Primer design for unidirectional sequencing

[soleulloa]

Hi all,

Sorry, my english is not perfect, but I can try to explain my question.
I want to sequence an amplicon by undirectional sequencing. I was looking the "Guidelines for amplicon experimental design" and that says I have to use Primers A and B (Lib-L) to amplify the DNA, but the sequencing will be just using Primer A.

So, Primer B will be used just to bind the DNA to the capture beads? Or exists another function for Primer B in this case (unidirectional sequencing)?
Can I forget the primer B and just design my fusion primers using only Primer A?

Thanks for your help!

[soleulloa]

Anyone?

[Nicole 454 Sequencing]

Hi Soleulloa

You will want to be sure to use both the A and B primer here, even though only sequencing in the forward direction. During emPCR, only amplicon targets containing both an “A” and “B” primer sequences will be amplified. The “B” primer, as you mentioned, will allow the DNA to anneal to the capture primers on the DNA Capture Beads. If only the “A” primer is used, you will not get any amplification during emPCR, resulting in no enriched beads. During sequencing, the sequencing primer is annealed to the “A” sequence, thus sequencing in the forward direction.
In addition to “Guidelines for Amplicon Experimental Design” this information can also be found in App. Note 001-2009 “Unidirectional Sequencing of Amplicon Libraries Using GS FLX Titanium emPCR Kits (Lib-L); http://454.com/downloads/my454/appli...-Amplicons.pdf. Please don’t hesitate to contact your local Roche representative with any additional inquiries.

Nicole
Technical Support Scientist
454 Life Sciences, A Roche Company

[ajthomas]

Yes, listen to Nicole. You must use both A and B. However, be careful not get confused with the A and B primers of the Lib-A chemistry.

By the way, Nicole, is there any chance of changing the nomenclature so that the two adapter sequences of the two different chemistries aren't both called "A" and "B"? More than one person I know has already screwed up experiments because of this.

[soleulloa]

Thanks Nicole and ajthomas,

But I disagree at one point... It's my opinion, I don't know.
For the emPCR I have to use my amplicon, so I wouldn't need 2 primers because the fragment lenght is already determined by the first PCR when I got my amplicon.
So, I think that emPCR would be succesfull just using primer A. The polymerase needs 3'OH (Primer A) to link it and the reaction will happen until the enzyme walk through the complete amplicon and then polymerase "falls down"...
Primer B will allow the amplification of the other strand, but I don´t need that strand because I want to sequence just one strand (the other).

Then, primer B will be necessary just for the enrichment using capture beads.
Or not?

Thanks Nicole and ajthomas

[ajthomas]

No, emPCR requires that you have the A sequence on one end and the B sequence on the other. The B sequence hybridizes to B oligonucleotides attached to the DNA beads, and that oligonucleotide serves as the reverse primer for PCR. Here's what will happen if it's not there: you will get linear amplification (not exponential amplification) of your amplicon, and the product will be single stranded and not attached to the DNA beads.

The enrichment primer is also the A sequence, except it is biotinylated.

[soleulloa]

Ohhhh you're right!!!
Thanks

Sometimes I can be so blind

Thank you ajthomas

[Nicole 454 Sequencing]

Ajthomas,

I appreciate your feedback and bringing this to our attention. I will forward your suggestions internally to see if the terminology within the documentation can be clearer.

---------------------
Nicole
Technical Support Scientist
454 Life Sciences, A Roche Company

[Dynamac]

Confused

As I understand unidirectional sequencing, one would ligate the adaptors AFTER aqueous-phase PCR, using the Rapid Library method (and subsequent Lib-L emPCR). So doesn't the RL adaptor mix contain both A and B sequences already, giving you no choice but to use both? And what if you are using MIDs? Wouldn't you have to prepare your own adaptor A and B mix?

Thanks.

Barry

[ajthomas]

You could do it that way, or you can integrate the adapter sequences in your PCR primers. It's much easier, cheaper, and more reliable to integrate the adapters with the PCR primers. You can integrate MIDs either way you do it. Either way, however, you have the A adapter on one and and the B adapter on the other end. The B adapter binds to the beads during emPCR and the A adapter is in solution. Both sequences are required for emPCR.

[Dynamac]

Ok, so if my fusion primers have the A and B sequences integrated there is no reason to do the RL procedure, where you ligate adaptors after doing fragment end repair. Correct?

The Roche bulletins are very confusing on this point.

[ajthomas]

Yes, that's correct. Just clean up your PCR products, quantify them, and sequence.

[Nicole 454 Sequencing]

Hi Barry,

Hopefully I can clarify some of your confusion. The use of Rapid Library Adapters for preparing amplicons is used for ‘Ligated Adapter Amplicon Sequencing’. This design is primarily useful when the amplicon set already exists. As Ajthomas points out, this does increase cost as it requires the GS FLX Titanium Rapid Library Preparation Kit. This method also lacks information on the directionality of reads unless known flanking sequences are present.

When generating Amplicon Libraries by ‘fusion primer design’ the Rapid Library Adapters are not used. The sequences required for annealing to the DNA Capture Beads, Amplification Primers and Sequencing Primer are inherent in the forward primer (Primer A, Lib-L) and reverse primer (Primer B, Lib-L).

More information on these can be found in the 454 Sequencing System Guidelines for Amplicon Experimental Design (pages 24, 32) found on our customer accessible website http://my454.com/my454/documentation...em/manuals.asp.

It is also important to note that these amplicon preparation methods are specific to the Lib-L chemistry. Using these primer sets with Lib-A chemistry will not yield amplification during emPCR.

Regards,

Nicole

Technical Support Scientist
454 Life Sciences, A Roche Company