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  • reads number in single-end vs paired end seq

    Hi all,

    probably a stupid question but, i'm going to use NextSeq500 and preparing my sample with TruSeq Nano library prep kit.

    Sequencing will be performed by the V2 kit, 75 cycles.

    So, the stupid question is:
    the reads count with single end seq is up to 400M, does it means that the paired end seq will produce up to 800M reads??

    Many thanks

  • #2
    If you count like Illumina does, then yes these would be 800M reads. You will still sequence only 400M library molecules though.

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    • #3
      Yes I'll sequence 400M molecules. But if my fragmented DNA is around 150 bp the difference compared single and paired is that in the latter I'll sequence all my fragments, isn't it?

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      • #4
        Nope you are unlikely to sequence "all" your fragments since you sequence only a fraction of the library that you load on the flowcell.

        You always sequence fragment)s). If you stop after sequencing one of the ends then that is all you get. If you happen to sequence from the other end they you get spatial information that tells you how long the original library fragment is. You can know this in two ways. If you have a reference genome you can map your reads to that genome and figure out how far apart they map. If you don't have a reference genome but your insert sizes are small then you can merge the two reads to create a longer representation that gives you the size if the insert. If you only start with 150 bp inserts and say do 2 x 100 bp paired end sequencing then your reads will overlap in the middle.

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