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  • several basic questions about adaptor and sequencing primers

    1. in the prepared library, only 5' P5 -> P7 3' strands bind to the flow cell and 5' P7 -> P5 3' strands do not. correct?

    2. in a dual index paired sequencing, the order of sequencing is Read 1, i7 index, i5 index, and then Read 2. it needs three or four sequencing primers. One is for Read 1. One is for i7. Does sequencing i5 need a sequencing primer since the synthesis is the extension from P5 adaptor binding oligo? The oligo can serve as the sequencing primer. The sequencing primer for Read 2 is the reverse complement of the i7 sequencing primer. Correct?

    3. An Illumina sequencing kit contains many sequencing primers to fit different library preparation kits. Correct?

    4. The reason to do adaptor trimming is that sequencing goes into the sequencing primer region, index region, and adaptor region because the insert is short. For the large fragment, sequencing stops before it goes into the regions. Correct? The adaptor trimming is to trim the sequencing primer region and it's 3' downstream. Correct?

  • #2
    I'll try my best to answer these questions for you szy0931

    1. This depends if you're doing a single-end or a pair-end sequencing run. I believe for single-end that only the one strand will bind, but in pair-end the 5'P7 -> P5 3' will bind. Check out this older blog that's still relevant but explains the process better than I can: https://kscbioinformatics.wordpress....are-sequenced/

    2. I believe it requires the 4 different primers because it's a different region for the Read 1, i7 index, i5 index, and Read 2. The indexes are below the region of interest (insert) so they need their own primers.

    3. The region where the index primers bind should be the same for Illumina-compatible adaptors, so as long as your library kit says it works with your sequencer, you can use it on your machine without problems.

    4. Yes, you do adaptor trimming because you don't want your adaptor sequences showing up in your final sequence results. Adaptors have contaminated sequences in the past and if not trimmed they can mess up your analysis.




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    • #3
      Hi,

      I would like to share some information and hope that it will be helpful.

      1. When library preparation, if you used the bead - based normalization, your library is single-strand DNA. It means that only one strand will be undergoing cluster generation steps on MiSeq. In case you used standard normalization, your library still is double-strand DNA. So both of 2 stands will come into flowcell and it could be binded to the flowcell when cluster generation step.

      2. Only 3 sequencing primers on MiSeq reagent cartridge: Read 1, Index 1 and Read 2. For index 2 sequencing, it oucurs during re-synthesis, which creates the reverse complement of the Index 2 (i5) index adapter sequence.

      3. No. It fits with all ILMN library prep. But for 3-party, it depends on which sequence is designed for this kit.

      4. Yes. You're correct.

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      • #4
        In regards to question 2, I believe it depends on whether your instrument does forward strand or reverse complement method for index 2 read. In the forward strand method, yes, the grafted oligo substitutes as a primer whereas in the reverse complement method you have a read 2 primer.

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