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rudzik79 08-11-2017 12:42 AM

Illumina 16s protocol: size

I've been having a hard time amplifying 16s DNA from active sludge with Illumina suggested primers v3-4.

I tried NebNext HIFI and iProof mixes, same results.
Anyone knows why?

I am using standard conditions, but I never get the 550 bp band after 1st PCR, nor the 630 bp band after 2nd. I tried qPCR, and after 25 cycles suggested by Illumina it was empty (started rising at about 27 cycels), so I switche do 32 cycles of 1st PCR. Getting a lot of different products but not the 550 band.

Do the active sludge bacteria have a different size of this region? Then maybe I am throwing it away with 0,8x AMPure X beads after 1st PCR (it is not well visible on the gel before purification either). Or do the primers simply not hybridise?

I would be grateful for a quick answer.

adam.geber 08-15-2017 09:54 AM

0.8x cleanups shouldn't be able to touch your 550bp amplicons. Are you using the same polymerase recommended by Illumina? If not, then you'll likely have to do some further optimization via gradient PCR to confirm the ideal conditions -- see this thread for some starting suggestions. Also, are you using a positive control in your amplifications? I don't know much about sludge bacteria but you need to rule out issues of limited starting template or inefficient DNA extraction from your samples. Zymo makes a very good microbial DNA standard that you can use for both these purposes but if you don't want to spend the money and you don't need a standard with known species you can always just use an oral swab as input for your DNA extraction.

rudzik79 08-16-2017 03:33 AM

Thank you. For a standard I used microbial DNA that has worked before and was 16s sequenced. I checked everything for DNA intergrity first, it's fine.
The polymerase is not the same, but it has proofreading abilities and was used by me to prepare other libraries (iProof bioRad) or was recommended online as a substitute for KAPA (NEBNext).

My GRADIENT TOUCH-DOWN PCR was completely empty, and I used 15 cycles for touch down (68-61 in one corner and 58 down to 51 i the other with 5 intermediate touch-downs in between) and 20 regular cycles in the lowest temperature. That's how I normally do optimisation for tough products. This time it was empty.

Now my friend is bringing me primers that are known to work, because I have completely no idea what to do next... KAPA is not readily available in my country now. Plus, it's twice the price of NEB. And I have plenty of iProof that is nearing expiry date so I could use a lot of that too.

adam.geber 08-16-2017 09:24 AM

Gotcha, so your control has worked well before (and is passing your standard QC) but isn't yielding anything in the context of your gradient touch-down PCR? What input mass are you using for your amplifications? And have your primers worked well in your hands in the past? Can you share your exact conditions for PCR (in your standard protocol and your gradient touch-down setup)?

I also use a highly processive/proofreading enzyme (NEB's Q5) for 16S PCR but I use the primers that were modified as part of the Earth Microbiome Project rather than Illumina's standard primers. Usually I think of the two-step PCR method as being more specific to the V3/4 locus, though -- my group had severe issues with mispriming at our standard annealing temperature in the past and we chalked it up to the one-step addition of adapter sequences. Can you share some gel images showing the products you're seeing besides your expected 550 bp band?

You may find this paper helpful for a comparison of primers and enzymes in the context of 16S PCR, by the way.

rudzik79 08-17-2017 12:59 AM

Wow, Adam, thank you very much for such a nice response and willing to get to the bottom of this. I am afraid I got a simple answer yesterday, and it isn't pretty.

It was the primers all along.

I asked my colleague from another institution to come with her primers. We compared the sequences, they were identical. Nobody made any errors in sequences during purchase.
So we ran four parallel PCRs - iProof and KAPA, with her primer pair and with mine.
The results were striking, as hers worked perfectly in both mixes, on the same PCR program. Samples with my primers were, as usual, empty.
I am doing this for 17 years, and was in institutions that have ordered thousands of primers by now. NOTHING LIKE THIS has ever happened, except for my very first pair - but back then the sequences were copied by hand, and a lady from purchase simply made an error in polyN stretch.

I will be filing a complaint and demanding compensation for lost time and kits. By the way, my NEBNext kit is finished and I am really furious.

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