Solexa Protocol

[Macki1x]

Has anyone had success generating Adaptor1-TAG-Adaptor2 products for Solexa Sequencing?

I am trying to get this working and am only able to make Adaptor 2 concatamers. Basically, I extract RNA from tissue with trizol. Bind the rna to polyT dynabeads. Perform first and second strand synthesis. Cut with NlaIII. Ligate Adaptor 1. Cut with MmeI. Ligate adaptor 2. Perform PCR with a primer pair that binds to sites in adaptor 1 and adaptor2, respectively.

I consistently a ladder starting at 80bp and increasing in ~40bp increments (eg, 85bp, 120bp, 155bp, 185bp, 225bp, 265bp, 300bp, etc).

Curious to see if anyone else has experienced this and if so, what they did to troubleshoot or get the adaptor2 to ligate to the Adaptor1-Tag oligo.

Thank you.

[ECO]

I haven't personally done this protocol, so take this for what it's worth...

Are you gel purifying after the second ligation? The group in this paper purify the double-adapter-ligated tags TWICE on an acrylamide gel prior to bead PCR. I would check out the supplementary methods for that paper (found on this page).

See page 5 to see how much adapter-dimer they have on the first round...

Double acrylamide gel purification!

[Macki1x]

Thanks for your suggestion regarding the gel purification step performed prior to pcr amplification.

So far, I have been following the Solexa protocol that accompanies their kit (Solexa Gene expression sample prep protocol, v2.1B) which can be found posted on the web. This protocol doesnot include a pre-pcr gel purification step of the Ad1-tag-Ad2 product.

My initial thinking is that one of the following has occurred:

1. In addition to Ad1-Tag-Ad2 products, Adapter2-adapter 2 concatamers are a possible final ligation product (particularly if the 2bp NN overhangs is chewed off to create blunt ends at each end) and can form in excess, and thereby they would outcompete the Ad1-Tag-Ad2 product in a subsequent PCR. Accordingly, gel purification of the Ad1-Tag-Ad2 would (presumably and hopefully) enrich for the target pcr template. Evidence in support of this is that I can recreate the 40bp ladder I described in the previous post using only primer 2, which binds the adaptor 2 sequence.

A second explanation may be that one of the enzymatic reactions (ie, cDNA synthesis, Ligation, Digestion, Phosphatase, etc) is not occuring and I am not generating a compatible 2bp overhang for adaptor2.

I will try and double gel purify to see if this helps and design ways to test for enzyme activity.

I appreciate any and all ideas or thoughts regarding this situation.

Cheers.

[pinghli]

Hi Macki1x,

What is going on with your before PCR gel purification? I am going to start my SOLEXA, and very curious about your results. Thanks!

[Macki1x]

solexa seq

I spent alot of time dealing with an adaptor-adaptor concatamer that formed during the reaction and swamped-out and/or killed the formation of the desired Ad1-Tag-Ad2 product.

I realize now that the main issue is that I was using adaptors that I designed based on discussions with another lab who is going to load my samples onto their genome analyzer. Turns out these adaptors are quite different from the ones provided with the kit.

The adaptors I originally used were longer than the ones provided by Solexa. For reasons that are too much trouble to explain here, these adaptors form side reactions that kill the desired reaction. I tried just about everything I could think of, and I could never get this version to work. So I bought the kit, which gave me access to the sequences of the actual Adaptors that come with the kit.

I then ordered my own oligos based on these sequences and ran them in parallel to a reaction using the set of adaptors that come with the kit. My homemade reagents and adaptors performed comparable to the kit-based reagents/adaptors.

So, if you plan to do this and are buying the kit, you should have no problems and sail through the entire procedure.

Also, there is no need to purify the Ad-Tag-Ad construct prior to the 15 cycle pcr amplification, and there is no need to gel purify the amplified Ad-Tag-Ad construct more than 1x provided you run the gel long enough to seperate the 86bp Ad-Tag-Ad from the 44 bp Ad2-Ad2 dimer that forms.

If you see a non specific smear above the 86 bp band, then back off on the concentration of template in the pcr and/or the number of cycles- this smear will go away if you do either or both of those steps,

good luck!

[pinghli]

Hi Macki1x,

Thank you so much! This is really helpful!

[SeqKing]

Hi Macki1x

Could you elaborate a little on the side reactions that kills the desired reactions?

Thanks