SOLiD Adapters

[exprofiler]

Hello,
I would like to sequence gene expression tags with the SOliD machine-does anybody have the adapter-sequences flanking the inserts or do you know where could I get them?
Also, is there a relaible service provider for the SOliD system out there ?
Thank you very much in advance!

[universalprimer]

Here are the sequences for most of the current solid oligos, I tried to display it like the post in the solexa forum. I don't know the sequences of the P1 primer on the bead, the actual sequencing primers, or all the "block" oligos.

SOLiD 2.0 Oligos

P1 Adapter:
5' -----------------CC ACTACGCCTCCGC TTTCCTCTCTATGGGCAGTCGGTGAT (-) -------------------- -------------------- - 3'
3' ---------------TTGG TGATGCGGAGGCG AAAGGAGAGATACCCGTCAGCCACTA (-) -------------------- -------------------- - 5'

P2 Adapter:
5' -------------------- -------------------- ------------------ (-) AGAGAATGAGGAACCCGGGG CAGTT--------------- - 3'
3' -------------------- -------------------- ------------------ (-) TCTCTTACTCCTTGGGCCCC GTC----------------- - 5'

Library PCR Primer1:
5' -----------------CC ACTACGCCTCCGC TTTCCTCTCTATG------------- (-) -------------------- -------------------- - 3'
3' -------------------- -------------------- ------------------ (-) -------------------- -------------------- - 5'

Library PCR Primer2:
5' -------------------- -------------------- ------------------ (-) -------------------- -------------------- - 3'
3' -------------------- -------------------- ------------------ (-) TCTCTTACTCCTTGGGCCCC GTC----------------- - 5'

Resulting Frag Library:
5' -----------------CC ACTACGCCTCCGC TTTCCTCTCTATGGGCAGTCGGTGAT (N) AGAGAATGAGGAACCCGGGG CAG----------------- - 3'
3' -----------------GG TGATGCGGAGGCG AAAGGAGAGATACCCGTCAGCCACTA (N) TCTCTTACTCCTTGGGCCCC GTC----------------- - 5'


Mate pair oligos

EcoP15I Cap Adapter (note AC 3' overhang on one end)
5' -------pCTGCTGTAC---------- - 3'
3' --------GACGACAp----------- - 5'

Internal Adapter (note: lowercase T in top strand has internal biotin, note GT 3' overhangs for sticky end circularization)
5' --------CGTACAtCCGCCTTGGCCGT----- - 3'
3' ------TGGCATGTAGGCGGAACCGG------- - 5'

[exprofiler]

thank you !!

[snetmcom]

there are a few service providers out there. Your best bet would be to contact ABI. Seqwright and Agencourt are the two big ones, but I wouldnt send my samples anywhere other than agencourt.

[atp]

Does anybody know the sequence of the multiplexing adapters (barcoding) for the SOLiD system?

[Chipper]

Yes, but you should contact ABI since it is not officaly released yet...

[mulligan]

Y-Based SOLiD Adaptors

Has anyone heard if AB was planning on switching to Y-based adaptors and A- tailing for fragment libraries?

[pmiguel]

Has anyone heard if AB was planning on switching to Y-based adaptors and A- tailing for fragment libraries?

I have not heard anything along those lines. Seems like there would be a down side to y-adaptors. They would introduce some self-complementarity into the amplicons that could make the template strands less available for replication. And if you plan on doing any pre-ePCR PCR at all, they are unnecessary because the PCR "suppression effect" drastically favors amplification of amplicons not flanked by the same adaptor type.

--
Phillip

[AdrianCarr]

Does anyone know the sequence of the 'LMP CAP Adapter' used in the long mate-paired protocol? I assume it is similar to the EcoP15I Cap Adapter...

Thanks

Adrian

[Nitrogen-DNE-sulfer]

I saw a slide at AGBT which suggested AB is making sequencing primers to allow customers to sequence their ILMN libraries on SOLiD. These would leverage the Y adaptors.
Speaking to the Invitrogen people, TA cloning is much more efficient than blunt cloning and can be leveraged with either approach. If you look at oligos above you can remove one A from them (near the N) and get them to be compliant with TA cloning. You probably need to tweak the adaptor to insert ratio to reduce dimers as blunt requires a lot more adaptor.
Also need to alter the end repair reaction to ensure PlusA. The Sequencing primers should be unaffected as the Plus A is just replicating the 3'A on the oligo so the end molecules is the same.

We've also seen the use of thioates on the TT overhangs to help. We see the adaptors over time or with different oligo lots have a different propensity to form dimers. We attribute this to the TT tails being hydrolyzed or exo'd and thus the dimer which forms is 60-70bp. The only way this dimer size makes sense is if there is tail to tail dimerization which then dimerizes again.
would love to know if anyone has sanger sequenced these to confirm what the dimers are as the size after PCR doesnt make sense unless more than 2 molecules are coming together.

I think the biggest pick up is in A-Tailing. The Y-adaptors on paper should help get 2 X more as every strand gets P1 and P2 where as the AB method is a punnett square with P1-P1 and P2-P2 molecules being formed but presumably not amplifying so 50% of the inserts are wasted. The reason I say on paper is I have heard the Y-adaptors have some suppression PCR effects as well and havent seen a direct comparison of the yield between the methods.

[pmiguel]

I think the biggest pick up is in A-Tailing. The Y-adaptors on paper should help get 2 X more as every strand gets P1 and P2 where as the AB method is a punnett square with P1-P1 and P2-P2 molecules being formed but presumably not amplifying so 50% of the inserts are wasted. The reason I say on paper is I have heard the Y-adaptors have some suppression PCR effects as well and havent seen a direct comparison of the yield between the methods.

2x seems like a lot--but is it? As we have discussed before, these protocols are so inefficient that they often have you start with a million-fold more sample molecules than actually end up making it through the full procedure. (With Illumina and SOLiD protocol it is hard to even estimate what the final molecular yield is, because they generally will entail at least some PCR amplification along the way.)

Are blunt end ligations really less efficient than single base 3' complementary base overhangs? Historically the T-tailed cloning vectors seemed to become popular as quick ways to clone PCR products. But this was because Taq polymerase added a non-templated 3' A base. And Taq is the very devil to get rid of (being thermostable and all) sufficiently that polishing enzymes could remove the base without the A just getting added back again. Basically you could row upsteam or downstream with your protocol.

--
Phillip

[volks]

Does anyone know the sequence of the 'LMP CAP Adapter' used in the long mate-paired protocol? I assume it is similar to the EcoP15I Cap Adapter...

I would also be interested. Also, does someone know which part is required for ligation primer hybridization (or the sequence of the ligation primer)?

[pmiguel]

I would also be interested. Also, does someone know which part is required for ligation primer hybridization (or the sequence of the ligation primer)?

The internal and cap adaptor sequences were posted up thread:

http://seqanswers.com/forums/showpost.php?p=1681&postcount=2

The internal adaptor+ CAP Adaptors ligate into:

5'-CTGCTGTACCGTACATCCGCCTTGGCCGTACAGCAG-3'

The 3' terminus, or there about, would be your ligation primer. I'm not sure the exact length.

--
Phillip

[volks]

The question was whether the 'EcoP15I Cap Adapter' and the 'LMP CAP Adapter' are the same. So far I couldn't find the 'LMP CAP Adapter' sequence.

[pmiguel]

The question was whether the 'EcoP15I Cap Adapter' and the 'LMP CAP Adapter' are the same. So far I couldn't find the 'LMP CAP Adapter' sequence.

They have the same sequence. They differ only in the 5' phosphorylation state of the shorter oligo. This is from p. 206 of the SOLiD 4 System Library Preparation Guide:

EcoP15I CAP Adaptor, 50 µM
5′ Phos-CTGCTGTAC-3′
5′ Phos-ACAGCAG-3′

LMP CAP Adaptor, 50 µM
5′ Phos-CTGCTGTAC-3′
5′ -ACAGCAG-3′

--
Phillip