Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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

    Comment


    • #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

        Comment


        • #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

            Comment

            Latest Articles

            Collapse

            • seqadmin
              Strategies for Sequencing Challenging Samples
              by seqadmin


              Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
              03-22-2024, 06:39 AM
            • seqadmin
              Techniques and Challenges in Conservation Genomics
              by seqadmin



              The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

              Avian Conservation
              Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
              03-08-2024, 10:41 AM

            ad_right_rmr

            Collapse

            News

            Collapse

            Topics Statistics Last Post
            Started by seqadmin, Yesterday, 06:37 PM
            0 responses
            10 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, Yesterday, 06:07 PM
            0 responses
            9 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-22-2024, 10:03 AM
            0 responses
            49 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-21-2024, 07:32 AM
            0 responses
            67 views
            0 likes
            Last Post seqadmin  
            Working...
            X