RAPID Libraries adaptor+key sequences

[dottomarco]

I was wondering if any of you know what the adaptors A and B sequences are (+ key sequence).
Even better if you can also provide the MID sequences of the kit that is shipped by Roche for the rapid library protocol

Thank you

[ulz_peter]

Maybe this file can help you,
However I've got no idea of the MID sequences (so far).

[dottomarco]

Hy ulz_peter. I have that exact pdf, but it is the TECHNICALM BULLETIN for the amplicon protocol titanium.
I am sewarching for the exact same correspondent for the RAPID LIBRARIES.
The picture of the adaptors etc is pre-rapid as you can see from the fact that adaptor A does not have FAM attached to it.
I assume both A and B adaptors, the key sequence and the MIDs have changed with the new protocol: RAPID. And that information is exactly the one I was searching for.
Thanks

[pmiguel]

Hy ulz_peter. I have that exact pdf, but it is the TECHNICALM BULLETIN for the amplicon protocol titanium.
I am sewarching for the exact same correspondent for the RAPID LIBRARIES.
The picture of the adaptors etc is pre-rapid as you can see from the fact that adaptor A does not have FAM attached to it.
I assume both A and B adaptors, the key sequence and the MIDs have changed with the new protocol: RAPID. And that information is exactly the one I was searching for.
Thanks

Roche is not releasing the sequence of their RAPID adaptors. I have heard company reps refer to them as "hairpin" adaptors, however. So it is possible you might be able to infer their sequence from a sequence run. That said, I gave it a shot with our first RAPID run and nothing obvious was leaping out at me. The B-adaptor sequence (non-barcoded in this case) seemed the same as the non-RAPID B-adaptor sequence.

As Roche now had a deal with IDT here in the US to provide extra barcodes if the limited number offered in the RAPID kit (12) are not sufficient, my anger at Roche for withholding this information is somewhat ameliorated.

--
Phillip

[dottomarco]

We need to amplify the rapid library and I really do not understand the point of not let the users know what the adaptor's sequences are?
Any idea on how to design a couple of primers to amplify the rapid libraries?

[AKroxy]

Here is the info I got from the Roche rep when I asked for the RAPID adaptor sequences so I could design PCR primers.

The adaptors are double stranded. The sequences TCAG at 3' end of each long adaptor strand are the general Ti shotgun key sequences. The key sequence is different with Rapid Library. The following figure will help in explaining better. You can order the Primers below to check your library.

emPCR Primer A 5' CCATCTCATCCCTGCGTGTC 3'
TI A Adaptor 5' CCATCTCATCCCTGCGTGTCTCCGACTCAG 3'
3' AGAGGCTGAGTC 5'

emPCR Primer B 5' CCTATCCCCTGTGTGCCTTG 3'
TI B Adaptor 5' CCTATCCCCTGTGTGCCTTGGCAGTCTCAG 3'
3' ACCGTCAGAGTC 5'

[dottomarco]

Thanks a lot AKroxy ! Roche reps around the word must have different conception of "confidential" information.
At list now we know that the adaptors are the same as the "standard" adaptors except for the key sequence that still remains unknown.

[pmiguel]

Thanks a lot AKroxy ! Roche reps around the word must have different conception of "confidential" information.
At list now we know that the adaptors are the same as the "standard" adaptors except for the key sequence that still remains unknown.

Rapid libraries use "GACT" as the key bases.

--
Phillip

[0Gen]

This Y adaptor variant might be of interest.

http://nar.oxfordjournals.org/cgi/content/full/gkq332

[Aero]

Rapid Library Adpators

I thought there should be a 3' A-overhang after the key sequence for TA-ligation of insert and adaptor...is that correct?

[AKroxy]

I was told that there was an overhang (shouldn't it be a T?) by someone at Roche that I spoke to on the phone. I don't know why it doesn't appear in the sequences above. Often the emails from my rep are cryptic and confusing.

[Aero]

Hi AKroxy,

Sorry for the typo and thank you for pointing it out...I actually meant a T-overhang for the adaptor. And it's a 3' A-overhang for the insert.

[0Gen]

Hi,

The RL adaptor seems to be a Roche secret. There is a recent paper uses an Y adatpor + Taqman-MGB qPCR + Poisson distribution.

..................5'C*C*A*T*C*T*CATCCCTGCGTGTCTCCGA*C*T*C*A*G*T3'
..............................................||||| | | | | |
3'G*G*A*T*A*G*GGGACACACGGAACAGATAGGGGACAACGCACAGGCT*G*A*G*T*Cp5'



A single Y adaptor has adavantages over two adaptors (A and B), as it showed in Fig.1.

http://nar.oxfordjournals.org/cgi/co...bstract/gkq332

[tokikake]

I don't know, if the sequence of the rapid adaptors is still of interest, but here is an official document from roche.

Cheers!

[orangekel13]

Thanks for posting that document, tokikake! I looked and looked and couldn't find that anywhere!