Hello Frédérique,

Sure it is. What kind of problems do you expect?

Hi Frédérique,

Like Rocco, I also see no obvious problem. What do you think your problem could be?

After your PCR amplification, all the samples consist of in-vitro generated DNA. As long as the amplicon length is not too different and you load the same amount of material, the MiSeq will sequence these samples with equal efficiency.

This is my first experience with NGS experimental design and if I have found some publications with 16S (both bacteria and archea) multiplexing, I have failed to find archeal and bacterial 16S and ITS PCR preamplifications (before index) in the litterature. Currently my doubts focus on primers design (best coverage, consistency in amplicon's lenght) . Indeed I hope to avoid any dimers between differents primers or lack of depth.

At the moment my choice heads for 338F/909R for Bacteria, 340F /915R for Archea and ITS1-F_KYO1/ITS4 2390–2409 for Fungi.

Is this choice getting my closer to my goals that's to say caracterise microbial community in a soil under different environmental pressure ?

Thanks

Frédérique

Hi Frédérique,

I assume that you will test the quality of your primers (i.e. the primers used to amplify your targets) before loading the products on the NGS instrument, e.g. simply by loading them on a standard agarose gel. If you see that the products are not the expected single bands you can either purify from gel or change amplification parameters or modify your primers. However, as long as the quality of your amplicons is ok there will be no problem.

Possibly, a bit of confusion arises from the concept of multiplex: when you say that you want to multiplex your samples in a Miseq run you mean that:

1) you will perform 3 independent PCR, generate libraries with 3 different indexes and then 'multiplex' them in a single run

2) or that you will co-amplify in a single PCR tube all your products (real PCR multiplex) ?

If your situation is #1 then there is no problem at all. If #2 then the problem is strictly a multiplex PCR optimization issue. The fact that you will sequence with NGS doesn't really modify things.

Dimers shouldn't be a major issue here, taking into account that you will have to gel-purify your amplicons and that their size is much bigger than the standard dimer size.

Finally, even if you will sequence with a 'small' NGS instrument, the typical coverage of small amplicons with NGS is so deep that I'm pretty sure you're experiment will just go fine. Happy sequencing!

Thanks a lot

Dear Frédérique,

Unfortunately I'm not an expert in 16S biology, so I can't help you with the choice of primer. Some of the points you mention though, are very important to generate biological material that can be transformed into good sequencing libraries:

- ensure that you have sufficient depth in your material. This you can ensure by amplifying your material using a limited number of PCR cycles. If you need many cycles to generate a signal, it means that you started with very few copies in your start material. If you can isolate sufficient material, you might want to amplify your material using varying numbers of cycles (e.g. 10, 12, 14, 16, 18 cycles) and see if (1) you get a measurable amount of your expected fragment and (2) if the amplification is linear (i.e. around 4 times more material after 2 extra cycles).

- non specific fragments (i.e of the wrong length) should definitely be avoided, particularly because the MiSeq (or any other Illumina machine) will preferably sequence shorter sequences. The AMPure beads purification should allow you to take out fragments of unwanted size. Keep a strict cut-off.

Good luck,

Daan

thanks for the advice. I have planned to amplify separately each taxon, add different index and read on the same run (case 1 rocco).

I also heard say (as Daan) that the AMPure is required for better removal of primers monomers and dimers...I wish to test this method.

Frederique

Hi Frédérique,

yes, I agree with Daan: in our experience the AMPure works very well.