Amplicon PCR Problems with Illumina MiSeq Sample Prep

[locon833]

Hi all,

My lab is new to Illumina sequencing, and as we have just obtained a MiSeq we are trying to get started!

We are using the 16s Metagenomics workflow/protocol laid out by Illumina. We are using the Greg Caporaso (515f and 806r) primers with the Illumina adapters attached to them for the amplicon PCR. We used the following thermocycler conditions which are from the Earth Microbiome protocol for these primers WITHOUT the adapter sequences on them:

Thermocycler Conditions for 96 well thermocyclers:

1. 94°C 3 minutes
2. 94°C 45 seconds
3. 50°C 60 seconds
4. 72°C 90 seconds
5. Repeat steps 2-4 35 times
6. 72°C 10 minutes
7. 4°C HOLD "

We have followed the protocol exactly, except for the thermocycler conditions and when I ran the products on a gel I got large amounts of high molecular weight smearing and a light non-target band.

Does anyone know if the reason for the smearing and non-target region bands could be due to the addition of the extra bp on the region specific primers? Has anyone run the 16s Metagenomics protocol with these primers and adaptor sequences with different thermocycler conditions and had success?

Any help would be appreciated!

[nucacidhunter]

As a general rule every protocol is designed to work with certain reagents under certain condition. Modifying any step often requires appropriate adjustments in other components for good results. Earth Microbiome protocol uses different DNA polymerase than Illumina protocol (if you are referring to Illumina’s two step 16S PCR with Nextera primers), so the PCR condition may not be exchangeable as buffer composition and proccessivity of polymerases can have significant effect on amplification. For optimum cycling condition it is best to follow the polymerase manufacturer guidelines.

We have followed the protocol exactly, except for the thermocycler conditions and when I ran the products on a gel I got large amounts of high molecular weight smearing and a light non-target band.

It is not clear to me which protocol you have followed. Caporaso and Illumina recommended primers have different structures and their amplification strategy is also different. It is possible that smear originates from non-specific amplification due to low annealing temperature or it could be excess template DNA which has been degraded during thermal cycling.

[locon833]

Hi nucacidhunter

Thank you for your response! I'm sorry if I was unclear. We followed the protocol for 16s Metagenomic sequencing from Illumina.

We used all of the same reagents and quantities that the Illumina workflow calls for such as: the Kapa HiFi mastermix, the 5 ng/uL DNA, and the 1uM primer concentration.

I wasn't sure if we would be able to use the thermocycler conditions that we had used when we were following the EMP protocol, because we are still using the Caporaso primers. With that being said, when we designed the primers for the Illumina MiSeq protocol we added the adapter sequences onto the Caporaso primers like the Illumina protocol specifies. I'm guessing that my best bet would be to run a gradient PCR to figure out the optimal temperatures for the Caporaso primers with the adapter sequences attached, but I didn't know if anyone had tried doing this before with any luck.

[nucacidhunter]

Following is comparison of 16S amplification primer by Caporaso and Illumina method:
Caporaso:

http://www.pnas.org/content/108/Supplement_1/4516/F1.expansion.html

Illumina:
16S Amplicon PCR Forward Primer = 5'
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG
16S Amplicon PCR Reverse Primer = 5'
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC

If your want to adapt Coparosa primers to Illumina protocol you should only have the reverse and forward and maybe linkers motives from Coparosa to replace 16S locus-specific sequences from Illumina. This should work for most designs if there is not adverse interaction between locus-specific sequences and Illumina adapters overhangs. If this is the design that has been used and is not working by using Illumina recommended reagents and cycling conditions, as you have suggested gradient PCR would be a good approach.

[cliffbeall]

We have noticed that 16S amplifications of mixed populations often produce smeared high molecular weight products - we are pretty sure they are due to heteroduplexes that migrate slowly on a gel. They should be ok since they get denatured before sequencing.