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Ayaka 05-07-2014 04:46 PM

Newbie:Use of kit for library prep.Why can I just amplify using index,adaptor seq.?
Hi there, I am very new in the sequencing world and I am gathering the needed info to start the firs sequencing project of 16S amplicons with MiSeq in our lab.
My question is regarding the library prep. I donīt understand why there is the need to use kits like Nextera or Truseq to attach the index or adaptors needed for pooling and sequencing with the illumina platforms, if those sequences are available. Could I just make those as primers and perform a PCR? without the need of the kit? if so, what is the point of using the kit?

I have an illumina manual were they sequence 16S amplicons and use the nextera kit and perform 2 PCR reaction. Why does it has to be done in 2 PCR reaction, instead of just 1? is it because of the Tm of the primer would be not adequate?

I am sure these are very simple questions! but I havenīt been able to find satisfying answers. I hope someone can shed some light into my querys.
Thank you in advance

ScottC 05-07-2014 10:21 PM

Yes, you can certainly synthesise your own primers if you want to do that. The kit is really only for convenience. Illumina will recommend it becase 1) that's what Illumina used when they tested the procedure and 2) they want you to buy their reagents.

You can make your own oligos and use your own PCR reagents if you wish. We use our own PCR reagents but we use the Illumina adapters, because we already have them in our lab (we use them with the rest of the Nextera XT kit when preparing genomic DNA libraries).

nucacidhunter 05-08-2014 01:56 AM

For 16S amplicon library prep for metagenomics studies there are few options. One is the manual you are referring from Illumina user developed protocol which is done in two steps. If you are just doing a project with no long term intention of doing similar work, following this protocol offers certainty that it is more likely to work saving time and money. It is validated, region specific primers sequences are provided and libraries can be sequenced in any Illumina system with standard Illumina sequencing reagents. Second option is designing region specific primers with Illumina adapter flow cell binding motif and barcodes which is more complicated and sequenced with custom sequencing primers. This option requires overhead expenses for synthesis of long barcoded primers and is more suitable for labs doing regular metagenomics work. Using custom primers also enables longer reads because the uninformative primers are not sequenced. Your first step should be deciding which region to amplify and sequence based on your project aims and published work. This subject including many other options has been previously discussed in this forum for instance here:

microgirl123 05-08-2014 05:39 AM

The reason for using the 2-step PCR for amplicon sequencing is so that you can use one set of primers to produce all your amplicons, and the Nextera dual-index primers to index/add the rest of the adapter. This cuts down on cost dramatically. It would be very expensive to order a different set of primers for every indexed sample.

Ayaka 05-08-2014 08:08 PM

Thank you all for your answers! I have a very clear understanding of my queries now. I am truly glad I found this forum!

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