I work in a core facility with that's just gotten into next-gen sequencing with the purchase of a MiSeq. It's been a learning experience! I'm trying to write a short, but detailed, introduction to how MiSeq sequencing actually works and what a constructed library looks like, including detailed explanations of what's going on with the TruSeq adapters.
I'm stuck at a couple of key points though and I hope someone will have some suggestions! The first concerns when and how the index sequence is read on the MiSeq. My understanding is that, after cluster generation, the clusters are all attached to the flow cell by the universal P5 (not indexed) adapter. The sequencing primer that is hybridized on is attached at the priming site on the indexed adapter (P7). Sequence is then generated downward toward the flow cell until Read 1 is complete.
Somewhere in between Read 1 and Read 2 the index read occurs and the Illumina website doesn't seem to have details on this. I assume that turnaround and cluster regeneration must happen first so that the index is now at the "top" of the cluster rather than just above the flowcell binding site. Is there a separate primer for the index read that is located within the P7 attachment region? That would mean that this primer would anneal, the index reads would occur, all that would be washed off, and the Read 2 primer hybridized? If anyone has some input on this or great websites that explain things, please post them!!!!!
Also, all of these questions came about due to difficulties people are having in removing adapter dimer sequences from their data. The current thought is that in Read 1 (originating from the priming site on the Universal Adapter), data should never begin with the reverse complement of the Indexed Adapter. In Read 2 (originating from the priming site on the Indexed Adapter), data should never begin with the reverse complement of the Universal Adapter.
Sorry this is so long, and thank you to anyone with any thoughts and ideas!
I'm stuck at a couple of key points though and I hope someone will have some suggestions! The first concerns when and how the index sequence is read on the MiSeq. My understanding is that, after cluster generation, the clusters are all attached to the flow cell by the universal P5 (not indexed) adapter. The sequencing primer that is hybridized on is attached at the priming site on the indexed adapter (P7). Sequence is then generated downward toward the flow cell until Read 1 is complete.
Somewhere in between Read 1 and Read 2 the index read occurs and the Illumina website doesn't seem to have details on this. I assume that turnaround and cluster regeneration must happen first so that the index is now at the "top" of the cluster rather than just above the flowcell binding site. Is there a separate primer for the index read that is located within the P7 attachment region? That would mean that this primer would anneal, the index reads would occur, all that would be washed off, and the Read 2 primer hybridized? If anyone has some input on this or great websites that explain things, please post them!!!!!
Also, all of these questions came about due to difficulties people are having in removing adapter dimer sequences from their data. The current thought is that in Read 1 (originating from the priming site on the Universal Adapter), data should never begin with the reverse complement of the Indexed Adapter. In Read 2 (originating from the priming site on the Indexed Adapter), data should never begin with the reverse complement of the Universal Adapter.
Sorry this is so long, and thank you to anyone with any thoughts and ideas!
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