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  • Why Sanger sequencing can have the forward and reverse reads?

    I read the page and get to know Paried end reads are most oriented to the NGS, like Illumina aiming to get longer sequencing read.



    But (anything gets a but), I received data set from Sanger sequencer. For each read, they have both forward and reverse reads (aka, paried-end).

    From what I read, paired reads are basically:

    fragment ================================
    fragment + adaptors ~~~================================~~~
    SE read --------->
    PE reads R1---------> <---------R2
    unknown gap .........................................

    But after alignment, my one looks like in the following img, they have some region in common



    Anyone knows why they are quite diff? thx

  • #2
    In my experience, typically you sequence amplified fragments in both directions (once using the forward primer, and once using the reverse primer) in Sanger sequencing mainly for checking the accuracy of the base calls in the overlapping regions.

    In your example above, you can see that the sequence quality gets really low as you get towards the end of the read, but the other one is high-quality at this point. If you just sequenced it using the forward primer then you would not have very much confidence in the base calls from around 1275-1325.

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    • #3
      Originally posted by atcghelix View Post
      In my experience, typically you sequence amplified fragments in both directions (once using the forward primer, and once using the reverse primer) in Sanger sequencing mainly for checking the accuracy of the base calls in the overlapping regions.

      In your example above, you can see that the sequence quality gets really low as you get towards the end of the read, but the other one is high-quality at this point. If you just sequenced it using the forward primer then you would not have very much confidence in the base calls from around 1275-1325.
      Thanks for your quick reply.

      You mean in sanger, the forward and reverse reads are JUST for base calling quality? So may I know more on this? I think actually we do not need to do this since the Sanger sequencing usually have a fair well quality on each base. What do you think?

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      • #4
        Well I'm not sure if they're really used for anything else, but nothing that I know of. I do know sometimes people will only sequence in one direction to save money. Your sequence there looks like it's good quality, but certainly not all Sanger sequences are of good quality.

        In your example above, starting at location around 1310 it looks like you don't have good quality. Having the reverse read allows you to call the bases from then until the end of the fragment.

        Sometimes the quality of the reads will deteriorate far before the end of the fragment, in which case having the other direction is essential for reading the full sequence.

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        • #5
          Originally posted by atcghelix View Post
          Well I'm not sure if they're really used for anything else, but nothing that I know of. I do know sometimes people will only sequence in one direction to save money. Your sequence there looks like it's good quality, but certainly not all Sanger sequences are of good quality.

          In your example above, starting at location around 1310 it looks like you don't have good quality. Having the reverse read allows you to call the bases from then until the end of the fragment.

          Sometimes the quality of the reads will deteriorate far before the end of the fragment, in which case having the other direction is essential for reading the full sequence.
          Thanks. It seems like more related with the quality.

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          • #6
            There's no real Sanger sequencing equivalent to paired-end reads in NGS. What you have are sequences originating from different primers run in different wells. There are two main reasons to do that. Firstly and as mentioned above, base-call quality decreases with the length (this makes sense if you visualize how Sanger sequencing works), so as the sequence from one primer starts becoming crappy, you then have a good quality sequence to use. Secondly, sequencing errors may occur in one sequencing reaction but not in the reaction going the opposite direction. Realistically, the sequencing reactions should either be given separate PCR amplifications as input or further replicated to also rule out false-positive variation calls due to PCR error as well. So, by having sequence reactions run in both directions, you're cutting down the error rate of variation calls.

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            • #7
              Originally posted by dpryan View Post
              There's no real Sanger sequencing equivalent to paired-end reads in NGS. What you have are sequences originating from different primers run in different wells. There are two main reasons to do that. Firstly and as mentioned above, base-call quality decreases with the length (this makes sense if you visualize how Sanger sequencing works), so as the sequence from one primer starts becoming crappy, you then have a good quality sequence to use. Secondly, sequencing errors may occur in one sequencing reaction but not in the reaction going the opposite direction. Realistically, the sequencing reactions should either be given separate PCR amplifications as input or further replicated to also rule out false-positive variation calls due to PCR error as well. So, by having sequence reactions run in both directions, you're cutting down the error rate of variation calls.
              That is a real fantastic answer! Thanks!

              Comment

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