to concentrate would be problematic due to the elution buffer used in SequalPrep. I tried a Zymo clean and concentrator column, but lost 60% of the yield. SequalPrep does have a sequential elution recommendation, which I have not tried yet, but at 5 min per well it will be time limiting. Any suggestions?

[thermophile]

This seems to be the really expensive way to add index and adapters. Why not just make the big indexed primers (a la Kozich or Caporaso) or 2 step PCR?

[kmcarr]

This seems to be the really expensive way to add index and adapters. Why not just make the big indexed primers (a la Kozich or Caporaso) or 2 step PCR?

It sounds like maddo1ck has already indexed and pooled, now just needs to add the Illumina bits to the end, i.e. just a single ligation reaction.

maddo1ck, try a Amicon centrifugal ultra filtration concentrators . They are simple to use and recovery is generally excellent.

[maddo1ck]

The idea was that since we are a fairly small lab and don't have hundreds of samples for sequencing this method would be flexible in allowing us to do both PCR amplicons and WGS for individual samples if we wanted.

Thanks for the suggestion kmcarr, I will look into those. I also realized we could do a serial dilution with sequal prep to reduce our volumes so we are going to try that first to avoid any potential loss thru a spin step.

[bunce]

Hi Maddoc1ck - we found this protocol helpful when using a 'home-made' PCR free approach. http://www.ncbi.nlm.nih.gov/pubmed/21431776

Good luck with your workflows.

[JBKri]

...

We would start the TruSeq protocol with the dAtailing step, which calls for 15ul of sample that I guess is around 800ng since you start the full protocol with 1100 (for a 350bp insert).
...

Definitely start with the end repair; it adds a phosphate group that probably is necessary!

We usually add about 100 ng of PCR product, and we get around 50% of these fragments with adapters successfully ligated at both ends, which is more than enough.

We have moved to the two-step PCR protocol, it has many advantages.