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  • Illumina Adapter and Primer preparation

    Hi!

    I have ordered the Illumina PE primers and PE adapters from IDT with the required modifications. I have combed through the forum, but have not been able to find answers to two of my questions. There are references to method sections from published papers, nevertheless, I would like to know what people are currently doing for the following:


    1) What solution do you re-suspend the (i) adapters and (ii) primers in?

    2) What protocol do you use for annealing the adapters? Specifically, what concentration are the adapters when you heat to 95 degrees? (Is it at the stock concentration of 100 uM or the final concentration of 15 uM?) Do you let the adapters cool down to RT in the PCR machine, or just take it out of the heat-block and leave it on the bench? Is there a way of confirming that the adapters annealed properly?

    I would truly appreciate hearing back. Thank you all VERY much!

    Regards,
    -Sushma

  • #2
    Hi Sushma,

    I have attached our lab protocol for annealing the individual adapters to create the y-shaped product. It is for home-brew indexed SR adapters but I have also used it for our PE stocks. It is based on the original Illumina patent and Nat Methods 2008 Oct;5(10):887-93 paper. While the working stock is stated as 15 uM (from original patent) - we have been using a 1:2 for our RNAseq or 1:20 dilution of the working stock for our ChIPseq libraries - cuts down the potential for adapter dimers. Best of luck.
    Attached Files

    Comment


    • #3
      Cool! Thank you so much!!

      May I ask also since you mentioned RNA-seq, how much RNA (/cDNA) you start with for library prep for RNA-seq, and if possible, can you also attach your RNA-seq protocol?

      Thanks a million again!
      Sushma

      P.S- Also, do you re-suspend primers also in the Tris-NaCl buffer or just in water?
      Last edited by S.Iyengar; 07-19-2011, 03:27 PM. Reason: Additional question

      Comment


      • #4
        Good for HiSeq?

        Hi Sushma and protist,

        Someone told me that the Genome Analyzer adapters and the HiSeq adapters are different. Is that true?
        Can I use this adapter for HiSeq?

        Thanks,

        Luis

        Comment


        • #5
          illumina adaptor (containing truseq adaptor) is compatible with Genome Analyzer and HiSeq.
          Your library can be perform sequence with both sequencers.

          Comment


          • #6
            How about paired end and multiplex? Do they have same read 2 sequencing primer? Because I can't find the similarity (not sure if the sequence I got for both adapters are the same).

            Paired end adapter (P7), apart from the 5' end (for flow cell binding) and the 12-13bp complement region (adapter fork), the middle part has different sequence with Multiplexing adapter (p7). This means they both have different read 2 sequencing primer?

            Does GA and HiSeq use different read 2 sequencing primer as well? If I were to make my own adapter, paired end multiplexing, what is the important region that I have to include in the adapter so that the read 2 primer can bind, and also the index can be read?

            Thanks.

            Comment


            • #7
              Originally posted by protist View Post
              Hi Sushma,

              I have attached our lab protocol for annealing the individual adapters to create the y-shaped product. It is for home-brew indexed SR adapters but I have also used it for our PE stocks. It is based on the original Illumina patent and Nat Methods 2008 Oct;5(10):887-93 paper. While the working stock is stated as 15 uM (from original patent) - we have been using a 1:2 for our RNAseq or 1:20 dilution of the working stock for our ChIPseq libraries - cuts down the potential for adapter dimers. Best of luck.
              Hi,

              I am new in this community and saw the protocol of protist concerning the home-brew forked adaptors. We will also make our own adaptors for Sequencing with a Illumina MiSeq. In the protocol there is the Thermocyling step explained. Do you perorm a PCR with Taq-Polymerase or only the cycling of the 2 oligos??
              It would be graet if you could answer , Thanks

              Comment


              • #8
                Originally posted by Akira View Post
                How about paired end and multiplex? Do they have same read 2 sequencing primer? Because I can't find the similarity (not sure if the sequence I got for both adapters are the same).

                Paired end adapter (P7), apart from the 5' end (for flow cell binding) and the 12-13bp complement region (adapter fork), the middle part has different sequence with Multiplexing adapter (p7). This means they both have different read 2 sequencing primer?

                Does GA and HiSeq use different read 2 sequencing primer as well? If I were to make my own adapter, paired end multiplexing, what is the important region that I have to include in the adapter so that the read 2 primer can bind, and also the index can be read?

                Thanks.
                Previously Illumina had two adapter sets, a standard paired end and a multiplex paired end one. The main difference was that the mulitplex libraries required a different read 2 primer (and also the index read primer). The Illumina TruSeq adapters contain the multiplex read 2 primer sequence so I would base your designs on the MP adapters. If you can use Google, you should be able to find the Illumina customer letter where they publish all their sequences.

                Comment


                • #9
                  Credit

                  Originally posted by protist View Post
                  Hi Sushma,

                  I have attached our lab protocol for annealing the individual adapters to create the y-shaped product. It is for home-brew indexed SR adapters but I have also used it for our PE stocks. It is based on the original Illumina patent and Nat Methods 2008 Oct;5(10):887-93 paper. While the working stock is stated as 15 uM (from original patent) - we have been using a 1:2 for our RNAseq or 1:20 dilution of the working stock for our ChIPseq libraries - cuts down the potential for adapter dimers. Best of luck.
                  Hello,
                  I used the protocol you posted here. I would like to know how to give credit to it. Jose

                  Comment


                  • #10
                    Originally posted by protist View Post
                    Hi Sushma,

                    I have attached our lab protocol for annealing the individual adapters to create the y-shaped product. It is for home-brew indexed SR adapters but I have also used it for our PE stocks. It is based on the original Illumina patent and Nat Methods 2008 Oct;5(10):887-93 paper. While the working stock is stated as 15 uM (from original patent) - we have been using a 1:2 for our RNAseq or 1:20 dilution of the working stock for our ChIPseq libraries - cuts down the potential for adapter dimers. Best of luck.
                    Hello,
                    Is it possible for you to re-upload the file? I was trying to download it and I am unable to do so.
                    Thank you.

                    Comment

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