I'm working on a special library prep scheme for a specialized project that uses a custom read 1 and read 2 sequencing primer. In principle, the first read will always work, but only the correctly formed fragments will have a read 2 complementary sequence. In both cases, it will still have the P5/P7 sequence for bridge/cluster amplification.
How would this look in a Miseq/Hiseq run? Would it give me a bunch of Ns in my read 2 since there would be no intensity associated with the location of the cluster (and probably kill my Q-score)? Would this affect any kind of registration issues for the other properly formed clusters? I'm guessing these types of reads would be easy to filter out post-run...
Or would it just be much easier to reverse the read on which an improperly formed fragment may form (eg. read 1 may fail sometimes)? In this case the cluster would just fail to register in RTA for the first few cycles. I've already made some preliminary libraries so it would be more work to "reverse" everything.
How would this look in a Miseq/Hiseq run? Would it give me a bunch of Ns in my read 2 since there would be no intensity associated with the location of the cluster (and probably kill my Q-score)? Would this affect any kind of registration issues for the other properly formed clusters? I'm guessing these types of reads would be easy to filter out post-run...
Or would it just be much easier to reverse the read on which an improperly formed fragment may form (eg. read 1 may fail sometimes)? In this case the cluster would just fail to register in RTA for the first few cycles. I've already made some preliminary libraries so it would be more work to "reverse" everything.
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