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  • #16
    I have a question about the adapter sequences; for one of the adapter sequences you have a single base difference between the SR adapter and the PE adapter. In the official communication from Illumina that lists all their sequences there is no difference in the unphosphorylated adapter sequence between SR and PE adapters they are both 33 bases long. Where did you get the sequences from (as it's not the first time I've heard about the single base difference but there's definitely none in the sequences that I have).

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    • #17
      I am a little confused about this.

      In fig 6, the DNA is attached to flow-cell by both red and purple adapters in the same cluster. However, in fig 7, it is attached only by red adapters.

      What causes it to happen?

      Both adapters would mean the DNA strands in clusters are not identical, but half of them are complimentary to the other half.

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      • #18
        When beginning the sequencing, first a cleaving step is carried out,
        removing the sequences bound with adapter2 to the cell-surface.

        For paired-end sequencing, after having reached the desired cycle-count the synthesized strand is removed, another step of bridge amplification is carried out, followed by a cleaving of sequences bound with adapter1 to the cell surface. Thus leaving the `other'/reverse strand bound to the flow cell for sequencing...

        Anything else?
        Last edited by Jonathan; 09-30-2009, 05:55 AM.

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        • #19
          why PE1 and SE1 adapter sequece has 1 base different?

          I found that they should be identical. The attached diagram is wrong.

          compare diagram 1 and 2, adaptor sequence is changed!!!


          Originally posted by greigite View Post
          I didn't really understand the Illumina library prep and sequencing procedure until I played around with aligning the actual sequences. This resulted in a diagram of the ligation/PCR/hybridization/sequencing process for both single read and paired end reads using the publicly available adapter, primer, and flow cell oligo sequences. Hope it is useful to others, and let me know if you find errors. Thanks to kmcarr for posting your diagram of part of the single read process earlier, it inspired me to work it out for myself. You can find all the sequences I used in the 2008 nature paper, doi 10.1038/nature07517.

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          • #20
            Software choices?

            Hi there,

            I have a question about the software I can use to analyze the Illumina sequencing data. I'm doing targeted resequencing of long PCR fragments to find some EMS mutations. I'm waiting for my run to finish right now and meanwhile I'm looking for a suitable software. Any suggestion would be of great help to me. Thank you.

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            • #21
              illumina bridge amplification

              Can anyone explain how successive rounds of pcr are carried out in the bridge amplification step to form a cluster? what is the primer and is it separate or different from the oligos attached to the cell? are the unattached strands of dna discarded between successive pcr steps? if not, can anyone explain what happens to them?

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              • #22
                Fluorophore removal?

                Originally posted by mccullou View Post
                How does Illumina remove the fluorophore? They can't use AgNO3 (like SOLiD ) because of no Thio backbone. Are they using the Columbia IP that Intelligent BioSystems is using? http://www.intelligentbiosystems.com...20mod%201.html.

                Any thoughts here?
                This would be of interest to me as well!

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                • #23
                  Originally posted by tobiwan View Post
                  This would be of interest to me as well!
                  You definitely need to read this review.

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                  • #24
                    Fluorophore removal?

                    Thanks for the hint! I am new to this field of work...

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                    • #25
                      thanks all. very useful information for me.

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                      • #26
                        the chemistry is proprietary.

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                        • #27
                          So are most buffers if you ask qiagen or invitrogen, but we are all smart people here, right? It is either photo or chemical cleavage and looking at all the steps in the sequencing process there doesn't seem to be a hint. I am just curious because I have been having increasing background and phase issues and I think it might be inefficient removal of that flourophore/blocking agent, but I can't figure it out my issue if I don't know how the base is removed. . .

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                          • #28
                            Hi All

                            I have just joined SeqAnswers and I am hoping to get some advise from those who are currently doing sequencing on the Illimina GA. I am hoping to do an infection profile for infected plants using the Paired-end muliplex system. Is this advisable? Or should I run each infection/control pair per lane?

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                            • #29
                              Thanks a lot!
                              Very interesting for me taking into cosideration my little experience!

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                              • #30
                                comparison of illumina/ ABIsolid/ 454

                                simple comparison
                                Attached Files

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