Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • De-multiplexing, carryover from sample with highest reads?

    Hello,
    I use in house indexes (5nt) to tag my amplicons and pool 10 such samples and subject the pooled "sample" to library prep on TruSeq without fragmentation.

    After demultiplexing for Illumina indexes on Miseq, FASTX barcode splitter is used to de-multiplex the samples to the original "samples". These reads are then aligned with Bowtie and the result we need is a proportion of reads aligned aganist a particular reference sequence.

    When the final alignment results are observed, in all samples there is a carry over of some reads from the sample with the highest number of reads to the rest of the samples in the same 10 sample pool.

    I checked to see if this is associated with any particular pattern of the in house barcodes, it is not. In all the samples, it is the reads from the sample with "highest number of reads" that are also found in other samples.

    Has anyone else experienced something similar in sample pooling and is there any other software that has a more stringent algorythm for barcode splitting.

    Not sure if the post is confusing

  • #2
    There is a past thread on this topic. You can find it here: http://seqanswers.com/forums/showthread.php?t=29110

    Illumina's official response was in post #38 in the thread above.

    Comment


    • #3
      Yes this is a common problem with pooling e.g. http://genomebiology.com/content/13/5/R34

      We have just had a paper accepted which outlines a method to deal with it. I could send it to you if you inbox me your email

      Comment


      • #4
        GenoMax, thank you, I read this earlier but more that that our problem is what Jackie has pointed out.

        Comment

        Latest Articles

        Collapse

        • seqadmin
          Exploring the Dynamics of the Tumor Microenvironment
          by seqadmin




          The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
          07-08-2024, 03:19 PM
        • seqadmin
          Exploring Human Diversity Through Large-Scale Omics
          by seqadmin


          In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
          06-25-2024, 06:43 AM

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by seqadmin, Today, 07:20 AM
        0 responses
        12 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 07-16-2024, 05:49 AM
        0 responses
        32 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 07-15-2024, 06:53 AM
        0 responses
        36 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 07-10-2024, 07:30 AM
        0 responses
        41 views
        0 likes
        Last Post seqadmin  
        Working...
        X