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  • Library prep question - ligation of P1 and P2 adapters

    Hi there,

    I'm trying to grasp how the nick translation works exactly after ligating the P1 and P2 adapters to A-tailed DNA fragments. Does the reaction at 72oC exchange the nucleotides of the insert DNA (5' to 3') or does it go in the other direction, only exchanging the nucleotides in the adapter sequences towards the 'outisde'? This is very important to us since we want to bisulfite-convert our libraries after ligation. Can anyone shed light on this?

  • #2
    Above question is about the Solid 5500 single-ended library prep

    Comment


    • #3
      After ligation wouldn't there be 4 nicks? 2 on each strand? if that is the case then it seems that which ever direction the reaction has the insert will be exchanged?

      Comment


      • #4
        There's a diagram in this paper (from Life Technologies) that suggests that only the adaptors are affected (and also that I am wrong about the '4 nicks' thing.......)

        Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.


        JPC

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        • #5
          Thanks JPC, it looks like only the adapters are in fact nick translated.

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