Unconfigured Ad

Collapse
X
 
  • Time
  • Show
Clear All
new posts
  • OllyBolly
    Junior Member
    • Jan 2011
    • 7

    #1

    454 problem with adapter trimming and MIDs

    We've had some odd trimming results and I thought I'd check whether anybody else has had a similar experience.

    I received raw 454 reads from our provider with the A & B adapters removed (presumably via the 454 pipeline). I have MIDs on both the 5' and 3' end of the reads to facilitate multiplexing. I've noticed, however, that reads with MID 16, 18, 19, and 22 on the 3' end of the read have their terminal (i.e. 3') T nucleotide missing. Depending on how you demultiplex, this can mean that MIDs are not correctly read & sorted. The problem seems restricted to the MIDs I've used that begin with T

    It seems something to do with trimming the B adapter because MIDs that I have on the 5' end of the read, such as MID 10 that also have a terminal T (i.e. 5'), retain the T.

    Anybody else come across this?
  • ajthomas
    Senior Member
    • Mar 2010
    • 167

    #2
    I haven't ever come across that, and I'm not sure why it might be happening either. My first thought was that the software might be thinking it is part of the adapter sequence for some reason and trim it off, although I can't think of why it would do that. Nevertheless, I'm not sure you need to figure out the cause in order to fix it. If you got the .sff files from your provider, the data is still there. You may be able to put those missing bases back on by changing the trim position with sfffile. If your provider gave you the whole D directory, you could try reprocessing the data, perhaps with the shotgun pipeline or changing other parameters, and see if you can get the data trimmed in the right position.

    Comment

    • OllyBolly
      Junior Member
      • Jan 2011
      • 7

      #3
      Thanks AJ.

      Actually the barcode splitter/sorter in Geneious that I've used seems to deal with the missing base anyway, so luckily no major disruption, but if not for that I could have ended up with skewed results. Just something for people to be aware of.

      Comment

      • JackieBadger
        Senior Member
        • Mar 2009
        • 385

        #4
        We had the same problem coming from outsourcing to a 454 Junior run.
        Lucky for you that the MIDs are distinguishable at 7bp. Just trim both MIDs down in your barcode file

        Comment

        • OllyBolly
          Junior Member
          • Jan 2011
          • 7

          #5
          Good idea -thanks.

          Comment

          Latest Articles

          Collapse

          • SEQadmin2
            Beyond CRISPR/Cas9: Understand, Choose, and Use the Right Genome Editing Tool
            by SEQadmin2



            CRISPR/Cas9 sparked the gene editing revolution for both research and therapeutics.1 But this system still showed severe issues that limited its applications. The most prominent were the heavy reliance on PAM sequences, delivery limitations, double-stranded breaks that prompt unintended edits and cell death, and editing inefficiency (both in targeting and in knock-in reliability).

            Despite this, “CRISPR helped turn genome editing from a specialized technique into
            ...
            07-31-2026, 11:01 AM
          • SEQadmin2
            Proteomic Platforms: How to Choose the Right Analytical Strategy to Improve Detection and Clinical Applications
            by SEQadmin2


            Proteomics platforms are evolving rapidly, with advances in mass spectrometry and affinity-based approaches expanding what researchers can detect and at what scale. As the field moves toward deeper proteome coverage and clinical applications, scientists face an increasingly complex landscape of tools. This article will explore how researchers are navigating these choices to find the right platform for their work.

            The systematic characterization of the human proteome has
            ...
            07-20-2026, 11:48 AM
          • SEQadmin2
            Advanced Sequencing Platforms Tackle Neuroscience’s Toughest Genomics Problems
            by SEQadmin2



            Genomics studies in neuroscience face a special challenge due to the brain’s complexity and scarcity of samples. Mapping changes in cell type and state using conventional next-generation sequencing methods remains challenging. Advances in technologies like single-cell sequencing, spatial transcriptomics, and long-read sequencing have opened the door to deeper studies of the brain and diseases like Alzheimer’s, amyotrophic lateral sclerosis (ALS), and schizophrenia.
            ...
            07-09-2026, 11:10 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by SEQadmin2, Yesterday, 10:13 AM
          0 responses
          14 views
          0 reactions
          Last Post SEQadmin2  
          Started by SEQadmin2, 07-31-2026, 02:55 AM
          0 responses
          27 views
          0 reactions
          Last Post SEQadmin2  
          Started by SEQadmin2, 07-24-2026, 12:17 PM
          0 responses
          20 views
          0 reactions
          Last Post SEQadmin2  
          Started by SEQadmin2, 07-23-2026, 11:41 AM
          0 responses
          19 views
          0 reactions
          Last Post SEQadmin2  
          Working...