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  • #16
    Yes, polyhedron, you can just change the adaptors you use to add an index onto your read. This is discussed in detail elsewhere in these forums. For PE reads, you only need to change PE adaptor 1. I'll give an example here:

    +adaptor 1 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCT*T 3'
    -adaptor 1 5' [Phos]-AGGTTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 3'

    where the * is a phosphotioate and [Phos] is a phosphate when you order the oligos. You then need to anneal these two oligos together by mixing them to a final concentration of 15 uM each in TBS, heating them to 95C and cooling them slowly to room temperature to get the adaptor. Then you go ahead with the regular SE (with this adaptor) or PE (with the regular PE adaptor 2) sample preparation.

    This essentially adds AACCT to the regular adaptors followed by the required T end, and thus your first six bases of read will be AACCTT. You need to be careful to balance out the A, C, G, T content of the first four bases of your adaptors so that the phasing and intensity levels are calculated correctly by the software. So, four indexes could be:

    AACCTT
    CCGGAT
    GGTTGT
    TTAACT

    Others, feel free to correct me or improve on this comment. I'm only just beginning to do multiplex runs.

    I will also add here (because I'm not a fan of how much Illumina charges for its kits) that New England Biolabs sells a kit that has everything in it for sample preparation except for the oligos for way cheaper than Illumina charges. Catalogue number E6000S or E6000L.

    Comment


    • #17
      Hi,

      We are about to order custom adaptors for SE (without barcodes). What exactly is the phosphotioate modification for?

      Thanks

      Comment


      • #18
        Hi andibody,

        The phosphothioate bond is supposed to prevent degradation at the end of the adaptor. It is not absolutely essential, but is supposed to help with higher yield. I have had fine luck with adaptors that lacked the phosphothioate, so it isn't essential.

        Comment


        • #19
          Originally posted by NextGenSeq View Post
          I would be suspicious of the gel purification. Exposing nucleic acids to UV can nick and mutate it. I try to avoid gel purification if at all possible.

          Has anyone substituted a size selection column such as a QIAQuick? This should remove short ligation artifacts.
          You can use a visible light stain and a white reader for a gel extraction- Nile Blue works for me and although I haven't tried it I think GelGreen with a dark reader might also work well.

          Comment


          • #20
            Didn't Illumina replace the gel size selection with AmPure beads from Agencourt in their new protocols?

            Comment


            • #21
              Attached is the Agilent Sure Select protocol with the AmPure replacing the gel size selection. They used to use gel size selection in the earlier protocol. Thus, obviously they had problems with gel size selection also.
              Attached Files

              Comment


              • #22
                Blocking needed?

                Originally posted by BearClaw View Post
                Yes, polyhedron, you can just change the adaptors you use to add an index onto your read. This is discussed in detail elsewhere in these forums. For PE reads, you only need to change PE adaptor 1. I'll give an example here:

                +adaptor 1 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCT*T 3'
                -adaptor 1 5' [Phos]-AGGTTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 3'

                where the * is a phosphotioate and [Phos] is a phosphate when you order the oligos. You then need to anneal these two oligos together by mixing them to a final concentration of 15 uM each in TBS, heating them to 95C and cooling them slowly to room temperature to get the adaptor. Then you go ahead with the regular SE (with this adaptor) or PE (with the regular PE adaptor 2) sample preparation.

                This essentially adds AACCT to the regular adaptors followed by the required T end, and thus your first six bases of read will be AACCTT. You need to be careful to balance out the A, C, G, T content of the first four bases of your adaptors so that the phasing and intensity levels are calculated correctly by the software. So, four indexes could be:

                AACCTT
                CCGGAT
                GGTTGT
                TTAACT

                Others, feel free to correct me or improve on this comment. I'm only just beginning to do multiplex runs.

                I will also add here (because I'm not a fan of how much Illumina charges for its kits) that New England Biolabs sells a kit that has everything in it for sample preparation except for the oligos for way cheaper than Illumina charges. Catalogue number E6000S or E6000L.
                If one uses this approach to barcode, and then uses Sureselect, is there still a need to block the barcode?
                Has anybody had success with this approach?
                Last edited by TYS; 06-11-2010, 11:41 AM. Reason: typo/error

                Comment


                • #23
                  The SureSelect kit comes with three blocking reagents. I added 0.6 uL of a fourth blocking reagent. The fourth blocking reagent was made by mixing equal volumes of each 100 uM index primer (+ and - strands). I didn't try this protocol without the added index primer in the block, so am only assuming that it is needed.

                  Comment


                  • #24
                    Originally posted by BearClaw View Post
                    I am interested in finding out what the components of Block #1, Block #2, and Block #3 are. Does anyone know? I am sure that one of them is probably something equivalent to Cot1 DNA and the others are probably blocking oligonucleotides.
                    In the article 'Enrichment of sequencing targets from the human genome by solution hybridization', Tewhey et al. 2009. They use the Agilent SureSelect baits and say in m&m:

                    "500 ng of each genomic DNA-Fragment library was mixed with 2.5 ug of human Cot-1 DNA, 2.5 ug of salmon sperm DNA, and 1 unit of blocking oligonucleotides complementary to the Illumina single-end adapter"

                    So my guess is these are Block#1,#2 and #3. Does anyone have an idea about the rationale of adding the salmon sperm?

                    Comment


                    • #25
                      People have used salmon sperm DNA for decades in southern blots to reduce background from false hybridizations. Same thing here.

                      Comment


                      • #26
                        Originally posted by NextGenSeq View Post
                        People have used salmon sperm DNA for decades in southern blots to reduce background from false hybridizations. Same thing here.
                        The salmon sperm binds aspecific to the baits and gets replaced by target DNA during hybridisation? Then you get lots of salmon sperm in your enriched fraction, since there is an excess of baits. Or is this removed during washing?

                        Comment


                        • #27
                          Both Cot-1 and SSDNA block repetitive regions, supposedly Cot-1 blocks more effectively though so I'm not sure why they use both.

                          Comment


                          • #28
                            Originally posted by JUdw View Post
                            The salmon sperm binds aspecific to the baits and gets replaced by target DNA during hybridisation? Then you get lots of salmon sperm in your enriched fraction, since there is an excess of baits. Or is this removed during washing?
                            It doesn't matter if salmon sperm DNA is present. It's not a substrate for PCR with adapter-specific primers, and it won't anneal to the flow cell (for the same reason).

                            Comment


                            • #29
                              I believe he salmon sperm is merely there to take up space to push other molecules of DNA together. It can also act sacrificially should a random nuclease wander in or if the tube material is sticky for DNA.

                              Comment


                              • #30
                                To BearClaw,
                                Before reading about multiplexing, i had imagined that the simplest way to do the job would indeed look how you describe it (answer to polyhedron) rather than the way Solexa protocol goes.
                                Have you actually sequenced some libraries this way, did you get good yields and homogenous distribution between indexes? When do you actually pool together your libraries?
                                Last edited by matlavmac; 09-13-2010, 01:31 AM.

                                Comment

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