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  • aushev
    replied
    ok, finally I found by myself
    according to published patent 20100279305, adaptor oligo mix contains
    1) 5'-CCUCUCUAUGGGCAGUCGGUGAU (3.1 μM)
    2) 5'-NNNNNNATCACCGACTGCCCATAGAGAGG (6.1 μM)
    3) 5'-PO4--CGCCTTGGCCGTACAGCAG (3.1 μM)
    4) 5'-CTGCTGTACGGCCAAGGCGNNNNNN (6.1 μM)

    Leave a comment:


  • aushev
    replied
    well, for the moment I am working with microRNA, i.e. don't care about polyA.
    (to tell the full story, I am trying to understand if there is a way to make miRNA->cDNA library compatible for both sequencing and qPCR - that's why my question about adapters arose)

    Leave a comment:


  • pmiguel
    replied
    Yeah, Ambion is playing that pretty close to their vest.

    Unfortunate because with the recent major drop in the cost of both Illumina library construction kits and vast increase in the amount of sequence produced per run, I fear the SOLiD may no longer be competitive for RNA seq.

    Reagent costs for the SOLiD RNA sequence library construction kit needs to drop down to around $50/sample. The Illumina kit is around $70/sample -- but that includes polyA isolation!

    --
    Phillip

    Leave a comment:


  • aushev
    replied
    What about adaptors used for RNA ligation in "Total RNA-Seq" kit? if I am not wrong, their sequenced are not disclosed here?
    Thanks in advance.

    Leave a comment:


  • harrike
    replied
    Originally posted by yksikaksi View Post
    You can refer to this appendix D (page 206 onwards) of Applied Biosystems SOLiD™ 4 System Library Preparation Guide which covers:
    1. Library construction oligonucleotides
    2. Adaptor sequences
    3. Multiplex adaptor and barcode sequences
    Thank you very much. That's exactly what i want.

    Leave a comment:


  • yksikaksi
    replied
    You can refer to this appendix D (page 206 onwards) of Applied Biosystems SOLiD™ 4 System Library Preparation Guide which covers:
    1. Library construction oligonucleotides
    2. Adaptor sequences
    3. Multiplex adaptor and barcode sequences

    Leave a comment:


  • KevinLam
    replied
    Is there an official download source for the updated SOLiD adaptor sequences?
    The one I am using is from the bioscope example dataset
    ./applications/wholeTranscriptome.run/.run/reads/human_filter_reference.fasta

    looking at this list I think that file which I had previously thought sufficient seems to be quite lacking!
    other than the human tRNAs and line repeats,
    it lists the 16 barcodes
    >adaptor + bc16 + P2
    and only
    >P1 adaptor.

    Leave a comment:


  • pmiguel
    replied
    Originally posted by volks View Post
    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

    Leave a comment:


  • volks
    replied
    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.

    Leave a comment:


  • pmiguel
    replied
    Originally posted by volks View Post
    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/showpos...81&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

    Leave a comment:


  • volks
    replied
    Originally posted by AdrianCarr View Post
    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)?

    Leave a comment:


  • pmiguel
    replied
    Originally posted by Nitrogen-DNE-sulfer View Post
    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

    Leave a comment:


  • Nitrogen-DNE-sulfer
    replied
    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.

    Leave a comment:


  • AdrianCarr
    replied
    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

    Leave a comment:


  • pmiguel
    replied
    Originally posted by mulligan View Post
    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

    Leave a comment:

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