Probably you already know this, but illumina have a 16S protocol: http://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.ilmn

Hi Péter, thank you for the pdf. I indeed have already read it. My concern is related to the possibility of making less libraries than samples through the pooling of barcoded samples together... This is not mentioned in the guide, nor in other guides or manual I have red. Do you have any idea about this challange?

Cheers,

G.

For 16S we have just designed primers that include illumina adapter sequences in them for the amplification step, avoiding "library prep" entirely. The PCR products are ready to be loaded on the instrument after quantification with no other steps. We have a set of forward primers with different indexes in them so we can pool amplicons from different sources on single MiSeq run. This would work equally as well for your other gene of interest, and you could use the same index as the 16S for each sample.

Hi Tim, this is very interesting for me, do you have any publication where the protocol you have adopted is described in detail? I am very interest to it because several sequencing services proposed me astronomic quotation for the preparation of the libraries for illumina MiSeq... I will be very happy if I can read you work.

Thank you very much in advance,

G.

G

We only started using MiSeq for 16S when the 600 cycle kits came out in September, so we do not have publication describing the process. Basically, you just need a set of forward primers that have a "tail" of illumina adapter+various index sequences and a single reverse primer with the other adapter sequence on it. Then you amplify your metagenomic DNA sample with as few rounds as possible (remember, you only need 2 nM final concentration of fragment for loading onto the machine and the less you amplify the less PCR-introduced bias). Often this is 10-12 cycles, sometimes as much as 20 if your sample comes from an animal or plant and has high contamination with host DNA.

I won't try to post specific sequences here, illumina is oddly protective of their adapter sequence and I have repeatedly seen on SeqAnswers.com that they have requested removal of the sequence from the posts. If you want to email me at [email protected] I will send you the primers we have successfully used.

Hi, I agree with Tim - their approach is least cost and time intensive.

I'd like to add that there might be problems with the read quality (we sometimes observed low quality of the reverse reads from PCR products amplified with primers containing the illumina adapters), or difficulties already in the amplification step due to the long fusion primers.

For this case, we chose to only fuse 5-8 bp long indices (optimized for illumina sequencing) to our original primers. We purified and pooled the PCR products containing the different front indices and then ligated barcoded (TruSeq) illumina adapters as for whole genome DNA shotgun libraries. From these libraries we got good quality reads for both directions. We then separated the indexed reads using the mothur pipeline.

This way only one or two library preparations from the PCR pool(s) would be needed.

Anyway, with the new v3 sequencing chemistry, the low quality read problems might not be an issue anymore, so if you get good amplification results, I'd say go for Tim's protocol first.

Halina

Dear Tim and Halina,

I very much appreciate your replies. I would be very happy to contact you both for further advices and indeas. Halina, can I also have your email address?

You gave me very usefull advices and help, I am pretty surprised about how this community of research people works... This is the spirit I suppose, the strongest help the weakest with his bigger knowledge.

Thank you very very much,

Gian

Hello,

if you'd like some documentation on fusion primer for the MiSeq (PCR primers that include MiSeq adaptor and barcodes) you could look at the following document:

http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&ved=0CEgQFjAF&url=http%3A%2F%2Ffiles.figshare.com%2F1260090%2F16S_amplicon_Illumina_sequencing_methods.pdf&ei=zUPSVP6EFonKaPTqgvAI&usg=AFQjCNH7_cyvou4pIPC5hxJgniX-Pxocvg&bvm=bv.85076809,d.d2s&cad=rja

you can multiplex up to 96 individuals using the Truseq barcodes (8 forward barcodes x 12 reverse barcodes):

http://supportres.illumina.com/documents/documentation/chemistry_documentation/samplepreps_truseq/truseqsampleprep/truseq_sample_prep_pooling_guide_15042173_a.pdf

Illumina adaptors are complementary so when using those fusion primers for PCR it is likely you will uncounter a lot of primer dimer.

you will have to get rid of them.

a convenient way is to do some size selection with AMPure beads because in a single step you get rid of the primer dimer (if the size difference between the primer dimers and the PCR product is big enough) and you clean up the PCR. Because your PCR products are barcoded you can pool them after the PCR which makes all the downstream steps much easier.

after that step it's ready to go on the MiSeq (after some QC on a Bioanalyzer).

cheers

Marc

http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&ved=0CEgQFjAF&url=http%3A%2F%2Ffiles.figshare.com%2F1260090%2F16S_amplicon_Illumina_sequencing_methods.pdf&ei=zUPSVP6EFonKaPTqgvAI&usg=AFQjCNH7_cyvou4pIPC5hxJgniX-Pxocvg&bvm=bv.85076809,d.d2s&cad=rja

http://supportres.illumina.com/documents/documentation/chemistry_documentation/samplepreps_truseq/truseqsampleprep/truseq_sample_prep_pooling_guide_15042173_a.pdf

I use 16S illumina primers included illumina adapter sequences in them. However, I get band on the negative controls at the positive size. I thought it was contamination, and I repeated my PCR many times with brand new set of primers and made new reagents. But I still get this band on the negative controls , and the strange thing that some of my samples did not show any band on the same PCR, so i expect that if that was a contamination it would show in all PCR products, any suggestion please? Thank you