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  • 454 GS FLX Titanium 'paired end' protocol

    Hi,
    I know this is an obsolete technology, but I came across some data produced on the 454 instrument (archived in SRA/ENA) which I'd like to use for assembly.
    According to the meta data, insert sizes are 8kb and 20kb, but I wasn't able to figure out the protocol used to produce the libraries. I gather that this is some variant on what we'd now call mate-pair, but I'm not sure if I can treat the data exactly the same way as data coming from Illumina mate-pair libraries, in terms of read orientation, insert size distribution etc.
    Had anyone worked with such data, or can direct me to some documentation about it? I failed to find anything in the current Roche docs.
    Thanks!

  • #2
    Originally posted by soungalo View Post
    Hi,
    I know this is an obsolete technology, but I came across some data produced on the 454 instrument (archived in SRA/ENA) which I'd like to use for assembly.
    According to the meta data, insert sizes are 8kb and 20kb, but I wasn't able to figure out the protocol used to produce the libraries. I gather that this is some variant on what we'd now call mate-pair, but I'm not sure if I can treat the data exactly the same way as data coming from Illumina mate-pair libraries, in terms of read orientation, insert size distribution etc.
    Had anyone worked with such data, or can direct me to some documentation about it? I failed to find anything in the current Roche docs.
    Thanks!

    Wow, time to fire up the WayBack Machine Mr. Peabody.

    Yes, 454 "Paired End" technology is similar in design to Illumina Mate Pair tech. A large fragment is circularized with a linker, the circular DNA is fragmented and the piece with the linker is enriched. It is then sequenced and the linker sequence is identified to separate the "forward" and "reverse" reads. I have attached a 454 data processing manual; look at section 4.6 for information about paired end data.

    Here are some links to previous thread on SeqAnswers discussing 454 paired end reads:

    Pyrosequencing in picotiter plates, custom arrays for enrichment/decomplexing. (Roche)

    Pyrosequencing in picotiter plates, custom arrays for enrichment/decomplexing. (Roche)


    This latter thread includes a post with the linker sequences (post #2). There are different linker sequencing depending which generation of the library prep kit was used, FLX or Titanium.
    Attached Files

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    • #3
      Is the 454 Paired End technology beginner friendly, so to speak? How difficult is it to get into?
      Experts are blown away by these golf rangefinders for many reasons.

      Comment


      • #4
        Originally posted by DonnellM View Post
        Is the 454 Paired End technology beginner friendly, so to speak? How difficult is it to get into?
        See the OP. The 454 is obsolete. Reagents no longer available to run the instrument. Hard to get less "beginner friendly" than "dead".

        --
        Phillip

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        • #5
          In the age of long read sequencing (PacBio, Nanopore) and linked reads (10X Genomics), "mate pair" sequencing is as obsolete as 454.

          Comment


          • #6
            I wouldn't go that far! Cheap (no-agarose-gel) Illumina mate-pair libraries are still legitimate, if trailing-edge, technologies.

            454 is *dead*, dead.

            --
            Phillip

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