Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Demultiplexing dual-indexed MiSeq fastq files

    Hi all. I received .fastq files from an overlapping-paires paired-end MiSeq run. I joined the pairs together using FLASH. However we used dual-indexing, so now I've got sequences that look like this

    5' barcode1-link-primer1-----sequence-----primer2-link-barcode2 3'

    (actually its more like
    5' barcode1-link-primer1-----sequence-----2remirp-link-2edocrab 3' because of the reverse & join!)

    So I need to demultiplex these (including primer removal). Does anybody know of any good programs that can demultiplex dual-indexed .fastq files? Any and all help much appreciated.

  • #2
    Sorry, but something is not right here or I'm not quite following...

    If you did Illumina dual indexing you should have the reads and indexes in 4 separate (FASTQ) files (read 1, 2, 3 and 4). You should not have any indexes in your reads, unless you did additional barcoding?

    If you did, then the barcode is supposed to be after the primer site (where the sequencing primer binds) or else it would not get sequenced.

    Here's a handy pic of standard dual indexing:


    In case you did barcoding, you only want to remove the barcode from the beginning of each read, which you can do with almost any trimmer, before overlapping the reads. If you already overlapped you can use something like http://chipster.csc.fi/manual/prinseq-trimmer.html

    Here's a demultiplexer for dual indexed reads:
    Last edited by Guest; 08-18-2013, 12:49 PM.

    Comment


    • #3
      Thanks lorendarith.

      Sorry. That's my fault. I used the word "primer" instead of "adaptor". So it should really read:

      barcode1-link-adaptor1-----sequence-----adaptor2-link-barcode2 3'

      We used our own barcodes, not Illumina.

      All I've got is 2 files, for reads 1 and 2 (no 3 or 4).

      The problem is that both barcodes are required to identify which sample a read came from (i.e. beginning for readfile1 AND beginning of readfile2). So if I trim before merging the two reads, I need to make sure that I keep all of the pairing information so that I can still tell which samples they are from. So I'm not sure these programs will help but I'll take a closer look.
      Thank you.

      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
      51 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